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Stimulation of map kinases and S6 kinases by sodium selenate and vanadyl sulphate Farahbakhshian, Sepehr 1994

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STIMULATION OF MAP KINASES AND S6 KINASESBY SODIUM SELENATE AND VANADYL SULPHATEbySEPEHR FARAHBAKIISHIANB.Sc., The University of British Columbia, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESFaculty of Pharmaceutical SciencesDivision of Pharmacology and ToxicologyWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1994© Sepehr Farahbalchshian, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)t,i1o/‘c1cec14/c,ecr)9epartment ofThe University of British ColumbiaVancouver, CanadaDate A/ 3j9C)t/DE-6 (2/88)ABSTRACTInsulin has a wide range of biological effects on mammalian cells including bothmetabolic and mitogenic actions. A phosphorylation cascade originating at the insulin-receptor and involving a number of different serine/threonine-protein kinases is believed tomediate, at least in part, some of these effects. The two best studied kinases within thisphosphorylation cascade, MAP kinases and S6 kinases, are believed to play pivotal roles ininsulin signal transduction. Both selenium and vanadium compounds have been shown tohave insulin-mimetic effects on isolated rat adipocytes. In providing further evidence fortheir insulin-mimetic properties, their effects on the activity of MAP kinases and S6 kinasesin isolated rat adipocytes were examined by measuring the phosphorylation of myelin basicprotein (MBP) and Ribosomal S6 Protein, respectively.Both MBP kinases and Ribosomal Protein S6 kinases were shown to be activated inresponse to insulin treatment of adipocytes thus confirming the suitability of this system forthe investigation of insulin-mimetic agents. Sodium selenate and vanadyl sulphate treatmentof cells led to dose and time-dependent stimulation of both MBP kinases and RibosomalProtein S6 kinases. Maximal stimulation of MBP kinases by sodium selenate was -‘2-foldcontrol while Ribosomal Protein S6 kinases were stimulated to over 8-fold control. Vanadylsulphate treatment led to higher levels of stimulation with MBP kinase activity being -‘5-foldcontrol and Ribosomal Protein S6 kinase activity reaching levels that were greater than 16-fold control. Anion-exchange chromatography of the crude cell extracts revealed severaldistinct peaks of MBP and Ribosomal Protein S6 kinase activity corresponding to previousreports in the literature, however no distinct kinase families were conclusively identifiedusing immunological techniques.Our results further confirm the insulin-mimetic properties of selenium and vanadiumcompounds. Both were shown to stimulate kinases within the signal transduction cascade ofinsulin to a greater degree than insulin itself. The distinct families of MAP- and S6 kinases111stimulated by these agents were not identified although the presence more than one family foreach group of kinases was indicated.ivTABLE OF CONTENTSPgeABSTRACT iiTABLE OF CONTENTS ivLIST OF.FIGURES ixLIST OF ABBREVIATIONS xiiACKNOWLEDGEMENTS xivDEDICATION xvINTRODUCTION II. VANADIUM 1A. Background 1B. Forms 1C. Insulin-mimetic Effects 21. In Vitro 22. In Vivo 3D. Mechanism of Action 4II. SELENIUM 5III. MAP KINASES 7IV. S6 KINASES 9A. rsk 10B.70S6K 11VV. UPSTREAM KINASES .12A. MAP Kinase Kinase 13B. rafKinase Family 14C. Ras 16VI. RATIONALE 18A. Hypothesis 18B. Objectives 19MATERIALS AND METHODS 20MATERIALS 20A. Animals 21II. METHODS 21A. Ribosomal Protein S6 kinase Substrate Preparation 211. Ribosomal 80S subunit preparation 212. Ribosomal 40S subunit isolation 22B. Adipocyte Isolation and Incubation 231. Adipocytes isolation 232. Time-course 243. Dose-response curve 244. Genistein and Rapamycin experiments 25C. Phosphocellulose Paper Assays 25D. Ribosomal Protein S6 Kinase Assays 26E. Protein Assays 26vi1. Lowry protein assays .262. Bio-Rad reagent 27F. Chromatography 27G. Western Blotting 271. BCIP/NBT coloring reagent method 272. ECL 28H. Statistical Analysis 28RESULTS 30MBP KINASES 30A. Time-Course 301. Insulin 302. Sodium selenate 303. Vanadyl sulphate 31B. Dose-Response 311. Insulin 312. Sodium selenate 313. Vanadyl sulphate 32C. Mono Q Anion-Exchange Chromatography and Immunoblotting 321. Sodium selenate 322. Untreated control 333. Vanadyl sulphate 334. Vanadyl sulphate time-course 34viiII. RIBOSOMAL PROTEIN S6 KINASES .34A. Time-Course 341. Insulin 352. Sodium selenate 353. Vanadyl sulphate 35B. Dose-Response 361. Insulin 362. Sodium selenate 363. Vanadyl sulphate 36C. Mono Q Anion-Exchange Chromatography 371. Sodium selenate 37a. S6-peptide kinase 37b. Ribosomal Protein S6 kinase 382. Untreated control 38a. S6-peptide kinase 38b. Ribosomal Protein S6 kinase 383. Vanadyl sulphate time-course 39a. S6-peptide kinase 39b. Ribosomal Protein 86 kinase 40D. Immunoblotting 40III. GENISTE1N EXPERIMENT 41A. MBP kinases 41VIIIB. Ribosomal Protein S6 kinases .42IV. RAPAMYCIN EXPERIMENTS 42A. MBP kinases 42B. Ribosomal Protein S6 kinases 43DISCUSSION 107I. MBP KINASES 107II. S6 K11’JASES 114111.120 CHROMATOGRAPHY AND IMMUNOBLOTTING 120A. MBP kinase Mono Q and Immunoblot Analysis 121B. S6 kinase Mono Q and Immunoblot Analysis 124CONCLUSIONS 129BIBLIOGRAPHY 130ixLIST OF FIGURESFigure PageI Insulin signal-transduction cascade 201 Time-course for the activation of MBP kinases by Insulin 452 Time-course for the activation of MBP kinases by Sodium Selenate 473 Time-course for the activation of MBP kinases by Vanadyl Sulphate 494 Comparison of the time-course of activation of MBP kinases by10 nM Insulin, 1 mM Vanadyl Sulphate, and 1 mM Sodium Selenate 515 Dose-response for the activation of MBP kinases by Insulin 536 Dose-response for the activation of MBP kinases by Sodium Selenate 557 Dose-response for the activation of MBP kinases by Vanadyl Sulphate 578 Elution profile of Sodium Selenate-stimulated MBP kinase activityResolved on a Mono Q anion-exchange column 599 Immunoblotting of Mono Q Fractions from Sodium-Selenate-stimulatedsamples with MAP kinase-specific antibodies 6110 Elution profile of Vanadyl Sulphate-stimulated MBP kinase activityresolved on a Mono Q anion-exchange column 6311 Mono Q elution profile of Vanadyl Sulphate-stimulated MBP kinaseactivity time-course 6512 Immunoblotting of Mono Q fractions from VanadylSulphate-stimulated samples 6713 Time-course for the activation of Ribosomal Protein S6 kinase by Insulin 69x14 Time-course for the activation of Ribosomal protein S6 kinase bySodium Selenate 7115 Time-course for the activation of Ribosomal protein S6 kinaseby Vanadyl Sulphate 7316 Comparison of the time-course of activation of Ribosomal ProteinS6 kinases by 10 nM Insulin, 1 mM Vanadyl Sulphate, and1 mM Sodium Selenate 7517 Dose-response for the activation of Ribosomal Protein S6 kinases by Insulin 7718 Dose-response for the activation of Ribosomal Protein S6 kinasesby Sodium Selenate 7919 Dose-response for the activation of Ribosomal Protein S6 kinasesby Vanadyl Sulphate 8120 Mono Q elution profile of Sodium Selenate-stimulatedS6-peptide kinase activity 8321 Mono Q elution profile of Sodium Selenate-stimulated RibosomalProtein S6 kinase activity 8522 Mono Q elution profile of Vanadyl Sulphate-stimulated S6-peptidekinase activity time-course 8723 Mono Q elution profile of Vanadyl Sulphate-stimulated RibosomalProtein S6 kinase activity time course 8924 Immunoblotting of Mono Q fractions from Sodium Selenate andVanadyl Sulphate-treated samples with S6K-CT antibody 91xi25 Immunoblotting of Sodium Selenate-treated samples using theECL detection system 9426 Immunoblotting of Vanadyl Sulphate-treated samples using theECL detection system 9727 Effects of Genistein on MBP kinase activity 10028 Effects of Genistein on Ribosomal Protein S6 kinase activity 10229 Effects of Rapamycin on MBP kinase activity 10430 Effects of Rapamycin on Ribosomal Protein S6 kinase activity 106xiiLIST OF ABBREVIATIONSATP adenosine triphosphateArg arginineBCIP 5-bromo-4-chloro-indolyl phosphateBSA bovine serum albuminCHO Chinese hamster ovaryCytPTK cytosolic protein tyrosine kinaseDMF N,N-dimethyl formamideDMSO dimethylsulfoxideDTT dithiothreitolEDTA ethylene diaminetetraacetic acidEGF epidermal growth factorEGTA ethylene glycol bis(b-aminoethyl ether-P-N, N, N’,N’-tetraacetic acid)ERK extracellular signal-regulated kinaseGAP GTPase activity proteinGDI GDP dissociation inhibitorGDP guanine diphosphateGDS guanine nucleotide dissociation stimulatorGrb2 growth factor receptor-bound protein 2GRF guanine nucleotide releasing factorGTP guanine triphosphateIR insulin receptorIRS-i insulin receptor substrate-iIRTK insulin receptor tyrosine kinaseLys lysineMAP mitogen-activated proteinXIIIMAP-2 microtubule-associated protein-2MBP myelin basic proteinMEK MAP kinase kinase or ERK kinaseMEKK MEK kinasemm minuteNBT nitroblue tetrazoliumNGF nerve growth factorp42erk 2 42-kDa MAP kinase encoded by ERK 2 genep44erk 1 44-kDa MAP kinase encoded by ERK 1 genep44mpk 44-kDa sea star MBP kinasePAGE polyacrylaniide gel electrophoresisPDGF platelet-derived growth factorPKA cyclic AMP-dependent protein kinasePKI peptide inhibitor of PKAPMSF phenylmethylsulfonyl fluoridePTPase protein tyrosine phosphataseRPM rounds per minuteRSK ribosomal S6 kinaseS6K S6kinaseSDS sodium dodecyl sulphateSOS son of sevenlessSTZ streptozotocinser serinesrc oncogene isolated from chicken Rous Sarcoma cellsthr threoninetyr tyrosinexivACKNOWLEDGEMENTSI would like to thank Dr. J.H. McNeilI for his support and advice throughout myproject.I am also indebted to my other committee members for their invaluable help.My special thanks goes to Dr. Y.J. Hei who was involved in every aspect of myresearch and without whose help this thesis would not be possible. His unending enthusiasmand energy made difficult experiments seem easy.The technical help of Mrs. X.S. Chen and Ms. V. Yuen was much appreciated. Inaddition, I am grateful to Dr. R. Thies for his timely help in my slide preparation and Mr. C.Weiz for general computer support. The preparation of the thesis could not be done withoutthe expert help of Mrs. S. Chan and Dr. S. Bhanot.I would also like to thank Dr. S. Pelech for his advice and for providing me with thematerials for certain experiments. In addition, I would like to thank Mrs. C. Palaty for herassistance in some experiments.My most heart-felt thanks go to my family for their unquestioning support and to allmy friends especially Mr. F. Mitschelle.xvDEDICATIONTo my family.the FAR4HBAKHSHIANSINTRODUCTIONI. VANADIUMA. BackgroundVanadium compounds were first recognized by Nils Gabriel Sefstrom in 1831. It isnow known that vanadium is one of the most abundant minerals in the earth’s crust with anaverage concentration of 100 ppm (Wilisky, 1990).Early on following its discovery, a significant amount of work was done on thephysiological actions of vanadium. By the late 1 800s vanadium was experimented with inthe treatment of diseases ranging from tuberculosis to diabetes (Wilisky, 1990). There wasgeneral lack of interest in the biological actions of vanadium for the greater part of thetwentieth century. In 1977, Cantely et al. demonstrated that vanadate was a potent inhibitorof NaJK ATPase which led to a resurgence of interest in the biological effects of vanadium.B. FormsVanadium is a group Vb element (Ramasarma and Crane, 1980). It may exist inoxidation states ranging form -3 to +5 (excluding -2), although only the +3 to +5 states areknown to exist in biological systems (Butler, 1990). The toxicity of vanadium formsincreases with increased valency (Wilisky, 1990). The two forms with the most biologicalrelevance are vanadate, +5 (V043),and vanadyl (VO+2). Both pH and concentration play arole in the determination of the forms present (Butler, 1990).Vanadate is the form most likely to be present at physiological pH (pH 6-8) (Willsky,1990). It exists as the monomericH2V04 and HV042ions in the jim concentration range,but can form the HV40123 at low mM concentrations (Willsky, 1990). Still higher2concentrations can lead to the formation of the V10028-6decavanadate ion. Vanadyl ismost stable in acidic pH (Ramasarma and Crane, 1980).The V-O bond in the vanadates closely resembles the P-O bond which may accountfor some of the observed inhibitory effects of vanadium on ATPases (Ramasarma and Crane,1980). The vanadyl ion, however, is not an effective inhibitor for ATPases. Following entryinto the cell, vanadate is converted to vanadyl (Nechay et al, 1986). This along with a seriesof studies showing insulin-mimetic properties of vanadium in adipocytes without inhibitionof Na/K ATPase, suggests that this inhibition is not largely responsible for the insulin-mimetic effects of vanadium (Dubyak and Kleinzer, 1980).C. Insulin-mimetic Effects1. In VitroTolman et a!. (1979) were the first to show the insulin-mimetic properties ofvanadium in isolated rat adipocytes. They reported the stimulation of glucose oxidation andtransport into adipocytes following vanadium treatment. In addition, they showed thestimulation of glycogen synthase and inhibition of gluconeogenesis in the rat liver. Both thestimulation of glucose transport and glycogen synthase in adipocytes by vanadium have beendetermined to be time and dose dependent (Dubyak and Kleinzeller, 1980; Tamura et a!.,1983). Vanadium has also been shown to inhibit lipolysis (Degani et at., 1981). In addition,vanadium compounds have been shown to stimulate amino acid transport (Munoz et al.,1992), DNA synthesis (Hon and Oka, 1980; Canalis, 1985), cell proliferation (Lau et at.,1988), and the cell cycle (Wice et a!., 1987). Vanadium compounds have also been shown toexert their insulin-mimetic effects on skeletal muscle preparations and isolated perfusedlivers (Clark et al., 1985). However, of even greater interest are the insulin-mimetic effectsof vanadium seen in intact animals.32. In VivoHeyliger et at. (1985) were the first to show the insulin-mimetic properties ofvanadium in the intact animal. Female wistar rats were made diabetic by a single I.V.injection of streptozotocin (STZ). STZ-diabetic rats were treated with vanadate over a fourweek period. Vanadium-treated STZ-diabetic rats had blood glucose levels similar to that ofcontrols whereas those of the untreated diabetics remained more than two-fold higher thancontrols. The anti-diabetic effects of vanadium in STZ-diabetic rats following chronictreatment, including reductions in blood glucose, triglyceride, and cholesterol levels, havesince been reported in numerous studies (Meyervitch et a!., 1987; Ramanadham et al., 1989;Pederson et al., 1989, Cam et aL, 1993). In addition, long-term vanadium treatment has beenshown to prevent or improve the tissue alterations seen in the chronic diabetic state, such asdecreased cardiac performance (Heylieger et aL, 1985; Ramanadham et a!., 1989), increasedglycerol output from adipose tissue (Ramanadham et al., 1989), and decreased glycogensynthase activity both in the muscle of partially pancreacttectomized rat (Rossetti andLaughlin, 1989) and in the liver of STZ-diabetic rats (Bollen et al., 1990).Vanadium treatment of STZ-diabetic rats also doubles glucose uptake in muscle andliver tissue of these animals (Schecter, 1990). The improvements in the various bloodparameters of the STZ-diabetic rats following vanadium therapy, correspond well to the invitro stimulation of glucose uptake (Cam et a?., 1993; Dubyak and Kleinzeller, 1980), andinhibition of lipolysis (Degani et a!., 1981) seen in isolated rat adipocytes. This suggests thatthe in vivo actions of vanadium are brought about, at least in part, by vanadium itself and arenot simply a result of the potentiation of insulin action. However, vanadium alone is notsufficient in keeping completely insulin-deficient rats alive (Battell et a?., 1992), indicatingthat not all of the effects of insulin are mimicked by vanadium or that not enough vanadiumcan be absorbed to fully replace insulin.4D. Mechanism of ActionIt has been suggested that the insulin-mimetic actions of vanadium may be mediatedvia the insulin receptor. Tamura et aL (1984) were the first to show the phosphorylation ofthe 95-kDa subunit of the insulin receptor on its tyrosine residues following the treatment ofintact cells with vanadium. In addition, they reported the stimulation of tyrosine kinaseactivity. Other groups have since reported dose-dependent activation of insulin receptortyrosine kinase, IRTK, following treatment of intact adipocytes with vanadium compounds(Fantus et al., 1990; Katoda et al., 1987; Gherzi et al., 1988). Vanadium was also shown toincrease JR autophosphorylation and tyrosine kinase activity in purified JR preparations(Gherzi et a!., 1988). There is growing evidence, however, that the effects of vanadiumobserved in intact cells are not mediated through the insulin receptor. Adipocytes with verylow numbers of insulin receptors are fully responsive to vanadium while being extremelyinsulin resistant (Green, 1986). A lack of correlation between vanadium’s insulin-mimeticproperties and JRTK activity in the rat adipocyte (Mooney et a!., 1989) and in the mousediaphram in vivo (Strout et a!., 1989) has also been shown. In addition, the presence of acytosolic protein tyrosine kinase (CytPTK) in rat adipocytes (Shishiva and Schecter, 1992)which is specifically activated by vanadate but not insulin (Shisheva and Sheeter, 1993) andwhose activation correlates well with vanadium’s stimulation of glucose oxidation andlipogenesis, suggests the presence of an JR-independent pathway for at least some of theeffects of vanadium. Some of the other insulin mimetic effects of vanadium such asstimulation of glucose uptake and inhibition of lipolysis are not facilitated via this kinase(Shisheva and Shechter, 1993), indicating the presence of more than one pathway throughwhich the insulin-mimetic actions of vanadium are brought about.The activity of protein tyrosine phosphatases (PTPases) has been shown to beregulated by insulin as a possible means of modulating insulin-stimulated metabolic andgrowth-related effects (Meyerovitch et al., 1992). Vanadium compounds are well known5PTPase inhibitors (Swamp et al., 1982; Lau et at., 1989). The dose and time-dependentinhibition of PTPases by vanadium compounds have been correlated with increased proteintyrosine phosphorylation and 3-0-methyiglucose transport in isolated rat adiocytes (Shishevaand Shecter, 1993). It is therefore a possibility that the enhanced tyrosine phosphorylation ofa series of proteins via the inhibitory effects of vanadium compounds on PTPases is involvedin mediating the insulin-mimetic properties of vanadium compounds.Protein tyrosine phosphorylations and tyrosine kinases are generally associated withthe early steps in the signal