REGULATION OF THE STRESS-ACTIVATED PROTEIN KINASE PATHWAYS IN HEMATOPOIETIC CELLS Ian Nevin Foltz B.Sc , The University of Guelph, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Experimental Medicine Program We accept this thesis as conforming Jp^fteoecjuired standard THE UNIVERSITY OF BRITISH C O L U M B I A March 1999 © Ian Nevin Foltz, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of r A ^ T - C x ^ e The University of British Columbia Vancouver, Canada Date ftf ; l [ T * " — - T ' T P » -200 •116 -97 -66 * .... ~ ' + - + - + Figure 3.1 - Analysis of phosphoproteins induced by IL-3 and S L F using 2D SDS-PAGE. Murine bone marrow-derived mast cells were lyzed after being treated with SLF or IL-3 for the indicated times. Whole cell extracts were analyzed using 2 dimensional SDS-PAGE, and the proteins were immunoblotted with an anti-phosphotyrosine antibody 4G10. The position of the phosphoprotein putatively corresponding to p38 M A P K is indicated by arrowheads. This figure was adapted from Welham et al., 1992. 7 7 Objectives: (1) We initiated a collaboration with Dr. Young to determine if p38 M A P K was indeed the same p38 phosphoprotein. Dr. Young provided us with antisera that recognized full-length p38 M A P K , that would be used to immunoprecipitate p38 and determine if it was tyrosine phosphorylated and activated by IL-3 and GM-CSF. (2) Dr. Young also provided us with the specific inhibitor of p38 M A P K , SB 203580. We would use this inhibitor to determine if M A P K A P kinase-2 was indeed a substrate of p38 M A P K or the E R K M A P K , which were previously identified as activators of M A P K A P kinase-2. (3) Hematopoietic growth factors are required for the proliferation, differentiation and the prevention of apoptosis in hematopoietic cells. We would test the role of p38 M A P K in these biological functions using the p38 M A P K inhibitor to acutely deprive a cell of p38 M A P K activity. 3.2 Results 3.2.1 Identification of p38 MAPK - an unknown phosphoprotein. To determine whether p38 M A P kinase was involved in responses to IL-3 or SLF, we stimulated primary bone-marrow derived mast cells with these factors, immunoprecipitated p38 M A P kinase and assessed its tyrosine phosphorylation by immunoblotting with the anti-phosphotyrosine specific antibody 4G10. As expected, both SLF and IL-3 induced tyrosine phosphorylation of p38 M A P kinase (Fig. 3.2). 3.2.2 Activation of p38 MAPK by IL-3, GM-CSF or SLF but not IL-4 or IL-13. To demonstrate that the tyrosine phosphorylation of p38 M A P kinase in cells treated with hematopoietic growth factors correlated with increased kinase activity, we immunoprecipitated p38 M A P kinase from either untreated MC/9 cells, or cells treated with saturating doses of GM-CSF, SLF or 0.2 M NaCl. We then assessed the activity of p38 M A P kinase in an in vitro kinase assay using a truncated form of ATF-2 as substrate (Fig. 3.3). p38 M A P kinase immunoprecipitated 78 - 2 10 10 5 m i n IP: a n t i - p 3 8 IB: 4G10 IB: a n t i - 3 8 Figure 3.2 - IL3 or S L F , but not IL -4 , induce tyrosine phosphorylation of