transduction of insulin and other growth factors. Proteinserine/threonine kinases, which account for a much greater percentage of the phosphorylationevents that occur within the cell, are believed to be more directly involved in the mediation ofthe effects of insulin (Czech et aL, 1988). It is therefore possible that an insulin-mimeticcompound such as vanadium may bring about some of its effects through the activation ofserine/threonine protein kinases. The Mitogen Activated Protein kinases (MAP kinases) andthe S6 protein kinases are two of the most widely investigated serine/threonine proteinkinases (Pelech and Sangera, 1992). MAP kinases have been shown to activate at least onefamily of S6 kinases (Sturgill et al., 1988). Vanadium compounds have been implicated inthe in vitro activation of MAP and S6 kinases (Tobe eta!, 1992).II. SELENIUMSelenium is a group VI element which can exist in +6, +4, +2, and -2 oxidation states(Ursini and Bindoli, 1987). There are strong physical and chemical similarities betweenselenium and sulphur allowing the former to substitute for the latter in certain biologicalsystems (Ursini and Bindoli, 1987). Selenium is considered to be an essential element withits deficiency resulting in a wide variety of effects ranging from compromised functioning ofthe immune system (Koller, 1986) to Keshan’s disease, a cardiomyopathy of unlcnownetiology prevalent in some selenium-deficient areas of China (Chen et al., 1980).6Selenium in association with glutathione peroxidase and vitamin E is believed to playan important anti-oxidant role (Ursini and Bindoli, 1987). A strong correlation betweenselenium levels and glutathione peroxidase activity and a negative correlation with lipidperoxide levels has been shown (Gromadzinka et al., 1988). Most studies on selenium havebeen centered around its anti-oxidant effects. Selenium, however, may also be necessary forthe proper functioning of the immune system (Koller, 1986), mitogenic stimulation oflymphocytes (Pamham et al., 1983; Sheffy and Schultz, 1983), and prevention of pancreaticatrophy (Bunk and Combs, 1986). In addition, selenium is an essential nutrient for thegrowth of human fibroblasts in cell culture (McKeehan Ct al., 1976) and can support cellproliferation in cell free media.Although the role of selenium in glutathione peroxidase activity is well establishedand a number of selenoproteins have been identified (Chen et a!., 1980), the effects ofselenium as an insulin-mimetic agent or as a growth factor are just beginning to beinvestigated. Ezaki (1990) was the first to report a number of insulin-mimetic effects ofsodium selenate in isolated rat adipocytes, which included: the time and dose-dependentstimulation of glucose transport, translocation of glucose transporters to the plasmamembrane, stimulation of cAMP phosphodiesterase activity, tyrosyl phosphorylation of anumber of cellular proteins, and the phosphorylation of the ribosomal S6 protein. Morerecently, Pillay and Makgoba (1992) have shown the phosphorylation of the epidermalgrowth factor (EGF) receptor and the stimulation of its kinase activity in A43 1 cells bysodium selenate. In addition, they have shown that incubation of intact NIH-3T3-HIR3.5cells with sodium selenate leads to enhanced phosphorylation of the insulin B-subunit and a185 kDa, protein believed to be the insulin receptor substrate-i (IRS-l), following insulintreatment. These in vitro insulin-mimetic effects are in part supported by work done on intactanimals. MeNeill et a!. (1991) have reported the in vivo insulin-mimetic effects of sodiumselenate in STZ-diabetic rats. They showed a decrease in plasma glucose levels, food intake,and fluid intake to near control levels in STZ-diabetic rats treated chronically with sodium7selenate. This appears to be the only report in the literature that is concerned primarily withthe insulin-mimetic properties of selenium in the intact animal at this time.III. MAP KINASESExtracellular signal-regulated protein kinases (ERK) also known as mitogen activatedprotein kinases (MAP kinases) are a family of serine/threonine-protein kinases (ser/thrprotein kinases) believed to be essential in intracellular signaling processes. They are highlyconserved and nearly ubiquitous in a diverse range of organisms from yeast to mammals(Nishida and Gotoh, 1993). Ray and Sturgill (1987) were the first to report the insulinstimulation of a soluble serine(ser)/threonine(thr)-protein kinase in serum-starved 3T3 -Liadipocytes that facilitated the phosphorylation of microtubule-associated protein 2 (MAP-2)in vitro. Further work resulted in the partial purification of a 40-42 kDa kinase requiringphosphorylation on both tyrosine and threonine residues for full activation (Ray and Sturgill,1988a;b). In addition, MAP kinase was shown to phosphorylate and activate S6 kinase II,another insulin-stimulated kinase, obtained from Xenopus laevis (Sturgill et al., 1988). Thefact that MAP kinases were phosphotyrosine activated and were able to stimulate S6K TIledsome investigators to believe that they might serve as “switch kinases” transferring signalsfrom tyrosine kinases associated with cell surface receptors to downstream ser/thr proteinkinases thereby bringing about the metabolic and/or mitogenic effects (Sturgill et a!., 1988).However, other reports identifring the MAP kinase activator as a ser/thr kinase have refutedthe “switch kinase” hypothesis (Kosaka et al., 1992; Seger eta!., 1992).At least six different MAP kinases ranging in molecular mass from 40 kDa to 64 kDahave been identified (for review see; Pelech and Sanghera, 1992). Extracellular signal-regulated kinase 1 (ERK 1) orp44erkl was first purified as a 43 kDa protein from rat 1 HIRcB cells stimulated with insulin (Boulton et a!., 1991). ERK 2, with nearly 90% homologywith ERK 1 was cloned from a rat brain cDNA library (Boulton et a!., 1990). A 54 kDa8MAP kinase was also obtained from the livers of rats treated with cycloheximide (Kryiakisand Avruch, 1990). It shares about 50% homology with ERK 2 in the catalytic domain(Pelech and Sanghera, 1992). Another MAP kinase, p44mPk (maturation-activated myelinbasic protein kinase), isolated from sea star oocytes (Sanghera et al., 1990), shares nearly77% homology with ERK 1 and ERK 2. In addition, there are several yeast protein kinaseswhich show a relatively high degree of homology with ERK 1 and ERK 2 suggestingsequence conservation (Pelech and Sanghera, 1992).As mentioned above, there are many members within the MAP kinase family presentin a wide variety of species. All, however, are believed to share the common requirement ofphosphorylation on both tyrosine and threonine residues for full activity, except for p44m$which is not threonine phosphorylated (Anderson et a!., 1990; Sanghera et a!., 1991; Scimecaet al., 1991). The phosphorylation sites generally have the “TEY” (threonine-glutamic acidtyrosine) amino acid sequence order with a threonine separated from tyrosine with a glutamicacid (Nishia and Gotoh, 1993). Thr-204 and Tyr-206 are phosphorylated in ERK 1 (Her eta!., 1991), whereas Thr-183 and Tyr-185 are phosphorylated for ERK 2 (Payne et a!., 1991).MAP kinases also share among themselves a similar substrate specificity and are regarded asproline-directed kinases (Hall and Vulliet, 1991). Myelin basic protein (MBP) and MAP-2serve as excellent substrates for MAP kinases with the recognition determinant having the -Pro-Xaa-Ser/Thr-Pro (where Xaa represents one or two basic andlor neutral amino acids)motif (Gonzales et a!., 1991; Alvarez et al., 1991). Both are often used as substrates formeasuring MAP kinase activity. A number of substrates for MAP kinases have beenidentified in physiological systems, such as:p9Orsk from Xenopus oocytes (Aim et al., 1990),c-jun and c-Myc proteins (Alvarez et al., 1991), Tau protein (Drewes et a!., 1992), EGFreceptor (Alvarez et al., 1991) cPLA2 (Lin et al., 1993), and many other proteins, most ofwhich are involved in signal transduction. From the list of substrates above, it can be seenthat MAP kinase might be involved in the regulation of protein kinases, proteins involved intranscription control, and membrane receptors. In addition, they are likely to play a role in9the induction of differentiation, expression of oncogenes, and G0 to G1 transition (Pelech andSanghera, 1992). Due to the eclectic nature of the effects of MAP kinases and their targets, itis not surprising that a wide variety of substances stimulate MAP kinase activity. Includedare most agonists whose receptors are coupled to tyrosine kinases such as growth factors andlymphokines, activators of protein kinase C, and inhibitors of protein-ser/thr and tyrosinephosphatases such as okadaic acid and vanadate (Pelech and Sanghera, 1992; Cobb et a!.,1991; Haystead eta!., 1990; Scimeca eta!., 1991).IV. S6 KINASESThe role of the ribosomal S6 protein in cellular functioning is not yet clear, althoughit is believed to be involved in the initiation of protein synthesis. It is one of the manyproteins that are activated through phosphorylation on serine residues following treatment ofintact cells with various growth-factors such as epidermal growth factor, platelet-derivedgrowth factor, and insulin (Smith et a!., 1980; Rosen et a!., 1981). The in vivophosphorylation sites of the S6 protein have been shown to be Ser 235, Ser 236, Ser 240, Ser244, and Ser 247 which reside in between Mg 231 and Lys 249 near the carboxyl terminus ofthe molecule (Bandi et aL, 1993). Two families of S6 kinases, the p9O’’ and the 70S6Khave been well characterized in a number of studies (for reviews see Mailer, 1990; Erikson,1991; Strurgill and Wu, 1991; Avruch et a!. ,1991). Recently, a 31 kDa insulin-stimulated S6kinase, distinct from p9OrSk and the7oS6K, has been purified and characterized from ratskeletal muscle (Hei et al., 1994). This kinase has been reported to be the main insulin-stimulated S6 kinase in rat skeletal muscle although it has not yet been cloned and sequenced(Hei eta!., 1993).10A. rskErikson and Mailer (1985) were the first to identify a protein kinase specific forribosomal S6 protein with a molecular weight of approximately 92,000 from Xenopus eggs.This protein kinase was able to phosphorylate all of the sites in the S6 protein associated withgrowth-promoting stimuli in contrast to other protein kinases such as cAMP-dependentkinase which only phosphorylated some of the S6 protein sites (Erikson and Maller, 1985).This kinase was shown to be activated in Xenopus oocytes by progesterone and insulin, aswell as by the microinjection of purified insulin-receptor tyrosine kinase (Cicirelli et al.,1990; Stefanovic et al., 1986). Subsequent purification of the kinase on a DEAE-Sephacelcolumn yielded two peaks of activity which were termed S6 kinase I (eluted at 90 mM NaC1)and S6 kinase II (eluted at 160 mM NaC1) based on the order of elution (Erikson and Maller,1986). Molecular cloning of S6 kinase II led to the discovery of two distinct eDNAfragments with 91 % homology, but more importantly, S6 kinase II was shown to have theunusual characteristic of having two apparent catalytic domains (Jones et aL, 1988). Theamino terminal catalytic domain, believed to be the main site involved in phosphotransferaseactivity, showed similarity to catalytic sites of protein kinase C, cAMP-dependent proteinkinase, and cGMP-dependent protein kinase, while the other catalytic domain resembled thatof phosphorylase B (Jones et aL, 1988). S6 kinase II homologues were then found in avianand murine eDNA libraries (Alcorta et a!., 1989) and a 90 kDa protein that was functionallyand immunologically related to S6 kinase II was found in chicken embryo fibroblasts (Sweetet al., 1990). These homologous enzymes were placed in one family and referred to asribosomal S6 protein kinases or rsk. Another member of this family isolated from insulin-stimulated rabbit skeletal muscle (Lavoinne et al., 1990), has recently been shown to be100% homologous to its mouse counterpart (Sutherland et a!., 1993). It is likely that morehomologues are in the process of being identified at the present time.11B.Although Xenopus rsk homologues are present in mammalian cells, another family ofS6 kinases, referred to as7oS6k, are believed to be the more dominant S6 kinase in thissystem. Early reports identified the presence of a 60-70 kDa insulin-stimulated kinase with avery high specificity towards ribosomal S6 protein in 3T3-Ll cells (Cobb, 1986). Thiskinase was determined to be distinct from several other protein kinases capable of in vitrophosphorylation of the S6 protein based on its substrate specificity and chromatic properties,in addition to its strong dependency on the presence of 3-g1ycerophosphate in thehomogenization buffer for the measurment of its activity (Cobb, 1986). An insulin-stimulated S6 kinase was also reported in rat hepatoma H4 cells (Nemenoff et a!., 1986).Blenis et al. (1987), reported the presence of a 65 kDa S6 kinase in developing chickenembryos and chicken embryo fibroblasts (CEF) which was activated by the tyrosine-specificprotein kinase of Rous sarcomma virus (pp60Sl), phorbal esters, and various growth factors.The above mentioned kinases are believed to be the same as the 70 kDa kinase identified andcharacterized in Swiss mouse 3T3 cells (Jeno et al., 1988). This kinase was activatedfollowing treatment of the cells with epidermal growth factor (EGF), sodium orthovanadate,and serum. The tryptic phosphopeptide maps of the in vitro phosphorylation of ribosomal S6protein by this kinase resembled those of the S6 protein phosphorylated in vivo, suggesting aphysiological role for the kinase. Subsequent reports have identified an approximately 70kDa kinase specific for ribosomal S6 protein in regenerating rat liver, cycloheximide-treatedrat liver, and insulin-treated rabbit liver (Nemenoff et al., 1988; Kozma et al., 1989; Gregoryet a!., 1989). In all cases, the enzymes had similar chromatographic properties, substratespecificity, and were extremely susceptible to inactivation by phosphatase 2A. In addition,activation of the enzyme was shown to be dependent on its specific phosphorylation (Price eta!., 1990). This family of approximately 70 kDa S6 kinases,7oS6K, were shown to bedistinct from the previously described p9O’ S6 kinase family based on molecular size,12chromatographic properties, phosphopeptide maps, lack of immunological cross-reactivity,and molecular cloning (Chen and Blenis, 1990; Banerjee et al., 1990). The molecularcloning of7oS6K revealed the presence of one catalytic domain resembling that of proteinkinase C which was 56% homologous to its rsk counterpart and showed that the two enzymefamilies are structurally quite distinct (Banerjee et aL, 1990).As previously mentioned, MAP kinases have been shown to be the upstreamactivators of the rsk family (Sturgill et a!., 1988; Chung et a!., 1991). Gregory et al. (1989)reported the partial reactivation of7oS6K inactivated with phosphatase 2A using activeMAP kinase. This, however, is contrary to a number of different reports which suggest thatMAP kinase is not the upstream activator of 70S6K (Blenis, 1991; Ballou et al., 1991;Chung et a!., 1992). Therefore the activator of the7oS6K family remains elusive, althoughevidence suggests the involvement of a ser/thr kinase (Price et a!. ,1990; Ferrari et aL, 1991).V. UPSTREAM KINASESGrowth factor signal transduction is believed to involve a number of proteins such asmembrane tyrosine kinases, Ras, Raf-l-kinase, MAP kinase kinase, MAP kinase, and rsk,interconnected in a complex and not necessarily linear network as shown in Figure I(Williams and Roberts, 1994). The activation of S6 kinases by MAP kinases (Sturgill et a!.,1988) was one of the first examples of a ser/thr kinase signal transduction cascade. Notsurprisingly, these two groups of kinases have been used as a pivotal point from which otherlinks in the signal transduction pathway of insulin and other growth-factors can be identified.13A. MAP Kinase KinaseMAP kinase kinases or ERK kinases (MEKs) are believed to be the immediateupstream activators of MAP kinases and are activated through the phosphorylation of theirtyrosine and threonine residues (Kosaka et a!., 1992; Seger et al., 1992; Anderson et al.,1990; Payne et a!., 1990). Previous experiments had already reported a factor whichactivated MAP kinase through tyrosine/threonine phosphorylation (Aim et a!., 1991).Subsequent experiments demonstrated the sequential activation of MAP kinase activator,MAP kinase, and a S6 peptide kinase in rat liver following insulin injection (Tobe et a!.,1992). MEK was first purified from insulin-stimulated rabbit skeletal muscle as a 45 kDaprotein (Nakienly et aL, 1992). Other homologues including Xenopus oocyte and yeasthomologues have also been identified suggesting that MEK is highly conserved (Matsuda eta!., 1992; Crews and Erikson, 1992). MEK is a novel enzyme in that it is the first dualspecificity tyr/thr kinase identified (Nakienly et a!., 1992; Crews and Erikson, 1992). It isinactivated following treatment with phosphatase 2A, suggesting that it is activated throughser/thr phosphorylation (Matsuda et a!., 1992). MEK is the only known physiologicalsubstrate of Raf-1 kinase, a ser/tbr kinase protooncogene (Kryiakis et aL, 1992). Raf-1kinase is believed to be the main upstream activator of MEK (Kryiakis et aL, 1992; Dent eta!., 1992). Recently, a Ras(a GTP-activated protooncogene)-dependent MEK kinase (REK)which activates MEK in a Raf-1-independent manner has been identified (Itoh et a!., 1993).MEK can therefore be activated through at least two separate Ras-dependent pathways whichmay work in conjunction or separately. There are also Ras-independent pathways. Forexample, the proto-oncogene product Mos can also phosphorylate and activate MEK (Posadaeta!., 1993).14B. rafKinase FamilyRaf-1 kinase, or c-raf-1, is the ubiquitously expressed —74 kDa cellular homologue ofthe v-raf oncogene believed to be a mediator of growth factor and differentiation signaltransduction ( Bonner et a!., 1985; Morrison et a!., 1990). Two other members of the rafkinase family have been isolated, A-raf, a 68 kDa protein generally found in the epididymisand urogenital tissue, and 95 kDa B-raf, most abundant in brain and testicular tissue (Beck eta!., 1987; Ikawa eta!., 1988; Nishida et a!., 1988).Raf-l is the best studied member of the raf kinase family. Raf-l kinase is a 648amino acid protein divided into three conserved regions CR1, CR2, and CR3 (Heidecker eta!., 1990). CR1 is a cysteine-rich region homologous to the lipid-binding site of proteinkinase C, while CR2 is a ser/thr-rich region possibly involved in raf-1 kinase activation(Ishikawa et a!., 1988). CR3 is the region responsible for the kinase activity (Hanks et a!.,1988). Truncation of both the CR1 and CR2 regions results in constitutively active kinaseactivity by CR3 (Stanton eta!., 1989; Heidecker et al., 1990) suggesting some regulatory rolefor CR1 and CR2.Morrison eta!. (1988) were the first to show that Raf-1 kinase was an intermediate inthe signal transduction cascade of growth factors. In their experiments they demonstratedthat treatment of NIH-3T3-HIR cells with PDGF, FGF, and EGF led to hyperphosphorylationof Raf- 1. In addition, they provided evidence that Raf- 1 was downstream of PKC and thetransforming oncogenes v-src, v-sis, and v-ras but upstream from nuclear oncogenes v-fosand v-myc. Insulin and other growth factors have also been shown to activate Raf-1 kinaseby increasing its phosphoserine content in HeLa, NIH-3T3-HIR, and Chinese hamster cellsoverexpressing the human insulin receptor (Kovacina et a!., 1990). Further evidence for theimportance of Raf-1 in signal transmission stemmed from experiments identifying Raf-1 asthe upstream activator of MEK (Kryiakis et a!., 1992; Dent et a!., 1992). Cells transformedwith the v-Rafoncogene displayed constitutively high levels of MEK activity while activatedL5Raf was able to phosphorylate and activate MEK over 30-fold in vitro (Kryiakis et al., 1992).Other evidence suggests that the role of Raf-l kinase in MEK activation and signalpropagation may be tissue and cell-type specific. For example, rat PC 12 cells, induced toexpress activated Raf, did not show significant activation of MAP kinases or rsk (Wood etal., 1992). In addition v-Raftransformed rat fibroblasts also did not show constitutively highlevels of MAP kinases while NIH-3T3-HIR mouse cells did (Gallepo et a!., 1992). In a morerecent experiment with RCR cells mutated to block Raf-1 kinase expression, ERK1 andERK2 were activated to the same extent as the NRK parent cell line discounting Raf-1 kinaseas the only upstream activator of MAP kinases (Kizaka-Kondoh and Okayama, 1993).Another MEK kinase which activates MEK has also been identified in Xenopus oocytessuggesting the presence of more than one activator of MEK (Matsuda et a!., 1992).Interestingly, in the same study MAP kinase was also shown to phosphorylate MEK,although no activation occurred. MAP kinases have also been shown to phosphorylate Raf-1kinase in vitro although no activation of Raf-1 was reported (Anderson et al., 1991).The cellular homologue of the oncogene v-ras, 21ras has been implicated in theactivation of Raf- 1 kinase (Blenis, 1993). Ras has been shown to lie upstream of Raf- 1 withcells transformed with v-ras showing Raf-1 hyperphosphorylation (Morrison et a!., 1988).Other investigators have shown, however, that although ras increases the sensitivity of Raf-1to growth factors, it alone is not sufficient to cause Raf-1 hyperphoshporylation (Reed et a!.,1991). Based on other experiments involving the activation of Raf-1 through v-src (atyrosine kinase oncogene) in a ras-independent manner, it seems possible that activation ofRaf-1 involves ras as well as a tyrosine kinase element (Williams and Roberts, 1994)16C. RasThe Ras group of proteins have been implicated as key mediators of signals fromreceptor-associated tyrosine-kinases to downstream ser/thr-protein kinases and are thereforebelieved to play an important role in the regulation of cellular proliferation anddifferentiation (Satoh et a?., 1992). At least four different highly homologous 21 kDa Rasproteins (ie. H-Ras, K4A-Ras, K4B-Ras, and N-Ras) have been found in humans (Satoh etal., 1992). Ras proteins show a high affinity for guanine nucleotides and are believed to be inan active state when bound to GTP (Ras-GTP), whereas the Ras-GDP complex is associatedwith the inactive state (Bourne et a?., 1990). Three classes of proteins are presently known tobe involved in the control of Ras function via their role in the interconversion between RasGTP and Ras-GDP complexes. The first class, GTPase activating proteins (GAPs),negatively regulate Ras by stimulating the endogenous Ras GTPase activity therebyfavouring the formation of the Ras-GDP complex (Bollag and McCormick, 1991). Thesecond class of Ras regulators are known as guanine nucleotide dissociation stimulators(GDS) which allow the release of GDP from the inactive Ras-GDP complex and favour theformation of the active Ras-GTP state (Downward, 1992). The most recently identifiedgroup of Ras regulatory proteins are the Ras guanine nucleotide dissociation inhibitors (RasGDI)(Bollag and McCormick, 1993).Two Ras-specific GAPs that have been identified in mammalian tissue are p120 GAPand NFl GAP (Trahey et a?., 1988; Xu et a?., 1990). Both GAPs have been shown tosubstantially increase Ras-GTPase activity but are believed to be differentially regulated(Bollaga and McCormick, 1991). Mutations in the GAP-binding region of Ras leading tolack of Ras binding to GAPs have led to the inhibition of Ras signal propagation suggestingthat GAPs may also serve as downstream effectors of Ras (DeClue eta?., 1991). The bindingof Ras to p120 is believed to cause conformational changes in the p120 molecule which arenecessary for the transmission of signals to downstream targets of Ras (Martin et a?., 1992).17It therefore seems that GAPs serve the dual function of being both regulators of Ras functionand mediators of its signal.Another important class of Ras regulators are the GDS proteins which favour therelease of GDP from the Ras-GDP complex and the formation of the active Ras-GTP form.A number of different GDSs have been identified in various organisms ranging from theyeast GDS, CDC25, and the Drosophila GDS, son-of-sevenless (SOS), to the human andmouse homologues of SOS, hSOS and mSOS1 and 2 (Jones et al., 1991; Bowtell et a!.,1992). The translocation of Ras to the inner face of the plasma membrane, brought aboutthrough a series of posttranslational modifications including proteolysis,carboxylmethylation, palmitoylation, and farnesylation, is essential to Ras biological activity(Khosravi-Far et al., 1992). It has therefore been suggested that the regulation of Ras viaGDSs involves the translocation of the GDS proteins via adapter molecules such as growth-factor receptor-bound protein 2 (Grb-2) (Lowenstein et al., 1992). Grb-2 is believed to beassociated with cytoplasmic GDSs via the association of its two SH3 domains and theproline-rich present in GDSs (McCormick, 1993). Following the stimulation of receptortyrosine kinases by their respective ligands and their subsequent autophosphorylation, theGrb-2-GDS complex associates with the autophosphorylated receptor or a membrane-boundsubstrate of the receptor, such as IRS-i for the insulin receptor, thereby allowing theactivation of membrane-associated Ras by GDS (McCormick, 1993; Tobe et al., 1993). Thethird class of Ras regulators, GDI, are believed to be inhibitors of GDS proteins which do notsignificantly affecting GAP activity (Bollag and McCormick, 1993).Ras-GTP formation has been shown to be brought about by the interaction of anumber of different growth-factors with their respective receptors which can affect either/orGAP and GDS activity depending on the ligand and the cell system (for review; Khosravi-Farand Der, 1994). Following Ras activation, the signal from receptor tyrosine-kinases canpropagate to the downstream ser/thr-protein kinases which have been discussed in theprevious sections. Raf- 1 kinase has already been mentioned as the initial ser/thr-protein18kinase in the cascade, and MAP kinase and rsk have been used as a pivotal point from whichother kinases were investigated. There are, however, other Ras-independent means ofactivating MAP kinases and Ras undoubtedly has downstream targets other than ser/thrprotein kinases.VI. RATIONAlEInsulin has a wide variety of biological functions ranging from its role in glucosehomeostasis in the intact animal to its growth-factor and mitogenic effects in cell culture.Although no consensus has been reached on exactly how the actions of insulin are broughtabout, a ser/thr-protein kinase cascade, also present in the signal transduction pathway ofother growth-factors, is believed to play a role in mediating at least some of the effects ofinsulin. MAP and S6 kinases are pivotal points within this pathway with their stimulationserving as a consistent step in growth-factor signal transmission. Both vanadium andselenium have insulin-mimetic properties in the intact animal as well as the isolated ratadipocyte. The mechanism of action of these two elements, however, has not yet been frillyelucidated. Therefore, by investigating the effects of vanadyl sulphate and sodium selenateon the ser/thr-protein kinase phosphorylation cascade of isolated rat adipocytes through themeasurement of MBP and Ribosomal Protein S6 kinase activity, we hope to gain someinsight into the mechanism of action of vanadium and selenium as well as to provide furtherevidence for the insulin-mimetic properties of these two elements.A. HypothesisThe insulin-mimetic compounds vanadyl sulphate and sodium selenate will stimulateMAP and S6 kinases, key enzymes involved in the ser/thr-protein kinase signal transductioncascade of insulin, in the isolated rat adipocyte.19B. Objectives1) To investigate the possible stimulation of MAP and S6 kinases in isolated ratadipocytes by sodium selenate and vanadyl sulphate in a time and dose-dependent manner.2) To gain insight into the relative contribution of the different MAP kinase and S6kinase families in the overall activity observed in the crude cell extracts by the use of MonoQ anion-exchange chromatography and immunoblotting.3) To gain insight into the role of tyrosine kinases in the activation of MBP andRibosomal Protein S6 kinases by vanadyl sulphate and sodium selenate using the tyrosinekinase inhibitor genistein.4) To gain insight into the level of activity of the 70S6K family of Ribosomal ProteinS6 kinases by using the specific70S6K inhibitor rapamycin.20I’,Insulin Signal-Transduction CascadeINSULINRECEPTOR‘I,TranslationV??Inacti Active RasI4-’TranscriptionNUCLEUS21MATERIALS AND METHODSI. MATERIALSAprotinin, ATP, benzamidine, bovine insulin, bovine serum albumin (fraction V 98-99% albumin), collagenase type II, D-glucose, dithiothreitol (DTT), Dulbecco’s ModifiedEagle Media Base, EDTA, EGTA, leupeptin, MOPS, myelin basic protein (MBP), N,Ndimethylformamide (DMF), pepstatin A, peptide inhibitor of cAMP-dependent protein kinase(PKI), phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, soyabean trypsininhibitor, B-glycerophosphate, and B-methyl-aspartic acid were purchased from SIGMAchemical company. Acrylamide, 5-bromo-4-choloro-indolyl phosphate (BCIP), ammoniumpersulfate, Bio-Rad protein reagent, Coomasie blue stain, L-glycine, N,N’-methylene-bisacrylamide, nitroblue tetrazolium (NBT), nitrocellulose membrane (0.45 micron), sodiumdodecyl sulphate, and B-mercaptoethanol were purchased from Bio-Rad Laboratories.Sodiumselenate and vanadyl sulphate hydrate were purchased from Aldrich Chemicals.[y32PjATPwas purchased from ICN chemicals. S6-10 peptide (AKRRRLSSLRASTSKSESSQK) wasgenerously provided by Dr. Ian Clark-Lewis (Biomedical Research Laboratories,Vancouver,Canada). Rapamycin was kindly provided by Dr. S. Sehgal of Wyeth-AyersthResearch.The following anti-kinase rabbit antibodies were made in the laboratory of Dr. StevePelech using the synthetic peptides shown below:GLAYIGEGAYGMVYKAC (GEGA);EFQDFVNKCLVKNPAERADLKC (MAPKK-9);PFEHQTYCQRTLREIQILLGFRHENVIGIRDILRAPGGC (Ri);CGGPFTFDMELDDLPKERLKELIFQETARFQPGAPEAP (R2);AMIVRNAKDTAHTKAERNILEEVKHPGGCC (S6K-III);CLVKGAMAATYSALNSSKPTPQLKPIESSILAQRRVRKLPSTTL (rsk-CT).22A. AnimalsMale Sprague-Dawley rats weighing 150-220g were used in all experiments.Animals were obtained either from Charles-River (Montreal, Canada) or U.B.C. animalbreeding facilities. All animals were food-deprived overnight prior to the day of theexperiment.II. METHODSA. Ribosomal Protein S6 kinase Substrate PreparationThe preparation procedure was based on that of Krieg et a!. (1988) and is describedbelow in detail.1. Ribosomal 80S subunit preparationOne-hundred grams of frozen liver from food-deprived male Sprague Dawley ratswere used in each preparation. The livers were ground to a powder using a mortar and pestlecooled with liquid nitrogen. The powder was then suspended in 200 ml of homogenizationbuffer (20 mM Tris-HC1 (pH 7.4) 100 mM KCI, 5 mM MgC12, 1 mM dithiothreitol, 1%Triton X-100, and 1% sodium deoxycholate), and disrupted with a polytron homogenizer(setting 5) for 20 second bursts until a homogeneous suspension was formed.The homogenate was centrifuged at 9000xg for 20 minutes at 4°C. The supernatantwas distributed in 29.5 ml aliquots into 38.5 Quick Seal Tubes (Beckman) containing 4 ml ofbuffer A (5 mM Tris-HC1 (pH 7.4), 500 mM KCI, 2.5 mM MgC12, 0.5 M sucrose, 1 mMdithiothreitol, 1% Triton X-100, and 1% deoxycholate) and 5 ml of buffer B (same as buffer23A except for the sucrose concentration which was I M) was then carefully added to the top ofthe homogenate using a long glass pipet. The tubes were sealed and centrifuged for 16 hoursat —‘200,000xg and 4°C.After the supematant was removed and discarded, the tubes were inverted andcentrifuged at 3000 RPM for 10 seconds in a table-top clinic centrifuge. This allowed for theseparation of the ribosomal pellet, which remained firmly fixed to the bottom of the tube,from the extraneous jelly-like material that slid to the top of the tubes and was removed withKimwipes. Following this removal, 1 ml of buffer C (20 mM Tris-HCI (pH 7.4), 100 mMKC1, 5 mM MgC1, and 1 mM dithiothreitol) and three glass beads were added to each tube.The tubes were placed on a table-top shaker and rotated at 100 RPM for 4 hours at 4°C inorder to resuspend the pellets. The 80S suspensions were then pooled and stored at -70°Cuntil they were used for 40S subunit preparation.2. Ribosomal 40S subunit isolationThe isolation of the 40S ribosomal subunits involved the dissociation of the 80Sribosomal subunits followed by sucrose gradient centrifugation. The process is describedbelow in detail. The dissociation cocktail was made by adding 3 ml of the 80S suspension to20 ml of buffer C, 894 mg KCJ, 30 mg puromycin and the volume made up to 30 ml withbuffer C. The cocktail was then incubated at 37°C in a shaking water bath for 30 minutes.Stock sucrose solutions containing 7.5% and 37.5% sucrose dissolved in dissociation buffer(500 mM KC1 (pH 7.4), 3 mM MgCl2,and 4 mM dithithreitol) were used to make six 32.5ml continuous sucrose gradients with the aid of a BRL gradient former. To each gradienttube, 4.8 ml of the dissociation cocktail was added and the tubes were centrifuged at87,000xg for 16 hours at 4°C.Following the centrifugation, a hole was made into the bottom of each gradient tubeand thirty 1 ml fractions were collected. From each fraction, 20 pi was added to 2 ml of24distilled water and absorbance measurements were made at 260 and 280 nm. When theA260/A8 ratio was plotted versus the fraction number, two distinct peaks corresponding to60S and 40S subunits were observed. The fractions containing the second and considerablysmaller peak were pooled and centrifuged at 225,000xg for 4 hours at 4°C. The pellets werethen resuspended in 1-3 ml of buffer C using a Teflon hand homogenizer. The proteincontent and the A260 of the suspension were measured in order to assess the purity of thepreparation. The suspensions were stored at -70°C until used as substrate for RibosomalProtein S6 kinase assays.B. Adipocyte Isolation and Incubation1. Adipocyte isolationRat adipocytes were isolated using a modified version of Rodbell’s method (1964).Male Sprague Dawley rats (weighing 150-220g) were fasted overnight before beingsacrificed either by a blow to the head followed by decapitation or with CO2 gas. Theirepididymal fat pads were quickly excised and placed in warm Dulbecco’s Modified EagleMedia (DMEM) containing 2% BSA and 2 mM glucose (pH 7.4, 37°C).The pads were then cut into pieces 5 mm in diameter and incubated in mediacontaining 0.75 mg/nil crude collagenase (type II). The pieces were digested for 3 5-40minutes while shaking at 80 cycles/minute in a 37°C water bath. The resultant slurry wasthen filtered through a 250 jim nylon mesh in order to remove undigested fat and otherdebris. The filtrate was centrifuged at very low speed (—300 RPM) for 30 seconds forming afat-cell layer above the aqueous infamate. The infarnate was removed using a 19 gaugeneedle attached to a 20 ml syringe and warm media was added to the cells. The adipocyteswere ‘washed’ twice more in this manner in order to remove any residual collagenase. The25cells were then equilibrated for 30 minutes in order to remove any effects that the digestionprocess may have had on them.2. Time-courseAll incubations were made in a 37°C shaking water bath using 290 jil of cellsuspension and 10 jil of stock solution; 300 nM insulin, 30 mM sodium selenate, or 30 mMvanadyl sulphate. The final concentration of the agents and their respective time-points wereas follows: 10 nM insulin; 0, 2.5, 5, 10, 15, 20, and 30 minutes; 1 mM sodium selenate; 0,2.5, 5, 10,13, 16, 19, and 27 minutes; and 1 mM vanadyl sulphate 0, 2.5, 5, 10, 20, and 30minutes. All time-points contained untreated controls. The incubations were stopped withthe addition of 1.25 ml of ice-cold homogenization buffer (25 mM MOPS (pH 7.2), 10, mMEGTA, 2 mM EDTA, 75 mM B-glycerophosphate, 1 mM sodium orthovanadate, 1 mMdithiotbriethol, and the following protease inhibitors; 1 mM PMSF, 3 mM benzamidine, 10iM leupeptin, 0.75 M aprotinin, and 100 g/ml soybean trypsin inhibitor) followed by thedisruption of the cells using a polytron (setting 5, 3 seconds). The cellular extracts werecentrifuged for 60 minutes at 19,000xg and the supematant was stored at -70°C to be assayedlater for kinase activity and protein content.3. Dose-response curveIsolated rat adipocytes were incubated with different concentrations of insulin,sodium selenate, and vanadyl sulphate for 5, 10, and 20 minutes, respectively. These optimaltime-points for each agent were based on the results obtained from the time-courseexperiments. As before, all incubations were done in a 37°C shaking water bath using 290 .tlcell suspension and 10 iil stock solutions and were stopped with the addition of 1.25 ml ofice-cold homogenization buffer followed by cell disruption. The final concentrations of26insulin used were 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM. The sodium selenateconcentrations used were 100 nM, 1 jiM, 10 jiM, 100 jiM, 1 mM, and 10 mM. The vanadylsulphate concentrations were 31.6 jiM, 100 jiM, 316 jiM, 1 mM, 3.16 mM, and 10 mM.4. Genistein and Rapamycin experimentsDuring these experiments, adipocytes were incubated for 30 minutes with 200 nMgenistein or 50 ng/ml rapamycin prior to a 20 minute incubation with either 1 mM sodiumselenate or 1 mM vanadyl sulphate. All other aspects of these experiments were the same asthose described above.C. Phosphocellulose Paper AssaysPaper assays were used for measuring MBP kinase and S6-peptide kinase activity.The assay cocktail contained 20 mM MOPS (pH 7.2), 25 mM 13-glycerophosphate, 5 mMEGTA, 2 mM EDTA, 20 mM MgCI2,2 mM sodium orthovanadate, 1 mM dithiothreiotol,500 nM protein kinase inhibitor, and one of the following substrates; myelin basic protein (1mg/mi) or S6-10 peptide (0.25 mg/ml). The following procedure was used in allphosphocellulose paper assays. Five jil of cellular extract (5-10 jig) was added to 15 ji.i ofthe cocktail. The reactions were initiated with the addition of 5 jil of[y32P]ATP (250 jiM,specific activity 2000 cpmlpmol) and allowed to proceed for 10 minutes at 30°C. Thereactions were stopped by spotting 20 jil of the reaction mixture onto a P-8 1phosphocellulose paper square (2X2 cm). The paper squares were then repeatedly washedwith 1 % phosphoric acid in order to remove non-specific ATP binding and counted usingliquid scintillation counting.27D. Ribosomal Protein S6 Kinase AssaysThe assay cocktail used was identical to that described above except for the use of0.35 jiM purified rat liver 40S ribosomal subunit as substrate. The reactions were startedwith the addition of 5 iil of[y32P]ATP (15 jiM, specific activity 10,000 cpmlpmol). Afterbeing allowed to proceed for 30 minutes at 30°C, the reactions were stopped with theaddition of 20 jil of SDS sample dilution buffer (10% SDS, 20% glycerol, and 5 % Bmercaptoethanol dissolved in 0.125 mM Tris-HC1, pH 6.8). The samples were then boiledfor 4 minutes and loaded onto 12.5% polyacrylamide mini-gels. Gel electrophoresis wasconducted using a Bio-Rad Protein II cell. The bands were visualized using Coomasiebrilliant blue stain and the 32-kDa S6 protein band was excised and counted using liquidscintillation counting.E. Protein AssaysAll protein assays were performed using either the Bio-Rad protein reagent or theSigma Lowry assay kit. In either case, a standard curve range between 13.9 ji.g-141 jig wasused and 20 jil of cellular extract were measured in duplicate for each sample.1. Lowry protein assaysAll tubes containing either protein standard or sample were diluted to 1 ml withdistilled water. One milliiter of Lowry reagent was then added and the tubes were allowed tostand at room temperature for 20 minutes. With rapid and immediate mixing, 0.5 ml of Folin& Ciocalteu’s Phenol Reagent Working Solution were added to each tube and colour wasallowed to develop for 30 minutes. Absorbance values were measured at 750 nm and proteinconcentrations were determined using a standard curve.282. Bio-Rad reagçntAll tubes were made into a final volume of 0.1 ml using distilled water. The reactionwas started with the addition of 5 ml of diluted Bio-rad dye reagent to each test tube. Thetubes were vortexed and allowed to stand for at least 10 minutes at room temperature beforeabsorbance measurements at 595 nm were made. Protein concentrations were againmeasured using a standard curve.F. ChromatographyA Pharmacia LKB Biotechnology inc. FPLC (fast protein liquid chromatography) anda Pharmacia MonoQ column (HR515) were used at 4°C for all adipocyte cell extractchromatographic fractionations. Two to three mg of protein were applied at a rate of 1ml/min to the column which was equiliberated with buffer A (10 mM MOPS, 25 mM Bglycerophosphate, 5 mM EGTA, 2 mM EDTA, 2 mM sodium orthovanadate, and 2 mMdithiothreitol). The column was developed at the same flow rate with a 15 ml linear NaClgradient (0-800 mM) in buffer A. Sixty 250 iil fractions were collected and used for kinaseactivity measurements and for immunoblotting.G. Western Blotting1. BCIP/NBT coloring reagent methodSelected fractions from MonoQ runs of 1 mM vanadyl sulphate, 1 mM sodiumselenate, or control crude extracts were digested with an equal volume of SDS sample bufferfor 4 minutes at 100°C and applied to 11% polyacrylamide gels. Gel electrophoresis was29conducted overnight using a Bio-Rad Protein II cell at 15 mA per gel. The proteins in thegels were then transferred onto nitrocellulose membranes in a Hoefer transfer cell at 300 mAfor 3 hours. The membranes were briefly stained with Ponseau stain in order to visualizemolecular weight standard proteins before being incubated for 3 hours at room temperaturewith blocking solution (20 mM Tris-base (pH 7.5), NaCl 500 mM, 5% Skim milk, and 0.1%NaN3) using a Belico orbital shaker. They were then washed 2 times with TTBS (20 mMTris-base (pH 7.5), 500 mM NaC1, and 0.05% Tween-20) before being incubated overnightwith one of the following primary antibodies; S6K70111, S6K-rsk-CT, Ri, R2, or GEGA.The membranes were again washed 2 times with TTBS and incubated with the secondaryantibody (Bio-Rad goat anti-rabbit IgG alkaline phosphatase diluted 1:3000) for 2 hours.Following this incubation, the membranes were washed 2 more times with TTBS and rinsedwith TBS (20 mM Tris-HC1 (pH 7.5) and 500 mM NaC1). A BCIP (5-bromo-4-choloro-indoyl phosphate dissolved first in N,N-dimethylformamide) and NBT (nitroblue tetrazoliumdissolved first in 70% N,N-dimethylformamide) containing color development solution (100mM NaHCO3 (pH 9.8) and 100 iM MgCl2.6H0)was used to incubate the membranes for2-3 hours. Following the colour development, the membranes were washed with distilledwater and dried in between two pieces of blotting paper.2. ECLImmunoblots were developed using reagents and protocol provided in the AmershamLife Sciences ECL starter kit.H. Statistical AnalysisSome data are expressed as mean ± standard error of the mean (S.E.M.). Statisticalsignificance was determined by one way analysis of variance (ANOVA) followed byFischers LSD test used for comparing results for a given set of groups or using the Student30“t” test. A probability of less than 0.05 (p<0.05) was used as the level of statisticalsignificance.31RESULTSI. MAP KINASESA. Time-Course1. InsulinA 40% suspension of isolated rat adipocytes was incubated with 10 nM insulin for 0-30 minutes in order to establish a time-course for the stimulation of MBP kinase activity(Fig. 1). Initial activation of MBP kinase activity appeared to occurr at 2.5 minutes with a -1.3-fold stimulation over control although this was not statistically significant. At 5 minutes,maximal stimulation at -4.7-fo1d over control was observed. MBP kinase activity was notobserved at any of the later time-points, thus confirming the transient nature of insulin actionin this system as previously reported in the literature (Haystead et al., 1990).2. Sodium selenateA time-course of the stimulation of MBP kinase activity by selenium was carried outby incubating adipocytes with 1 mM sodium selenate for 0-30 minutes (Fig. 2). Initialactivation occurred at 2.5 minutes with a —1.3-fold increase over control. Maximalstimulation was observed after 10 minutes of incubation with nearly a two-fold increase overcontrol. In contrast to insulin, the MBP kinase stimulation by sodium selenate was relativelylong-lasting with —4.5-fold increase over control remaining even after 27 minutes ofincubation.323. Vanadvl sulphateRat adipocytes were treated with 1mM vanadyl sulphate for 0-30 minutes. After 10minutes of incubation activity was -‘4.5-fold control and at 20 minutes maximal stimulationoccurred with a ‘—5.5-fold increase over control (Fig. 3). Activity decreased after 30 minutesof incubation, however it remained over 2.5-fold that of control, and was higher than that ofeither insulin or sodium selenate at any time-point (Fig. 4).B. Dose-Response1. InsulinBased on the time-course, 5 minutes was chosen as the optimal time-point forobserving MBP kinase activation in isolated rat adipocytes by insulin. To obtain a dose-response curve, adipocytes were incubated for 5 minutes with the following concentrations ofinsulin; 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM (Fig. 5). Significant stimulationwas only observed at 10 and 100 nM with a —‘1.5-fold increase over control.2. Sodium selenateAdipocytes were incubated for 10 minutes with the following concentrations ofsodium selenate; 100 nM, 1 tiM, 10 1iM, 100 jtM, 1 mM, and 10 mM (Fig. 6). The 10minute time-point was chosen because significant stimulation of both MBP kinases andRibosomal Protein S6 kinases was present. MBP kinase activation by sodium selenate wasonly observed at 1 and 10 mM. At both concentrations the stimulation was approximately 2-fold higher than control.333. Vanadyl sulphateThe following concentrations of vanadyl sulphate were used in obtaining a dose-response curve for the effects of vanadyl sulphate on MBP kinase activity; 10 jiM, 36 jiM,100 jiM, 316 p.M. 1 mM, 3.16 mM, and 10 mM. Maximal stimulation of MBP kinases byvanadyl sulphate occurred at 1 mM with a ‘-‘5-fold increase over control (Fig. 7). No effectswere observed at lower concentrations. At 3.16 mM, MBP kinase activity dropped to -.4-foldcontrol and at 10 mM there was a further drop of activity to slightly more than 2-fold control.Therefore, there appears to be a very narrow range of concentrations at which the effects ofvanadyl sulphate on adipocyte MBP kinases can be observed.C. Mono Q Anion-Exchange Chromatography and ImmunoblottingCellular extracts from adipocytes stimulated with 1 mM sodium selenate, 1 mMvanadyl sulphate, or untreated controls were applied to a Mono Q anion-exchange columnand the collected fractions were assayed for MBP kinase activity using myelin basic proteinas the substrate. In addition, the fractions were probed with MAP kinase-specific antibodies.1. Sodium selenateFive peaks of MBP kinase activity were present in the Mono Q elution profile ofsodium selenate-stimulated samples (Fig. 8). The first peak was centered around fraction 17,226 mM NaCl, with 9 pmol/minlml of activity. Peak two was centered around fraction 26(346 mM NaC1) with 45 pmollminlml of activity. Immediately following it was peak three,which was centered around fraction 29 (386 mM NaC1) with 27 pmol/minlml of activity.Peaks four and five were 9 pmol/minlml each and were centered around fractions 38 (505mM NaCl) and 40 (532 mM NaCl), respectively.34Peak fractions with MBP kinase activity from the sodium selenate-stimulated sampleswere probed with the Ri antibody against MAP kinases (Fig 9). A 52 kDa band was presentin fractions 26, 27, 28, and 29 corresponding to the two major peaks of MBP kinase activity.A 50 kDa band was also present in fractions 26 and 27. No other bands were present in anyother fractions. A separate set of immunoblotts were subjected to the much more sensitiveECL detection system, although no bands were visualized (data not shown).2. Untreated controlThe Mono Q profile of extracts from untreated adipocytes had four peaks of interest(Fig. 8). Peak one corresponded with the second peak of sodium selenate-treated sampleswith 10 pmol/minlml of activity centered around fraction 27 (359 mM NaC1). Peak two with9 pmol/min!ml of activity was centered around fraction 32 (426 mM NaC1). Peak three,centered around fraction 48 (638 mM NaCI) was the most prominent peak in the controlMono Q profile with 31 pmollmin/ml of MBP kinase activity. This peak was not present inthe sodium selenate-treated samples. Peak four, centered around fraction 58 (771 mN1 NaC1)was the second largest control peak with 16 pmollminlml of activity.3. Vanadyl sulphateFour major peaks of MBP kinase activity were present in the Mono Q profile of thevanadyl sulphate-stimulated samples (Fig. 10). Peak one was centered around fraction 18(239 mM NaCl) with 10 pmol/minlml of activity. Peaks two and three both had 56pmol/min!ml of activity and were centered around fractions 26 (346 mM NaC1) and 29 (386mM NaCl), respectively. Peak four was centered around fraction 41(545 mM NaCI) with 9pmol/minlml of activity. All four peaks corresponded to the major peaks observed in thesodium selenate-stimulated samples.354. Vanadvl sulphate time-courseCellular extracts from adipocytes incubated for 10, 20, or 30 minutes and untreatedcontrol cells were applied to a Mono Q anion-exchange column (Fig. 11). A single majorpeak of activity was present for all time-points which were centered around fraction 26 (345mM NaC1). The 10 minute samples had 125 pmol/minlml of activity while at 20 minutesmaximal MBP kinase stimulation was present with 174 pmollminlml. MBP kinase activitywas 118 pmollminlml for the 30 minute samples.In the control samples, a broad peak ranging from fraction 6 (69 mM NaCl) tofraction 24 (166 mM NaCl) was observed with fraction 23 (152 mM NaC1) having themaximal MBP kinase activation, 79 pmol/minlml, was present. This peak was notreproduced in other experiments suggesting that it represented an experimental artifact.Mono Q fractions of vanadyl sulphate-stimulated samples from the time-course wereprobed with Ri and R2 antibodies against MAP kinase (Fig. 12). With the Ri antibody, adark 44 kDa band and a light 42 kDa band were visible in the pooled fractions 17-23. Inaddition, a 42 kDa band was present in the pooled fractions 24-29. Two bands were visiblefor the pooled fractions 17-23 with the R2 antibody, a very dark relatively broad 42 kDa bandand a lighter 38 kDa band. No bands were visible for the pooled fractions 24-29. With theuse of the ECL detection system, no relevant bands were visualized (data not shown).36II. RIBOSOMAL PROTEIN S6 KINASESA. Time-Course1. InsulinIsolated rat adipocytes were incubated with 10 nM insulin for 0-30 minutes. A slightstimulation of Ribosomal Protein S6 kinase activity was observed after 2.5 minutes ofincubation (Fig. 13) and increased activity continued with longer incubation times until amaximal was reached at 10 minutes, with a greater than 2-fold increase over control. At 15and 20 minutes of incubation, a 1.5-fold control level of activity remained, but after 30minutes of incubation no difference from control samples was apparent.2. Sodium selenateAdipocytes were incubated with 1 mM sodium selenate for 0-27 minutes. Initialactivation of Ribosomal Protein S6 kinases by sodium selenate occurred after 10 minutes ofincubation with a 3-fold increase over controls (Fig. 14). By 13 minutes, activity had risenslightly and it continued to increase at 16 minutes with nearly a 5-fold increase over control,but it was not until 19 minutes that the maximal stimulation of 8.5-fold control wasobserved. The activity decreased after 27 minutes but remained over 4-fold that of control.3. Vanadyl sulphateThe time points chosen for adipocytes treated with 1 mM vanadyl sulphate were 0-30minutes. Significant activation of Ribosomal Protein S6 kinases by vanadyl sulphate was notobserved until 10 minutes of incubation with an 8-fold increase over control (Fig. 15).37Maximal stimulation occurred after 20 minutes with a 17-fold increase over control. Activitydecreased after 30 minutes of incubation but remained 11-fold higher than control. As withMBP kinases, the Ribosomal Protein S6 kinase stimulation of vanadyl sulphate was higherthan insulin and sodium selenate (Fig. 16).B. Dose-Response1. InsulinIsolated rat adipocytes were incubated with the following concentrations of insulin; 1pM, 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM (Fig. 17). Ribosomal Protein S6 kinaseactivity was present at insulin concentrations of 1, 10, and 100 nM with 1.5, 1.6, and 1.9-fold increase over control, respectively. The lowest concentration at which RibosomalProtein S6 kinase activity could be detected was 10 times lower than that for which MBPkinase activity could be observed. This seems an unlikely possibility if the MBP kinaseswere directly upstream of the activated Ribosomal Protein S6 kinases activated and mayperhaps point to the7oS6k family as being responsible for the activity observed.2. Sodium selenateAdipocytes were incubated with 100 nM, 1 jiM, 10 1iM, 100 jiM, 1 mM, and 10 mMsodium selenate. The lowest concentration of sodium selenate causing Ribosomal Protein S6kinase activation in adipoctes was 100 jiM with a stimulation of over 2-fold control (Fig. 18).At the two higher concentrations used, 1 and 10 mM, a nearly 3-fold control level of activitywas present. As was the case with insulin, Ribosomal Protein S6 kinase stimulation occurredat a 10 times lower concentration for MBP kinase.383. Vanadyl sulphateThe following concentrations of vanadyl sulphate; 10 .tM, 36 riM, 100 jtM, 316 1iM,1 mM, 3.16 mM, and 10 mM, were used in obtaining a dose-response curve of RibosomalProtein S6 kinase activity (Fig. 19). Maximal stimulation occurred at a concentration of 1mM with a 1 4-fold increase over control and decreased to 9-fold control at 3.16 mM. Unlikethe MBP kinase dose response curve, which retained a 2-fold control activity when theconcentration was increased to 10 mM, no activity was detected at this high concentration.In addition, as was the case with insulin and sodium selenate, Ribosomal Protein S6 kinaseactivation occurred at a 10 times lower concentration, 316 tiM, than that required to stimulateMBP kinase activity with a 2-fold increase over control.C. Mono Q Anion-Exchange ChromatographyCellular extracts from adipocytes incubated with 1 mM sodium selenate, 1 mMvanadyl sulphate, and untreated controls were applied to a Mono Q anion-exchange column.Collected fractions were assayed for S6-peptide kinase and Ribosomal Protein S6 kinaseactivity using a synthetic S6 peptide and 40S ribosomal subunits as the respective substrates.1. Sodium selenatea. S6-peptide kinaseFour peaks of S6-peptide-kinase activity in the Mono Q profile of the Selenium-stimulated samples were of interest (Fig. 20). Peak one, centered around fraction 17 (226mM NaC1) was the largest with 22 pmollminlml of activity. Peaks two and three werecentered around fraction 28 (372 mM NaCl) and 31(412 mM NaC1) with 10 pmol/minlml of39activity each. Peak four was centered around fraction 45 (599 mM NaC1) with 9pmol/minlml of S6P-kinase activity.b. Rjbosomal Protein S6 kinaseFour relevant peaks of Ribosomal Protein S6 kinase activity were present in the Mono Qprofile of selenium-treated samples (Fig. 21). Peak one, corresponding to peak one of theS6P-kinase profile, had the highest activity with 357 fmollminlml centered around fraction17 (226 mM NaCl). Peaks two and three had 107 fmollminlml of activity each and werecentered around fractions 24 (319 mM NaCl) and 27 (359 mM NaCl), respectively. Peakfour was centered around fraction 36 (479 mM NaCl) with 142 fmol/minlml of activity.2. Untreated controla. S6peptide kinaseThe predominant peaks of S6P-kinase activity for the control samples were centeredaround fractions 44 (585 mM NaC1) and 46 (612 mM NaC1 ) with 16 pmollminlml of activityeach (Fig. 20). These peaks correspond to peak four in the Mono Q profile of the selenium-stimulated samples although their level of activity was nearly 2-fold higher.b. Ribosomal Protein S6 kinaseSimilar to the sodium selenate-treated samples, untreated control samples also had apeak centering around fraction 17 with 71 fmol/minlml of activity and at fraction 24 with 143fmol/minlml (Fig. 21). A 71 firiol/minlml peak corresponding to peak three of seleniumstimulated samples was centered around fraction 28, 372 mM NaC1. The largest control peak40was centered around fraction 44 (585 mM NaC1) with 286 fmol/min!ml of activity and wasnot present in selenium-treated samples.3. Vanadvl sulphate time-coursea. S6-peptide kinaseAt least six major peaks were present in the S6P-kinase Mono Q profiles of cellularextracts from adipocytes incubated with 1 mM vanadyl sulphate for 10, 20, or 30 minutes(Fig. 22). Peak one was centered around fraction 5 (55 mM NaCl) with the 20 minutesamples having the highest activity (48 pmol/minlml) followed by the 10 minute samples, 24pmol/minlml, and the 30 minute and control samples with 12 pmol/minlml of activity each.Peak two was centered around fraction 8 (97 mM NaCl) for the 20 minute samples with 42pmol/minlml of activity. It centered around fraction 9 (110 mM NaC1) with 28 pmollminlmlof activity for the 30 minute samples and was not present for the 10 minute and controlsamples. The 20 minute samples had the highest activity for peak three with 48pmol/minlml, centered around fraction 19 (249 mM NaC1). The 30 minute samples had thenext highest activity with 42 pmollminlml centered around fraction 29 (235 mM NaCl). Peakthree was centered around fraction 22 (290 mM NaC1) for the 10 minute samples with 27pmol/minlml of activity. Peak four, was centered around fraction 26 (345 mM NaCl) for thevanadyl sulphate-stimulated samples with 31 pmol/minlml for the 10 minute samples, 49pmol/minlml for the 20 minute samples, and 37 pmol/minlml for the 30 minute samples.Peak four was not present in control samples. Peak five, centered around fraction 28 (373mM NaCl) for all groups, was the largest S6P-kinase peak with 15 pmol/minlml for controls,27 pmol/minlml for the 10 minute samples, 53 pmol/minlml for the 20 minute samples, and22 pmol/minlml for the 30 minute samples. Peak six was only present in the 20 and 3041minute samples, centering around fraction 32 (428 mM NaC1) with 33 pmollminlml ofactivity.Ribosomal Protein S6 kinaseThe Mono Q profile of the vanadyl sulphate-treated adipocyte extracts had a singlemajor peak of activity encompassing fractions 17-24 (Fig. 23). The 10 minute samples hadthe largest peak with 892 fmol/minlml of activity centered around fraction 21(276 mMNaC1). The 20 minute samples had 654 thiol/minlml also centered around fraction 21. The30 minute samples had 536 fmol/minlml of activity centering around fraction 20 (262 mMNaC1).D. ImmunoblottingSelected Mono Q fractions from sodium selenate-stimulated and vanadyl sulphate-stimulated samples were probed with S6K-III and S6K-CT antibodies against S6 kinases.Bands were visualized using the BCIPINBT coloring reagents or the enhancedchemiluminiscense (ECL) detection system.No bands were present in the blots of Mono Q fractions from sodium selenate orvanadyl sulphate-stimulated samples probed with S6K-III antibody and developed usingBCIPINBT coloring reagents (data not shown). A series of bands were observed on theselenium blot probed with the S6K-CT antibody (Fig. 24A). Fraction 18, associated withpeak one of S6 kinase activity in the Mono Q profile, had a band at 52 kDa. Fractions 19 and20, also associated with peak one, had a band at 55 kDa. A 64 kDa band was present in allother fractions.With the vanadyl sulphate-stimulated samples probed with S6K-CT, a 55 kDa bandwas visible for fractions 17-23 using the BCIP/NBT coloring reagents (Fig. 24B). Due to the42detection limits of this system, large amounts of protein were loaded onto gels which led tooverloading and poor resolution.Much lower concentrations of proteins were used for the series of immunoblottsvisualized using the ECL detection system. Selenium-stimulated samples probed with theS6K-III antibody had a 68 kDa band present for fractions 17,18,19, and 20 with fraction 19having the heaviest band (Fig. 25A). Using the S6K-CT antibody, a 62 kDa band which washeavy for fractions 17, 18, and 19 and light for fractions 20 and 21 was present (Fig. 25B).In addition, a light 54 kDa band was present for fractions 17-20. Vanadyl sulphatestimulated samples yielded a 70 kDa band for fractions 17-2 1 when probed with the S6K-IIIantibody (Fig. 26A). Fraction 18 had the heaviest band while fraction 21 had the lightest.Using the S6K-CT antibody, a 69 kDa band was present in fractions 17, 18, 19, and 20 withfraction 18 having the darkest band (Fig. 26B).. In addition, a faint 73 kDa band was presentin fraction 16 and a 59 kDa band in fractions 18 and 39. The —70 kDa bands present in figure28A-D are similar enough in molecular mass and their pattern of appearance as to suggestthat they represent one band. These bands were visualized with the Ponzo stain used to markthe molecular weight standards suggesting that they may represent a contaminating proteinsuch as BSA (molecular mass 66.2 kDa) present in high concentrations in all the samples asopposed to trace amounts of S6 kinases.III. GENISTEIN EXPERIMENTGenistein, a tyrosine-kinase inhibitor, was used to gain further insight into theactivation of MAP (Fig. 27) and Ribosomal Protein S6 kinases (Fig. 28) by insulin, sodiumselenate, and vanadyl sulphate.43A. MBP kinasesThe minor activation of MBP kinases by insulin, ‘-1.5-fold control, was not affectedby the presence of 200 nM genistein (Fig. 27). Sodium selenate alone had modest effects,1.5-fold control, which were abolished with genistein treatment. There was also a —36%reduction in the stimulation of MBP kinases by vanadyl sulphate from 2.5-fold control to 1.6-fold control in the presence of genistein.B. Ribosomal Protein S6 kinasesInsulin stimulation of Ribosomal Protein S6 kinase activity was reduced from 2-foldcontrol to control levels in the presence of genistein (Fig. 28). There was only a smallreduction in the effects of sodium selenate. The 6-fold control Ribosomal Protein S6 kinaseactivity following vanadyl sulphate treatment was reduced to only 2-fold control in thepresence of genistein.IV. RAPAMYCIN EXPERIMENTSThe macrolide rapamycin selectively blocks the activation of the‘7oS6K kinasefamily without affecting pp9orsk kinases or the MBP kinases (Fingar et a!., 1993). In orderto help elucidate the contribution of the two above mentioned Ribosomal Protein S6 kinasefamilies in extracts from adipocytes stimulated with sodium selenate and vanadyl sulphate,rapamycin was used in the next series of experiments.44A. MBP kinasesRapamycin did not block the activation of MBP kinases incubated with either 1 mMsodium selenate or 1 mM vanadyl sulphate (Fig. 29). Sodium selenate alone caused a 1.8-fold increase over control and in the presence of 50 ng/ml rapamycin a 1.7-fold increase overcontrol was observed. Likewise, vanadyl sulphate caused a 1.5-fold increase in the absenceand a 1.4-fold increase in the presence of rapamycin.B. Ribosomal Protein S6 kinasesRibosomal Protein S6 kinase activity was affected by the presence of rapamycin.Sodium selenate caused a 1.9-fold increase in Ribosomal Protein S6 kinase activity whichwas reduced to 1.4-fold in the presence of rapamycin (Fig. 30). Vanadyl sulphate produced a2.2-fold increase over control which was not significantly reduced with rapamycin.45Figure 1Time-Course for the Activation of MBP kinases by Insulin.Isolated rat adipocytes were prepared by the method of Rodbell (1964) using collagenasedigestion of epididymal fat pads. A 40% suspension of the cells was incubated with 10 nMinsulin for 0-30 minutes. The incubations were terminated with the addition of ice-coldhomogenization buffer and cellular extracts prepared and assayed for MAP kinase activity,as described in Methods, using MBP as substrate. Each time-point was expressed as mean± S.E.M. of fold-control activity (n=3). The actual control value was 24 pmollminlmg. *denotes significant difference from control (p<0.05)46Activation of MBP Kinasesby 10 nM Insulin2*lit ii.0 II I0 5 10 15 20 25 30Time (minutes)47Figure 2Time-Course for the Activation of MBP kinases by Sodium Selenate.Adipocytes were prepared and assayed for MBP kinase activity as described in Fig. 1following their incubation with 1 mM sodium selenate for 0-30 minutes. Each time-pointwas expressed as mean ± S.E.M. of fold-control activity (n=3). The actual control valuewas 27 pmollminlmg. * denotes significant difference from control (p.cz0.05)48Activation of MBP Kinasesby 1 mM Sodium Selenate30I I I0 5 10 15 20 25 30Time (minutes)49Figure 3Time-Course for the Activation of MBP kinases by Vanadyl Sulphate.Adipocytes were prepared and assayed for MBP kinase activity as described in Fig. 1following their incubation with 1 mM vanadyl sulphate for 0-30 minutes. Each time-pointwas expressed as mean ± S.E.M. of fold-control activity (n=3). The actual control valuewas 16 pmol/minlmg. * denotes significant difference from control (p<O.O5)50Activation of MBP Kinasesby 1 mM Vanadyl Sulphate76 T-le 40QoL4 32Tii0I I0 5 10 15 20 25 30Time (minutes)51Figure 4Comparison of the Time-Course of Activation of MBP kinaseby 10 nM insulin. 1 mM Vanadyl Sulphate, and 1 mM Sodium Selenate.Compilation of figures 1-3. * denotes significant difference from control (p<O.05)52IVIBP Kinase Time-CourseComparisons70 Insulin*6- I __ Vanadyl• SulphateSodium5- * SelenateC 4CC-)*o 3-****2T T_/•___1 I —-—----—-- —0 I I I0 6 12 18 24 30Time (minutes)53Figure 5Dose-response for the Activation of MBP kinases by Insulin.Isolated rat adipocytes were incubated for 10 minutes with an insulin concentration rangeof 1 pM to 100 nM. Cellular extracts were prepared and assayed for MBP kinase activityas previously described. Each time-point was expressed as mean ± S.E.M. of fold-controlactivity (n=3). The actual control value was 24 pmollminlmg. * denotes significantdifference from control (p<O.05)54Activation of MBP kinasesby Insulin2.502.00*I— 1.50 -00c-)01.00 -0.500.00I I I I I I-13 -12 -11 -10 -9 -8 -7 -6LogM55Figure 6Dose-response for the Activation of MBP kinases by Sodium Selenate.Isolated rat adipocytes were incubated for 10 minutes with sodium selenate concentrationsranging from 100 nM to 10 mM. Cellular extracts were prepared and assayed for MBPkinase activity as previously described. Each time-point was expressed as mean ± S.E.M.of fold-control activity (n=3). The actual control value was 27pmol/minlmg. * denotessignificant difference from control (p<0.05)56Activation of MBP Kinasesby Sodium Selenate2.50**2.00 T— 1.5000C-)0,-1.00TI I,0.500.00 I I • I-8 -7 -6 -5 -4 -3 -2 -1LogM57Figure 7Dose-response for the Activation of MBP kinases by Vanadyl Sulphate.Isolated rat adipocytes were incubated for 20 minutes with vanadyl sulphate concentrationsranging from of 10 iiM to 10 mM. Cellular extracts were prepared and assayed for MBPkinase activity as previously described. Each time-point was expressed as mean ± S.E.M.of fold-control activity (n=3). The actual control value was 13 pmollminlmg. * denotessignificant difference from control (p<0.05)58Activation of MBP Kinasesby Vanadyl Sulphate6.004.80 LL\I \*I\TII’3.60*2.40 /I1.20 —0.00 I I I-5 -4 -3 -2 -1LogM59Figure 8Elution Profile of Sodium Selenate-stimulated MBP kinase ActivityResolved on a Mono Q Anion-Exchange Column.Extracts made from isolated rat adipocytes incubated for 20 minutes with 1 mM sodiumselenate and untreated controls were applied to a Mono Q anion exchange column anddeveloped using a linear NaC1 gradient from 0 to 800 mM in 60 fractions with a volume of0.25 ml each. Fractions were assayed for MBP kinase activity. The experiment wasrepeated two times although the data points represent a single experiment.C) C)MBPKinaseMonoQProfileSodiumSelenate—•—sodiumselenate—0—control60 50 40 30 20 100051015202530354045505560Fractionnumbera’ C61Figure 9Immunoblotting of Mono Q fractions from Sodium Selenate-stimulated sampleswith MAP kinase-specific Antibodies.Selected fractions from the elution profile of sodium selenate-stimulated samples wereresolved on 11% SDS-polyacrylamide gels, transferred to nitrocellulose paper, and probedwith the Ri antibody against MAP kinases. The blots were developed using alkalinephosphatase as the secondary antibody and BCIP/NBT colouring reagents. A 52 kDa bandwas present in fractions 26-29 corresponding to peaks one and two of MBP-kinase activityfrom Fig. 8. Lane assignments are shown below.Lane Fraction Number1 molecular weight standards2 163 174 185 256 267 278 289 2910 3011 3112 3213 4014 4115 42624SkDa-*31 kDa-+21.5 kDa +14.4 kfla +5 6 7 8 9 10 11 12 13 14 1563Figure 10Elution Profile of Vanadyl Sulphate-stimulated MBP kinase Activityon Mono Q anion exchange Column.Extracts of isolated rat adipocytes incubated for 20 minutes with 1 mM vanadyl sulphate oruntreated controls were resolved and assayed as described in Fig. 8. Two major peaks at346 and 386 mM NaC1, corresponding to the peaks seen for the sodium selenate-stimulatedsamples, were present with 56 pmollminlml of activity each. The experiment was repeatedtwo times although the data points represent a single experiment.I 0 0Ivanadylsulphate0conlrolMBPKinaseMonoQProfileVanadylSulphate70 60 5040E20 10 0051015202530354045505560Fractionnumber65Figure 11Mono Q Elution Profile of Vanadyl Sulphate-stimulatedMBP kinase Activity Time-Course.Extracts from isolated rat adipocytes incubated with 1 mM vanadyl sulphate for 10, 20, 30,or untreated controls were applied to a Mono Q anion-exchange column and developedusing a linear NaCl gradient of 0-800 mM ( 60 fractions with 0.2 ml volume each).Fractions were assayed as previously described. Data points represent a single experiment.C) a) 0ActivationofI\4BPKinasesbyVanadylSulphate200 1’ 100 001020304050Fractionnumber——10minutes—20minutes—+—30minutes0—Control60C’67Figure 12Immunoblotting of Mono Q Fractionsfrom Vanadyl Sulphate-stimulated Samples.Selected Mono Q fractions from the 20 minute sample of the time-course experiment wereresolved as described in Fig 9. Both Ri and R2 primary antibodies were used to probe thefractions. With the Ri antibody, a light 42 kDa band was present in pooled fractions 17-23and 24-29. In addition, a heavy band was present at 44 kDa for pooled fractions 17-23.With the R2 antibody, a heavy 42 kDa band and a lighter 38 kDa band were present forfractions 17-23. Both sets of pooled fraction corresponded to the peak of MBP-kinaseactivity seen in Fig. 11.0069Figure 13Time-Course for the Activation of Ribosomal Protein S6 kinases by Insulin.Isolated rat adipocytes were incubated with 10 nM insulin for 0-30 minutes. Theincubations were stopped with the addition of ice-cold homogenization buffer and cellularextracts prepared and assayed for Ribosomal Protein S6 kinase activity, as described inMethods, using 40S ribosomal subunits as substrate. Time-points represent the average oftwo measurements.70Activation of Ribosomal Protein S6 kinasesby 10 nM Insulin32—ii0I I I0 5 10 15 20 25 30Time (minutes)71Figure 14Time-Course for the Activation of Ribosomal Protein S6 kinases by Sodium Selenate.Isolated rat adipocytes were incubated with 1 mM sodium selenate for 0-27 minutes.Cellular extracts were prepared and assayed for Ribosomal Protein S6 kinase activity aspreviously described.. Each time-point is expressed as mean ± S.E.M. of fold-controlactivity (n=3). The actual control value was 51 firiol/minlmg. * denotes significantdifference from control (p<0.05)72Activation ofRibosomal Protein S6 kinasesby 1 mM Sodium Selenate1086-*1 \*\jj4 - F*17±20I I I I0 5 10 15 20 25 30Time (minutes)73Figure 15Time-Course for the Activation ofRibosomal Protein S6 kinases by Vanadyl Sulphate.Isolated rat adipocytes were incubated with 1 mM vanadyl sulphate for 0-30 minutes.Cellular extracts were prepared and assayed for Ribosomal Protein S6 kinase activity aspreviously described. Each time-point represents the measurement of three separatesamples. Each time-point is expressed as mean ± S.E.M. of fold-control activity (n3).The actual control value was 87 finollminlmg. * denotes significant difference from control(p<0.05)74Activation of Ribosomal Protein S6 kinasesby 1 mM Vanadyl Sulphate20 *1A*12 /1/1114-0I I I0 5 10 15 20 25 30Time (minutes)75Figure 16Comparison of the Time-Course of Activation of Ribosomal Protein S6 kinaseby 10 nM insulin. 1 mM Vanadyl Sulphate. and 1 mM Sodium Selenate.Compilation of figures 13-15. * denotes significant difference from control (p<O.O5)76Ribosomal Protein S6 kinaseComparisons20*Insulin I• SodiumSelenate• Vanadyl10- Sulphate/!**00510 2030Time (minutes)77Figure 17Dose-Response for the Activation of Ribosomal Protein S6 kinases by Insulin.Isolated rat adipocytes were incubated for 10 minutes with the insulin concentrations 1pM-100 nM. Extracts were prepared and assayed as previously described in Fig. 13.Significant stimulation of Ribosomal Protein S6 kinase activity was present at 1, 10 and100 nM concentrations. Each time-point was expressed as mean ± S.E.M. of fold-controlactivity (n=3). The actual control value was 4 fmol/minlmg. * denotes significantdifference from control (p<0.05)78Activation of Ribosomal Protein S6 kinasesby Insulin2.502.00-I1*11.50 11.00 T IIT I0.50_1_0.00 I I I I-13 -12 -11 -10 -9 -8 -7 -6LogM79Figure 18Dose-Response for the Activation ofRibosomal Protein S6 kinases by Sodium Selenate.Isolated rat adipocytes were incubated for 10 minutes with 100 nM-10 mM sodiumselenate. Extracts were prepared and assayed as previously described. Ribosomal ProteinS6 kinase activity was present at 100 M, 1 mM, and 10 mlvi sodium selenateconcentrations. Each time-point was expressed as mean ± S.E.M. of fold-control activity(n3). The actual control value was 157 fmollminlmg. * denotes significant differencefrom control (p<zO.05)80Activation of Ribosomal Protein S6 kinasesby Sodium Selenate4**IIi..2 I1TII I I I I-8 -7 -6 -5 -4 -3 -2LogM81Figure 19Dose-Response for the Activation ofRibosomal Protein S6 kinases by Vanadyl Sulphate.Isolated rat adipocytes were incubated for 20 minutes with vanadyl sulphate concentrationsranging from 10 jiM-lU mM. Ribosomal Protein S6 kinase activity followed a similarpattern to MBP kinase dose-response curve in that it was not present at less than 1 mMvanadyl sulphate. Higher concentrations again resulted in lower activity. Each time-pointis expressed as mean ± S.E.M. of fold-control activity (n=3). The actual control value was87 pmol/min/mg. * denotes significant difference from control (p<0.05)82Activation of Ribosomal Protein S6 kinasesby Vanadyl Sulphate1812/ \*10 /\j8642 —0 I I I-5 -4 -3 -2 -1LogM83Figure 20Mono Q Elution Profile of Sodium Selenate-stimulated S6-peptide kinase Activity.Mono Q fractions from both sodium selenate-treated and control samples obtained in Fig. 8were assayed for S6-peptide kinase activity using a phosphocellulose paper assay with asynthetic S6-peptide as substrate. Peak one was the largest peak observed with 22pmol/min!ml of activity. Data points represent a single experiment.30:g 0 Cl) 0 Cl) 00S6-peptideKinaseMonoQProfileSodiumSelenate051015202530354045505560Fractionnumber—I—sodiumselenate—0--ConUtI0085Figure 21Mono Q Elution Profile of Sodium Selenate-stimulatedRibosomal Protein S6 kinase Activity.The fractions from figure 20 were assayed for Ribosomal Protein S6 kinase activity usingribosomal 40S subunits as substrate. Data points represent a single experiment.:300C,)I 0 0‘.0 C))——sodiumselenate—0—control400ActivationofRibosomalProteinS6kinasebySodiumSelenate200100 001020304050Fractionnumber600087Figure 22Mono Q Elution Profile of Vanadyl Sulphate-stimulatedS6-peptide kinase activity Time-Course.Mono Q fractions used in Fig. 11 were also assayed for S6-peptide kinase activity asdescribed in Fig. 20. Data points represent a single experiment.60:50a),4OActivationofS6-peptideKinasebyVanadylSulphateFractionnumber——10minutes——20minutes——30minutes—0—Control6001020304050co89Figure 23Mono 0 Elution Profile of Vanadyl Sulphate-stimulatedRibosomal Protein S6 kinase Activity Time-Course.The fractions from Fig. 22 were assayed for Ribosomal Protein S6 kinase activity usingribosomal 40S subunits. Data points represent a single experiment.C) 0 U, 0 CI)Activationof RibosomalProteinS6kinasebyVanadylSulphate0500400300200100 001020304050Fractionnumber—10minutes——20minutes—30minutes—0ControlC91Figure 24Immunoblotting of Mono Q Fractions from Sodium Selenateand Vanadyl Sulphate-treated samples with S6K-CT Antibody.Selected Mono Q fractions from the sodium selenate (A) and vanadyl sulphate (B) treatedsamples were resolved on 11 % SDS-polyacrylamide gels, transferred to nitrocellulosemembranes and probed with S6K-CT antibodies. For the sodium selenate-treated samples(A) a 52 kDa band was present in fraction 18, associated with peak one of RibosomalProtein S6 kinase activity, as well as a 55 kDa band in fractions 19 and 20, also associatedwith peak one. With the vanadyl sulfate-treated samples (B) a 55 kDa band was present inthe pooled fractions 17-23. Lane assignments are shown below.Sodium selenate-treated samples (A) Vanadyl sulphate-treated samples (B)Lane Fraction No. Lane Fraction No.1 molecular weight standards 1 molecular weight standards2 16 2 24-293 17 3 17-234 18 4 3-55 196 207 218 229 2610 2711 2812 3113 3714 3815 3992A)66 kDa45 kfla31 kDa21.5 kfla93B)66 kDa45 kDa31 kDa21.5 kDa94Figure 25Immunoblotting of Sodium Selenate-treated samples using the ECL detection system.Mono Q fractions from sodium selenate-treated samples were resolved as in Fig. 24 but thebands were visualized using the ECL detection system. A) A 68 kDa band was present infractions 17-20 probed with the S6K-III antibody. B) Using the S6K-CT antibody on thesame fractions, a 62 kDa band was present in fractions 17-21. Lane assignments are shownbelow.S6K-III Antibody (A) S6K-CT Antibody (B)Lane Fraction No. Lane Fraction No.1 16 1 162 17 2 173 18 3 184 19 4 195 20 5 206 21 6 217 22 7 228 26 8 269 27 9 2710 28 10 2811 molecular weight standards 11 molecular weight standards12 31 12 3113 37 13 3714 3815 3995A)68 kfla1 2 3 4 5 6 7 8 9 10 11 12 13 14 1596B)62 kDa1 2 3 4 5 6 7 8 9 10 11 12 1397Figure 26Immunoblotting of Vanadvi Sulphate-treated samplesusing the ECL Detection System.Mono Q fractions from vanadyl sulphate-treated samples were resolved as in Fig. 24 butthe bands were visualized using the ECL detection system. A) Samples probed with S6K-III antibody had a 70 kDa band in fractions 17-21. B) Using the S6K-CT antibody, a 69kDa band was present in fractions 17-20. Lane assignments are shown below.Lane Fraction No.1 162 173 184 195 206 217 228 269 2710 2811 molecular weight standards12 3113 3714 3815 3998A)70 kDa1 2 3 4 5 6 7 8 9 10 11 12 13 14 1599B)66 kfla45 kDa31 kDa69 kDa123456789101112131415100Figure 27Effects of Genistein on MBP kinase Activity.Extracts from adipocytes incubated with 10 nM insulin (5 minutes), 1 mM sodium selenate(10 minutes), 1 mM vanadyl sulphate (20 minutes), or untreated controls in the presence(diagonal stripes) or absence (solid) of 200 nM genistein were assayed for MBP kinaseactivity.101Effects of Genistein onMBP kinase Activity4I llnsulin3 -___SodiumSelenateVanadylSulphate21-0 ——__—__________ _____Genistein + + +102Figure 28Effects of Genistein on Ribosomal Protein S6 kinase Activity.The extracts prepared in figure 27 were assayed for Ribosomal Protein S6 kinase activity.* denotes significant difference from samples not treated with genistein (p<O.05)103Effects of Genistein onRibosomal Protein S6 kinase Activity87__6 SodiumSelenate5 - VanadylSulphatec_) 4-C3*2*_________Genistein + + +104Figure 29Effects of Rapamycin on MBP kinase activity of isolated rat adipocytesstimulated with 1 mM sodium selenate or 1 mM vanadvi sulphate.Isolated rat adipocytes pre-incubated for 30 minutes with 50 nglml rapamycin or vehiclewere stimulated with sodium selenate or vanadyl sulphate for 20 minutes. Cellular extractsfrom these cells were then measured for MBP kinase activity.105Effects of Rapamycin onMBP kinase Activity3___SodiumSelenate— VanadylSulphate21-0Rapamycin + +106Figure 30Effects of Rapamycin on Ribosomal Protein S6 kinase activityof isolated rat adipocytes stimulatedwith 1 mM sodium selenate or 1 mM vanadyl sulphate.Cellular extracts prepared in figure 29 were also assayed for Ribosomal Protein S6 kinaseactivity. * denotes significant difference from samples not treated with rapamycin (p<0.05)107Effects of Rapamycin onRibosomal Protein S6 kinase Activity3SodiumSelenate—Sulphate2*C-)01-0Rapamycin + +108DISCUSSIONIn this study, we have used the isolated rat adipocyte as the system in which theinsulin-mimetic properties of vanadium and selenium were investigated. Rodbell (1964) wasthe first to establish a consistent protocol for the isolation of rat adipocytes using collagenasedigestion. The isolated rat adipocyte has since been used as a valuable tool in numerousstudies involving the investigation of the biological effects of insulin and insulin-mimeticcompounds. Adipocytes are sensitive to a wide variety of insulin-mimetic effects whichinclude stimulation of glucose uptake, increased lipogenesis, and inhibition of lipolysis.Furthermore, the effects observed in isolated cell studies are not subject to the interferencewhich might be present in the intact animal. In addition, the observations are made in arelatively uniform group of cells and are not compounded by the response of other cell typesand systems to insulin or the insulin-mimetic agents being investigated. However, thissystem is not without its limitations. The observations made in isolated cells may not befully representative of the physiological responses, which in the whole animal are broughtthrough the concerted effects of neuronal, hormonal, and chemical factors that cannot beduplicated in isolated cells. In addition, the isolated cell may not be identical to its intactcounterpart as it exists in situ. For example, the collagenase treatment of adipocytes maycause desensitization to insulin through the partial digestion of their insulin receptors.Although isolated adipocyte studies would be greatly complimented by intact animal studies,they but still remain a valuable tool in the initial investigation of the mechanism of action ofcompounds affecting complex systems provided that only a limited number of parameters areinvestigated.109I. MBP KINASESIsolated rat adipocytes were incubated with relatively high concentrations of insulin(10 nM), sodium selenate (1 mM), and vanadyl sulphate (1 mM), for increasing amounts oftime to 30 minutes in order to establish a time-course of activation for MBP lcinase. Insubsequent experiments we also established a dose-response for the above agents. MAPkinases have been shown to incorporate into MBP in a specific and linear fashion undercontrolled experimental conditions, thus making the phosphorylation of MBP an effectivemeans of measuring MAP kinase activity (Erikson eta!. , 1990; Boulton eta!. , 1991). In allof our MBP kinase experiments we have used the phosphorylation of MBP as a means ofquantitation of MAP kinase activity. This was done by scintillation counting of p-81phosphocellulose which can bind phosphorylated MBP and other phosphoproteins throughionic interactions.We observed maximal MBP kinase stimulation after 5 minutes of incubation withinsulin, similar to that reported by Haystead eta!. (1990) although in work done with 3T3-L1adipocytes maximal MBP kinase activation occurred after 10 minutes (Ray and Sturgill,1987). Other investigators working with Chinese Hamster Ovary (CHO) cells and rat 1 HIRcB fibroblasts have also shown a 5 minute maximal activation time (Dickens et a!., 1992;Boulton et a!., 1990). Tobe et a!. (1992) injected insulin into the liver of the intact ratdirectly via the portal vein. They reported maximal stimulation of MAP kinase activity 4.5minutes after injection. In our own laboratory, Hei et al. (1993) observed maximal MAPkinase activity in rat skeletal muscle 10 minutes after an i.v. injection of insulin into theintact rat. In a similar recent study, Zhou et al. (1993) have suggested the sequentialactivation of MAP kinase isoforms in rat skeletal muscle with one isoform, believed to beERK1, being maximally stimulated 3 minites after insulin injection while the other, believedto be ERK2, being activated after 10 minutes. The magnitude of the maximal MBP kinasestimulation in our study was —P1 .7-fold-control which again was similar to that reported by110Haystead et a!. (1990) who also used isolated rat adipocytes. In addition, in both our studyand that of Haystead et al. (1990) the response of the cells was of a transient nature whereasthe effect of insulin on cultured cells seemed to be of longer duration. Ray and Sturgill(1987) reported a 3.4-fold increase in 3T3-Ll adipocytes while in rat fibroblasts and CHOcells, both of which were induced to overexpress human insulin receptors, maximal MAPkinase stimulation was over 5-fold above control (Boulton eta!., 1990; Dickens et a!., 1992).The higher response of cultured cells could perhaps be due to a higher number of intactinsulin receptors, which in the case of isolated adipocytes may have been damaged due totheir exposure to collagenase and have therefore made the cells somewhat resistant to insulin.We used insulin concentrations ranging from 1 pM to 100 nM but only saw stimulation ofMBP kinase activity at the 10 and 100 nM concentrations. Other investigators working withcultured cells, which are not affected by collagenase treatment, have also observed optimaleffects on MBP kinase activity at 10 nM insulin with no effects present at concentrationsbelow 1 nM (Boulton eta!., 1990; Dickens eta!., 1992). Therefore, we are within the insulinconcentration range and time-course necessary to stimulate MAP kinase activity as reportedpreviously in the literature for both isolated rat adipocytes and cultured cells making ourisolated rat adipocytes a suitable tool to investigate the effects of sodium selenate andvanadyl sulphate on the insulin signal transduction pathway.It is our understanding that the effects of selenium on MAP kinases have not beenpreviously reported in the literature and that our study represents novel findings. We havedemonstrated that incubation of isolated rat adipocytes with sodium selenate results inincreases in MBP kinase activity in a time and dose-dependent manner up to a maximum of2-fold-control at 10 minutes of incubation with 1 mM sodium selenate. Ezaki (1990) was thefirst to report the in vitro insulin-mimetic effects of selenium. He demonstrated significantincreases in glucose-transport, glucose-transporter translocation, cAMP phosphodiesteraseactivity, ribosomal S6 protein phosphorylation, and the tyrosyl phosphorylation of a numberof cellular proteins in isolated adipocytes following their incubation with 1 mM sodium111selenate. Both our study and that of Ezaki report the presence of the insulin-mimetic effectsof selenium on adipocytes after nearly 30 minutes of incubation which is in contrast to thetransient nature of effects seen with insulin. In addition, maximal MBP kinase stimulation inour study occurred after 10 minutes which corresponds to maximal glucose transport activityreported by Ezaki although these two events have not been linked. The possibility that theinsulin-mimetic effects of sodium selenate are mediated via the IR through conformationalchanges caused by interactions with receptor suithydryl groups has been suggested.Although selenium compounds have been reported to interact with glucocorticoid receptorsthrough their suifflydryl groups, they were shown to play an inhibitory role. Also thesereceptors are structurally and functionally different from insulin receptors (Tashima et al.,1988). In addition, although Ezaki (1990) reported the tyrosine phosphorylation of a numberof unidentified proteins in adipocyte cellular extracts following sodium selenate stimulationno stimulation of IRTK activity or inhibition of tyrosine phosphatases was observed. Pillayand Makgoba (1992) who reported the phosphorylation of the EGF receptor in A431 cellsfollowing sodium selenate treatment saw no tyrosine phosphorylation of IRS-i in NIH 3T3-HIR-3.5 cells, although they also observed tyrosine phosphorylation of a number of proteinsin response to selenium. The above two studies suggest that the insulin-mimetic properties ofselenium are perhaps mediated through a post insulin-receptor mechanism and are mostlikely not due to inhibitory effects of sodium selenate on tyrosine phosphatases. The aboveinvestigators have suggested that, based on the lack of tyrosine phosphatase inhibition bysodium selenate and the large number of tyrosine-phosphorylated proteins observed in thesestudies, sodium selenate may be activating an unidentified tyrosine kinase(s) which, based onour study, may result in the activation of MBP kinases and other downstream ser/thr-proteinkinases.We constructed a dose-response curve for the stimulation of MBP kinase activity withselenium by incubating isolated adipocytes for 10 minutes with concentrations of sodiumselenate ranging from 100 nM to 10 mM. The optimal concentration of sodium selenate in112our study was 1 mM, the same as that reported by Ezaki (1990) for both glucose uptake andcAMP phosphodiesterase activity, although Ezaki reported effects at lower concentrations(100 iiM) than we observed. We found no stimulation at lower concentrations and nosignificant increases in MBP kinase activity at 10 mM. Our results are also consistent withthose of Pillay and Makgoba (1992) who constructed a dose-response curve for tyrosinephosphorylation of the EGF receptor by incubating A43 1 cells with sodium selenate(concentrations ranging from 10 M to 10 mM). They also saw effects only at I and 10 mMsodium selenate with no stimulation of phosphorylation at lower concentrations.Interestingly, a recent reported has shown the inhibition of the EGF receptor in A43 1 cells byMAP kinases through the activation of an unidentified phosphatase (Griswold-Prenner et a!.,1993). If sodium selenate also activates MAP kinases in A43 1 cells as we have seen in theadipocytes, the results of Pillay and Makgoba (1992) might have been somewhat attenuated.The sodium selenate concentrations used in both our study and the others mentioned wouldbe considered high in a physiological setting. However, there are a number of compounds inthe cell incubation buffer, including large amounts of BSA, which may bind to selenate andsignificantly decrease the free amounts available. Furthermore, unless sodium selenate isworking through the IR or some other cell surface interaction, it must pass through thehydrophobic outer membrane of the adipocyte in order to interact with the intracellularmachinery. This may require high levels of sodium selenate to drive the reaction and mayalso explain the longer incubation, as compared to insulin, necessary for the manifestation ofmaximal MAP kinase stimulation.We also investigated the effects of vanadyl sulphate on MBP kinases in the isolatedrat adipocyte. We found more than double the maximal MEP kinase stimulation observed ineither the insulin or the sodium selenate samples with vanadyl sulphate maximal activationexceeding 5-fold control. The initial activation of MBP kinase activity by vanadyl sulphateoccurred at 2.5 minutes of incubation, similar to that observed for insulin and sodiumselenate, but like sodium selenate and unlike insulin, the stimulation was long-lasting.113Vanadium has been studied extensively both in vivo and in vitro as an insulin-mimetic agent.Numerous studies have shown its stimulation of glucose-transport and other insulin-mimeticeffects on the isolated rat adipocyte. The stimulation of MBP kinase activity by vanadylsulphate has not previously been shown in the rat adipocyte, although Scimeca et a!. (1991)have conducted some work complementary to our study in the NIH-3T3-HIR 3.5 cells withsodium orthovanadate. They immunoprecipitated, using ERK- 1-specific antibodies, a 44kDa protein which was phosphorylated in vivo in response to a 5 minute incubation with 10mM sodium orthovanadate, as opposed to the 1 mM concentration we used in our study withvanadyl sulphate. This protein was also shown to have significant MBP kinase activityfollowing its immunoprecipitation from cells exposed to insulin or sodium orthovanadate.Therefore in agreement with the work of Scimeca et a!. (1991) in the NIH-3T3-HIR 3.5 cellswe also report the presence of a vanadyl sulphate-stimulated MAP kinase in the cytoplasmicextracts of isolated rat adipocytes. In our study we constructed a time-course of MBP kinaseactivation with vanadyl sulphate with 20 minutes as the optimal incubation period whereasScimeca et a!. (1991) presented a time-course for phosphorylation and activation only forimmunoprecipitated protein with a longer incubation leading to increased phosphorylationand activation. In addition, they only used two concentrations of sodium orthovanadate, 10mM for the intact cell incubations and 200 iM for their time-course experiment. Weconstructed a dose-response curve ranging from 100 iM to 10 mM vanadyl sulphate but didnot observe any stimulation of MBP kinase activity at concentrations less than 1 mM. Aswith sodium selenate the presence of various substances within the media could beinteracting with the vanadyl sulphate thus making it unavailable to the cell. Otherinvestigators working with the insulin-mimetic effects of vanadium compounds on isolatedrat adipocytes have also reported optimal insulin-mimetic effects with the use of highconcentrations of vanadium compounds whereas low concentrations have been shown toprolong insulin-binding and action possibly through their inhibition of tyrosine phosphataseactivity (Green, 1986; Heffez et at, 1990; Fantus eta!., 1990). Interestingly, as we increased114the vanadyl sulphate concentration above 1 mM, we saw corresponding decreases in MBPkinase activity. There are a number of possibilities that can help explain this trend. One isthat vanadyl sulphate is toxic at higher doses leading to cell death which would decrease thenumber of viable cells responsive to the effects of vanadyl sulphate. Another possibility maybe that higher concentrations of vanadyl sulphate could potentially be forming polymericforms of vanadium compounds (Wilsky, 1990) which may not be able to enter the cell inorder to manifest their effects or may not have the biological effects that might be present inthe monomeric forms. A third possibility may involve the non-specific stimulation orinhibition of unknown factors involved in the regulation of the signal transduction pathwaywith higher concentrations of vanadyl sulphate which may lead to a decrease in the overallresponse.Although not yet fully proven, vanadium compounds are believed to exert theirinsulin-mimetic effects through a post-receptor mechanism (Green, 1986). Vanadiumcompounds have been well established as potent phosphatase inhibitors (Swarup et a?., 1982;Lau eta?., 1989), which has been suggested as a potential mechanism for the phosphorylationand activation of cellular proteins. There is increasing evidence for a more active role forprotein phosphatases in cellular functioning (Cohen and Cohen, 1989). Vanadiumcompounds may be mediating their effects in part via the inhibition of certain proteinphosphatases. In a recent paper, Peraldi and Van Obberghen (1993) have provided evidencefor the existence of a insulin-regulated tyrosine phosphatase acting as an inhibitor of MAPkinases in NIH-3T3-HIR fibroblasts. They have suggested that this phosphatase isconstitutively active in unstimulated cells and is subject to ser/thr phosphorylation anddeactivation following insulin or vanadate treatment. The stimulation of MAP kinases byokadaic acid (Haystead eta?., 1990; Gotoh eta?., 1990), an inhibitor of phosphatases 2A and1 which are more specific towards phosphorylated serine and threonine residues, suggests arole for other potential phosphatases in the signal transduction cascade of insulin and growthfactors. It would be very difficult to speculate the exact mechanism through which vanadyl115sulphate stimulated MBP kinase activity in the adipocytes in our study. The large magnitudeof MBP kinase stimulation and the long duration of MBP kinase activation by vanadylsulphate resemble that seen with okadaic acid, although vanadium compounds are consideredprimarily to be phosphotyrosine phosphatase inhibitors whereas okadaic acid is more specifictowards ser/tbr phosphatases. Nevertheless, the contribution of the phosphatase inhibitoryeffects of vanadium compounds should be taken into account with regard to the overalleffects of vanadium. Based on the numerous in vitro and in vivo studies conducted onvanadium compounds, it seems likely that the insulin-mimetic effects of vanadium arebrought about by more than one route. We have shown that MBP kinase which are involvedin the ser/thr phosphorylation cascade of insulin are stimulated in a dose and time-dependentmanner by vanadyl sulphate. Whether the activation of these MBP kinases is related to themetabolic effects of vanadium on the adipocyte remains to be shown.II. S6 KINASESThe cellular extracts obtained for the MBP kinase time-course and dose-responseexperiments for insulin, sodium selenate, and vanadyl sulphate were also assayed for S6kinase activity. Two different methods of measuring S6 kinase activity, both involving thephosphorylation of the substrate with from radio-labeled ATP, were used in ourexperiments. The first was a paper assay similar to that used for the measurement of MBPkinase activity except for the substrate used which was a synthetic peptide resembling partsof the ribosomal S6 protein (see methods for details). The major disadvantage with the useof this substrate is the possibility that it might be phosphorylated non-specifically by otherkinases recognizing the ser/thr phosphorylation motif that it contains. In addition, the70S6K kinase family, one of the two major families of S6 kinases, are highly specifictowards the S6 protein contained within ribosomal 40S subunits and may not phosphorylatethe S6-peptide to the same extent as they would the 40S subunit. This, however, may also be116of benefit since it would allow us to somewhat distinguish the contribution of the two S6kinase families in the overall S6 kinase activity of the samples. The S6-peptide also offersthe advantage of allowing us to perform assays on a large number of samples in a shortperiod of time. The S6-peptide substrate was only used to assay S6 kinase activity of theMono Q fraction (to be discussed later). The second assay technique involved the use 40Sribosomal subunits purified from rat liver as substrate which are then isolated using gelelectrophoresis and counted using liquid scintillation counting(see methods). This method ismore specific towards S6 kinase activity than the first due to the presence of a physiologicalsubstrate whose phosphorylation is more specifically measured. It, however, requiresconsiderable time and effort in both substrate preparation and the actual assay. All crudecellular extracts and Mono Q fractions were assayed for Ribosomal Protein S6 kinase activityusing this technique.In our study, we have demonstrated the time and dose-dependent stimulation ofRibosomal Protein S6 kinase activity following treatment of intact adipocytes with insulin,sodium selenate, and vanadyl sulphate.The phosphorylation of ribosomal S6 protein through the activation of S6 kinases hasbeen established as one of the most consistent biological effects of insulin (Smith et a!.,1980; Rosin eta!., 1981; Stefanovic eta!., 1986). We therefore felt it necessary to show andcompare with others the effects of insulin in our system prior to the investigation of sodiumselenate and vanadyl sulphate. With the time-course, we observed maximal stimulation after10 minutes of incubation with more than a 2-fold increase over control. Our results wereagain similar to those of Haystead et a!. (1990) who also reported a maximal stimulation of2-fold over control between 10-20 minutes. They reported increased activity over controleven after 60 minutes of incubation whereas the Ribosomal Protein S6 kinase activity of ourinsulin-stimulated samples returned to basal levels after 30 minutes. We, however, usedribosomal 40S subunits as substrate in our assays, making them highly specific forRibosomal Protein S6 kinase activity. The synthetic S6-peptide used by Haystead et al.117(1990) could potentially be phosphorylated by other ser/tbr-protein kinases which may havelonger duration of action than S6 kinases. The intact liver shows maximal S6 kinase activity7.5 minutes after insulin injection which is also similar to our results (Tobe et al., 1992). Inrat skeletal muscle, S6-peptide kinase activity is maximally stimulated after 10 minuteswhereas S6 kinase activity reaches its highest levels after 30 minutes. This could perhaps bedue to the differential activation of the S6 kinase families present. In 3T3-L 1 adipocytes,maximal p9oC activation was shown after 5 minutes of incubation with insulin whichreturned to basal levels after 15 minutes, whereas 70S6K activation was highest after 15minutes and remained high for 60 minutes (Fingar et a!., 1993). It is possible, therefore, thatthe two S6 kinases would have a different time-course of activation in other systems as well.The p905* family has been shown to be downstream of the MAP kinases (Sturgill et a!.,1988). This is consistent with our time-course data in which the Ribosomal Protein S6kinase activity detected was maximal at a later time-point than MBP kinase activity thereforesuggesting the possibility that MBP kinase activation may be necessary for RibosomalProtein S6 kinase stimulation, which may indicate a role for the9oi’sk family. However, inour dose-response experiments with insulin, Ribosomal Protein S6 kinase activation occurredat a 10 times lower concentration, 1 pM, than that necessary to stimulate MBP kinaseactivity, 10 pM. Therefore, at least some component of our observed Ribosomal Protein S6S6Kkinase activity is stimulated in a MBP kinase-independent manner. The p70 is believedto be the major S6 kinase family stimulated in mammalian cells and has been shown not to bedownstream of MAP Kinases, making it a possible source of some of the Ribosomal ProteinS6 kinase activity that we have observed (Blenis et a!., 1991; Chung et a!., 1991). OurRibosomal Protein S6 kinase dose response is consistent with others showing detectable S6kinase activity and S6 protein phosphorylation at 1 nM and maximal activation at 100 nM(Stefanovic eta!., 1986; Ezaki, 1990). Based on the similarity of our insulin time-course anddose-response to other investigators, our isolated rat adipocytes seem to be a suitable system118with which to investigate the effects of sodium selenate and vanadyl sulphate on RibosomalProtein S6 kinase activity.We have shown the stimulatory effects of sodium selenate on the Ribosomal ProteinS6 kinase activity of isolated rat adipocytes in a time and dose-dependent manner. Moststudies with selenium compounds are centred around their antioxidant role with only a fewreports available on their insulin-mimetic properties. Ezaki (1990) has shown thephosphorylation of ribosomal S6 protein following the treatment of intact cells with 1 mMsodium selenate for 10 minutes in the presence of32P. We have shown an initial stimulationof Ribosomal Protein S6 kinase activity of approximately 3-fold control after 10 minutes ofincubation with sodium selenate. Maximal stimulation of over 8-fold control occurred after19 minutes of incubation. Ezaki (1990) did not construct a time-course or dose-responsecurve for S6 protein phosphorylation and did not measure Ribosomal Protein S6 kinaseactivity. In addition to our time-course, we also established a dose-response by measuringthe Ribosomal Protein S6 kinase activity of the same extracts used for the MBP kinase dose-response. As with insulin, we observed Ribosomal Protein S6 kinase stimulation, —‘2-foldcontrol, at a 10 times lower concentration, 100 jIM, than that needed for MBP kinaseactivation, 1 mM. This again suggests a MBP kinase-independent component to our totalRibosomal Protein S6 kinase activity which may in part be attributed to 70S6K kinaseactivity based on chromatographic and immunological evidence as well as our studies withrapamycin, a specific blocker of the 70S6K kinase family.As mentioned in the discussion of MBP kinases, the insulin-mimetic effects ofsodium selenate on glucose-transport and glucose-transporter translocation in isolated ratadipocyte have already been demonstrated (Ezaki, 1990). In our study we have shown theeffects of sodium selenate on the signal transduction cascade of insulin in terms ofRibosomal Protein S6 kinase activation. The connection between the two effects remains tobe elucidated. Fingar et al. (1993) have shown a dissociation of glucose transport and70S6Kactivation in 3T3-L1 adipocytes treated with insulin. It would therefore be119reasonable to assume that the two systems may also not be linked in isolated rat adipocytesstimulated with sodium selenate or other insulin-mimetic agents. We, however, cannotattribute our observed Ribosomal Protein S6 kinase activity solely to 70S6K We observeda decrease in the stimulation of Ribosomal Protein S6 kinase activity by sodium selenate inadipocytes pre-incubated with rapamycin, a specific inhibitor of the kinase family,but enough activity remained to suggest the presence of other Ribosomal Protein S6 kinase(s)within our system. The link between of the rsk S6 kinase family, already known to bepresent in rat adipocytes, and glucose transport has not yet been elucidated. In addition, Heiet a!. (1994) have identified an insulin-stimulated S6 kinase in the skeletal muscle of the ratwhich is distinct from the two main families of S6 kinases identified so far. The presence ofthis new kinase in adipocytes and its potential role in the mediation of the biological effectsof insulin also need to be investigated. The possibility also exists that the stimulation ofglucose-transport in adipocytes by sodium selenate is brought about through a signalingpathway other than the ser/thr-phosphorylation cascade.Perhaps the most dramatic results in our study are those of the stimulation ofRibosomal Protein S6 kinase activity with vanadyl sulphate. We observed a 15-fold controlstimulation of Ribosomal Protein S6 kinases at the maximal points for both our time-courseand dose-response experiments. Unexpectedly, the optimal time-point and concentration forRibosomal Protein S6 kinase activation were the same as that for MBP kinases. The effectsof vanadyl sulphate on Ribosomal Protein S6 kinases were of sustained duration with overli-fold control stimulation remaining after 30 minutes of incubation. The dose-response ofRibosomal Protein S6 kinase stimulation was also similar to that of MBP kinases in thatconcentrations above the optimal 1 mM actually led to a decrease in activity which we feelcan partly be explained by similar reasons to those mentioned in the MBP kinase discussion.Unlike the MBP kinase dose-response curve however, initial activation occurred at 313 1iM,less than one-third the 1 mM concentration necessary for MBP kinase activation, andRibosomal Protein S6 kinase activity returned to normal levels at the 10 mM concentration at120which MBP kinase activity remained 2-fold above control. These observations as with thosefor insulin and sodium selenate suggest that not all of the Ribosomal Protein S6 kinaseactivity we measured is downstream of MBP kinases. This again suggests a role for theS6Kp70 kinases or other S6 kinase(s) which are not downstream of MAP kinases. In fact, anS6Kearly study in the identification and characterization of the p70 kinase family in theSwiss mouse 3T3 cells involved the use of vanadium compounds as activators (Jeno et aL,1988). Although a time-course or dose-response curve were not constructed in this study,they chose 1 mM as the optimal concentration and 30 minute as the optimal time-point, bothof which would be consistent with conditions in which we observed high Ribosomal ProteinS6 kinase activity. In a later study by the same group, vanadium compounds, at aS6Kconcentration of 3.8 mM, were shown to induce a 10-fold stimulation of p70 kinaseactivity in Swiss mouse 3T3 cells (Ballou et a!., 1988). Vanadium compounds have sincebeen shown to stimulate S6 kinase activity even in the intact animal. Vanadate injecteddirectly via the portal vein into intact liver was shown to increase S6 kinase activity 3-foldover control, although this could not necessarily be attributed to the stimulation of any oneS6 kinase family. We also conducted studies with rapamycin to determine the role of the70S6Kfamily in the overall stimulation observed in cellular extracts from vanadyl sulphate-treated cells. Contrary to our expectations, we did not see a significant decrease inRibosomal Protein S6 kinase activity of cells pretreated with rapamycin following vanadylsulphate treatment. However, it must be noted that even the samples which were not treatedwith rapamycin had a low level of activity compared to our previous results suggestingpotential problems with the experiment. These results must therefore be examined carefullywith only a limited emphasis being placed on them. We also conducted some experimentswith genistein, an inhibitor of tyrosine-kinases in order to gain some insight into the routethrough which MEP kinases and Ribosomal Protein S6 kinases in our study are activated.Due to the relative non-specificity of genistein and the potential effects that it may have inaddition to its tyrosine-kinase inhibition, we must be careful of our interpretation of the data.121We therefore did not discuss the effects of genistein on MBP kinases because of the lowmagnitude of its effects. As expected, genistein prevented the stimulation of RibosomalProtein S6 kinases by insulin with some minor effects on sodium selenate effects. Theeffects of genistein were most prominent with the vanadyl sulphate-treated cells withRibosomal Protein S6 kinase activity decreasing from 6-fold control to less than 2-foldcontrol. This suggests that tyrosine kinases play an important role in the activation ofRibosomal Protein S6 kinases by vanadyl sulphate but are not the only route through whichthe effects of vanadium are mediated. The suggestion that the effects of vanadiumcompounds are mediated through the insulin-receptor tyrosine kinase has been made butremains an area of dispute because of contrary evidence provided by other investigators(Fantus et a!., 1991; Green, 1986). Shisheva and Schecter (1993) have suggested that theinsulin-mimetic effects of vanadium compounds in isolated adipocytes are mediated in partthrough the inhibition of a constitutively active cytosolic phosphotyrosine phosphatase.Based on this reasoning, the inhibition of phoshotyrosine phosphatases would have littleeffect if the tyrosine kinases were already inhibited. It seems highly likely however, that theactions of vanadyl sulphate are brought about through more than one mechanism. As withsodium selenate, we cannot link the many other insulin-mimetic actions of vanadiumcompounds with the activation of Ribosomal Protein S6 kinases which we have observed.However this remains a potential route through which these events may be mediated.III. CHROMATOGRAPHY AND IMMUNOBLOTTINGTo further analyze the MBP kinase and S6 kinase activities in our crude cellularextracts, we subjected the sodium selenate, vanadyl sulphate, and control extracts to Mono Qanion-exchange chromatography and assayed the obtained fractions for MBP kinase and S6kinase activity. In addition, we probed selective fractions with MAP kinase and S6 kinaseselective antibodies. Mono Q columns are often used in the purification of MAP kinases and122S6 kinases from crude cellular extracts. The column has a high affinity for these enzymesallowing extraneous material to be washed away. The kinases are then eluted usingincreasing concentrations of NaC1.A. MBP kinase Mono Q and Immunoblot AnalysisWe observed five peaks of MBP kinase activity in the Mono Q fractions of thesodium selenate-treated samples and four peaks for our control and vanadyl sulphate-treatedsamples. All of these peaks are not necessarily due to specific MAP kinase activity sinceother ser/thr-protein kinases can also potentially phosphorylate MBP. The two mostprominent peaks for the sodium selenate-treated and vanadyl sulphate-treated samples, elutedat 346 and 386 mM NaC1. We will limit our discussion to these two peaks which will bereferred to as peak 1 (eluted at 346 mM NaC1) and peak 2 (eluted at 386 mM NaC1). For thesodium selenate samples, peak 1 had the highest MBP kinase activity with 45 pmollminlmlwhile peak 2 had 27 pmol/min/ml of activity. For the vanadyl sulphate samples, both peakshad —56 pmollminlml of activity. The control samples did not contain the two above-mentioned peaks but had a small peak (10 pmol/minlml) at 359 mM NaCl. They alsocontained a large peak of 31 pmollminlml eluting at 638 mM NaC1 which was not present inthe other samples. This peak most likely represents an experimental anomaly since it was notpresent in the control sample for the vanadyl sulphate time-course control samples and elutedat very high NaCl concentration, nearly double the NaC1 concentration necessary to elutepeak 1 and peak 2. Previous reports of MAP kinase purification using Mono Qchromatography mainly report the presence of two peaks attributable generally to the 42 kDaERK2 and the 44 kDa ERK1 eluting in the order mentioned although at least six differentMAP kinases have been identified (Ahn et al., 1991; Cobb et al., 1991; Pelech andSanghera, 1992). Our peaks of MBP kinase activity appear to elute later than those reportedby Haystead et al. (1990), who observed two peaks of MBP kinase activity eluting at —240123mM and —270 mM NaCI, from extracts of isolated adipocytes stimulated with okadaic acid.Other reports, using different systems, also show two peaks of MBP kinase activity whichagain elute at lower NaC1 concentrations than we have observed such as —150 mM and —200mM in PC12 cells and -100 mM and —200 mM in rat skeletal muscle (Boulton et al., 1991;Zhou et a!., 1993). In a review of MAP kinases by Pelech and Sanghera (1992) ERK1 andERK2 are listed as eluting at —330 mM NaC1 and —320 mM NaC1, respectively, which arecloser to the value we have obtained. The elution profile of ERK1 and ERK2 is slightlydifferent for each particular report based on the various conditions inherent to the particularstudy. Even in our own study, the Mono Q profile of the samples from the vanadyl sulphatetime-course was slightly different from our other profiles. In all three time-points of vanadylsulphate incubation, only one prominent peak of MBP kinase activity eluting at 345 mMNaC1 was present. This peak most likely encompasses the two peaks of MBP kinase activityseen in other vanadyl sulphate Mono Q profile and the sodium selenate Mono Q profile andagain elutes at a NaC1 concentration similar to what has been reported in the literature(Pelech and Sanghera, 1992). In the control Mono Q sample profile from the time-courseexperiment an early eluting peak broad peak was present which was not observed in thevanadyl sulphate-treated time-course profile or any of the other MBP kinase Mono Q profilesincluding the mentioned control profile. It is unknown whether this peak represents a MAPkinase or another ser/thr-protein kinase active in unstimulated adipocytes.We probed the fractions containing the peaks of MBP kinase activity from thevanadyl sulphate time-course and sodium selenate samples and probed them with antibodiesspecific towards MAP kinases. We used both Rl and R2 antibodies with the vanadylsulphate-treated samples but decided to only use the Ri antibody for the sodium selenatefractions, which were probed later, based on our results with the vanadyl sulphate fractions.The R2 antibody detected a 42 and a 38 kDa band in the fractions immediately preceding themain peak of MBP kinase activity for the vanadyl sulphate samples but failed to detectanything for the actual peak fractions. With the Rl antibody we detected a 42 kDa band for124both the peak fractions and those preceding them which also contained a 44 kDa band. Thephosphorylation of the MAP kinases will increase their affinity for the Mono Q columnmaking activated MAP kinases elute slightly later than inactivated MAP kinases. The peaksof activity could therefore be due to a small amount of activated MAP kinases with theremaining unstimulated MAP kinases eluting earlier. The antibodies would not necessarilydiscriminate between stimulated or unstimulated MAP kinases and could therefore show astronger reaction with the fractions before the peaks because of the greater amount of MAPkinases they could contain. We have observed bands within the molecular weight rangeexpected for MAP kinases from fractions near peaks of MBP kinase activity using specificantibodies against MAP kinases. We therefore feel that there is a strong possibility that theMBP-kinase activity we have seen both in our crude samples and in the Mono Q profile ofvanadyl sulphate-stimulated samples could potentially be due to the presence of MAPkinases. Because we have seen the presence of two bands and based on previous reportsreporting the presence of both ERK1 and ERK2 in similar tissues, it seems likely that morethan one family of MAP kinases would be present in these samples. We felt that we obtainedbetter results with the Ri antibody because it allowed us to detect a 42 kDa band in the peakfractions which was not present with the R2 antibody. We therefore only used the Riantibody for probing the fractions from the sodium selenate Mono Q samples. With sodiumselenate samples, we observed a 52 kDa band in fractions encompassing both peaks whereasa 50 kDa band was present in two fractions from peak 1. The molecular weights of the bandwe observed are not consistent with that reported for ERK1, 44 kDa, and ERK2, 42 kDa,although the bands are present in peak regions of MBP kinase activity. Based on ourimmunoblot data alone, we cannot make any assumptions on the presence of MAP kinases.We did however, observe consistent MBP-kinase activity both in our crude samples and inour Mono Q fraction which corresponded to the fractions from the vanadyl sulphate samples.We therefore feel that the activity we have observed with our sodium selenate samples is dueto MAP kinases, although this was not immunologically verified. We originally tried125probing the crude extracts (data not shown) but found interference from the large amounts ofBSA present. We therefore decided to probe the fractions from the Mono Q column whichwould contain lesser amounts of interfering proteins. The main difficulty with theseimmnoblot experiments including those involving S6 kinases, to be discussed below, was thelow amounts present of the kinases in Mono Q fractions. To increase the amounts of kinasepresent, we increased the amount of samples which again led to difficulties. Firstly, weoverloaded our polyacrylamide gels used in the SDS-PAGE electrophoresis which led todecreased resolution of our proteins making our molecular weight determinations inaccurate.In addition, increased samples again led to increased presence of extraneous proteins whichdirectly interfered with our immunoblots. We also used the more sensitive ECL detection butdid not detect any bands possibly due to the an incompatibility of this system to ourantibodies (data not shown). Logistical considerations prevented clarification ofexperimental anomalies potentially due to technical difficulties or the further explorations ofother options which might potentially have improved our results such as theimmunoprecipatation of the MAP kinases in the crude extracts.B. S6 kinase Mono Q and Immunoblot AnalysisThe Mono Q fractions from the sodium selenate-treated, vanadyl sulphate-treated, andcontrol samples were assayed for S6 kinase activity using both a synthetic S6-peptide andpurified ribosomal 40S subunits as discussed previously for the crude samples. A significantdifference in the substrate specificity of the 70S6K and the 90rsk S6 kinase families hasbeen an apparent low affinity for the synthetic S6-peptide RRLSSLRA, patterned after thephosphorylation site of the S6-protein, by the 70S6K S6 kinase family (Avruch et al.,1991). Previous studies have used the phosphorylation of this peptide as a measure of S6kinase activity (Haystead et aL, 1990). This could potentially have led to a selection againstS6K . . . .the contnbution of the p70 S6 kinase family in the overall S6 kinase activity. We,126however, used the synthetic peptide AKRRRLSSLRASTSKSESSQK which may serve as abetter substrate for 70S6K S6 kinases based on the increased number of upstream residuesto the palindrome sequence (Price et a!., 1989). This corresponds to our results in which thepeaks observed using ribosomal 40S subunits were also present using our synthetic peptide.More peaks of S6 kinase activity were apparent with the S6-peptide substrate than theribosomal 40S subunits which could in part be attributed to the action of other ser/thr-proteinkinases. We will therefore limit our discussion to only some of the peaks present which wereof interest to us.The most prominent S6-peptide kinase peak of activity for the sodium selenatesamples, eluted at —226 mM NaC1, corresponded to a peak in the vanadyl sulphate time-course samples as well as a small peak in the control samples. This peak elutes at a similarNaCl concentration to that reported by Haystead et al. (1990) in the S6-peptide-kinase MonoQ profile of insulin-stimulated isolated rat adipocytes and that of Erikson and Maller (1986)in their early work on the purifióation of p90” from Xenopus eggs. This peak was alsopresent in the Ribosomal Protein S6 kinase profile of the sodium selenate samples. Wetherefore feel that this peak likely represents an important Ribosomal Protein S6 kinase in theoverall activity observed in the cellular extracts. Peak four of the sodium selenate S6-peptide-kinase profile was also present in the control S6-peptide-kinase Mono Q profile. Thepeak for the control samples however, was nearly double the magnitude than the sodiumselenate samples. This peak was also present in the control Ribosomal Protein S6 kinaseprofile. As with the MBP kinase Mono Q profile, this peak seems to represent a kinase thatis inactivated following sodium selenate treatment although this remains to be shown.In further analysis of our sodium selenate Mono Q fractions, we used the antibodiesS6K-III, recognizing sub domain III of 70S6K S6 kinase family, and S6K-CT or rsk-CTwhich recognizes the carboxyterminal of the90rsk S6 kinase family. The S6K-III peptideused to make the antibody has a homologous region to the N-terminal catalytic region of the90rsk the S6K-III antibody could therefore potentially cross-react with p90’ family127whereas the S6K-CT would not be expected to react with the 70S6K S6 kinase family basedon the significant differences that exist between the carboxyterminals of the two S6 kinasefamilies (Banerjee, 1990). We, however, found bands which were recognized by bothantibodies in some of our immunoblot experiments. As with the immunoblot experiments forthe MAP kinases, both BCIP/NBT colouring reagents and the ECL detection system wereused. We did not obtain satisfactory results with the BCIP/NBT colouring reagents. Whenusing the S6K-III antibody, no bands were evident and the bands present using the S6K-CTantibody were difficult to interpret. The low amounts of kinase versus total protein onceagain forced us to use high concentrations of protein which again led to overloading of thegel and poor resolution. Bands with an apparent molecular mass range of —52-55 kDa werepresent in the major Ribosomal Protein S6 kinase peak region while a —64 kDa band waspresent in all the fractions probed. No clear conclusions can be made with these results sincethe molecular weights are most likely inaccurate due to gel overloading and the antibodyreacts with other proteins present. With the ECL system, we were able to use much lowerconcentrations of proteins which prevented the overloading of the gels. However, bandscorresponding to our molecular weight standards were also evident possibly due to a nonspecific reaction of these proteins with the secondary antibody. A 68 kDa band was presentin the major Ribosomal Protein S6 kinase peak fractions using the S6K-III antibodysuggesting the potential presence of7oS6K kinases. With S6K-CT antibody we were ableto detect two bands within these fractions, a relatively dark —62 kDa band and a light —54kDa band. The 62 kDa band could again represent a7oS6K kinase cross-reacting with theS6-CT antibody with the —54 kDa bands representing the same enzyme in a lowerphosphorylation state. Both, however, could represent other kinases or proteins reacting nonspecifically to our antibody as could the 68 kDa band detected using the S6-III antibody.One possibility could be that the bands present are due to the non-specific reaction of thesecondary antibody to BSA. Our crude samples contain large amounts of BSA some ofwhich could still be present in the Mono Q fractions. The molecular weight of BSA is —66128kDa and ECL has been shown to visualize the bands for the molecular weight standards. Wetherefore cannot make any definite conclusions using our immunoblot data on its own. Butbased on the fact that these bands correspond to peak fractions of Ribosomal Protein S6kinase activity, we feel that it would be valid to view them as potential S6 kinases. Thepossibility that the observed Ribosomal Protein S6 kinase activity present both in these andthe crude samples is due to a different Ribosomal Protein S6 kinase stimulated by sodiumselenate but not recognized by these antibodies also remains such as an adipocyte homologueof the recently reported insulin-stimulated S6 kinase present in rat skeletal muscle (Hei et aL,1993).The S6-peptide kinase Mono Q profile of the vanadyl sulphate time-course samplescontained a number of peaks some of which are likely due to the phosphorylation of ourpeptide by kinases other than S6 kinases since corresponding peaks in the Ribosomal ProteinS6 kinase Mono Q profile are completely absent. The major peaks of Ribosomal Protein S6kinase activity were present for all vanadyl sulphate-stimulated samples but varied inmagnitude based on the incubation time. The 20 minutes samples generally had the highestpeaks in accordance to that observed for the crude samples in which the 20 minutesincubation time resulted in the highest Ribosomal Protein S6 kinase activity. Peak 3 of theS6-peptide kinase profile corresponds to the major peak discussed for the sodium selenatesamples and is also the present as the major peak of the Ribosomal Protein S6 kinase MonoQ profile. This peak elutes at a NaC1 concentration range of 235-290 mM which differsslightly for each time-point. The levels of activity are also different for each time-pointpossibly indicating different phosphorylation states of the kinases which could be in part beresponsible for the difference in the elution for the different time-points. Peaks 4 and 5,eluting at —345 mM and —375 mM NaC1, respectively, were also major peaks present in theS6-peptide kinase Mono Q profile but corresponded to a minor peak in the RibosomalProtein S6 kinase profile. Insulin-stimulated p901th in rat skeletal muscle has also beenshown to elute at similar concentrations to that seen for peak 4 (Hei et al., 1993). These129peaks also correspond to the major MBP kinase peak of these samples discussed earlier andcould therefore represent non-specific phosphorylation of the S6-peptide by MAP kinases.They could also potentially be due to the activity of the 70 S6K kinase family. The 70S6Kkinases are believed to elute later than the kinases in the Mono Q column and studiesusing their specific inhibitor rapamycin have shown them to be the more important S6kinases in mammalian tissue although this is the subject of some controversy based on morerecent evidence (Dickens et a!., 1992; Chung et al., 1991; Hei et al., 1994). These peaks werenot, however, present in the Ribosomal Protein S6 kinase Mono Q profile of the insulin-stimulated adipocytes reported by Haystead et al. (1990). But as discussed earlier, the S6-peptide used in that study was not a good substrate for thep7056K kinase family whichcould have led to the absence of a70S6K S6 kinase peak.Selected fractions from the vanadyl sulphate samples were subjected toimmunological analysis as described for the sodium selenate samples. Once again highlevels of protein necessary for band detection using BCIP/NBT colouring reagents led to geloverloading and poor resolution. A 55 kDa band was evident for the major peak ofRibosomal Protein S6 kinase activity observed for the Ribosomal Protein S6 kinase and theS6-peptide-kinase profile using the S6K-CT antibody. Using the ECL development system,a 69 kDa and a 70 kDa band were present in the peak fractions using S6K-CT and S6K-IIIantibodies respectively. As with the sodium selenate samples the presence of this band maysuggest a role for the 70S6K kinase family in our overall Ribosomal Protein S6 kinaseactivity. But again the possibility exists that these bands are unrelated to the observedRibosomal Protein S6 kinase activity and are a result of non-specific interactions since bothantibodies show similar bands for both sodium selenate and vanadyl sulphate in the samefractions. However, these are the fractions in which Ribosomal Protein S6 kinase activitywas observed for both agents. Although based on our results, we cannot make any majorconclusions on the identity of the S6 kinase family observed in our experiments.130CONCLUSIONS1) Our isolated rat adipocyte preparation contained MBP kinases and Ribosomal ProteinS6 kinases which were stimulated by insulin in a time and dose-dependent manner and werethus a suitable system in which to investigate the insulin-mimetic compounds sodiumselenate and vanadyl sulphate.2) Sodium selenate was able to stimulate MBP kinases and Ribosomal Protein S6kinases to a degree equal to or higher than insulin in a time and dose-dependent manner.This suggests that sodium selenate has effects on the signal transduction pathway of insulinalthough the precise link to the metabolic effects is yet to be made.3) Vanadyl sulphate stimulates both MBP kinases and Ribosomal Protein S6 kinases to ahigher degree than either insulin or sodium selenate in isolated rat adipocytes. 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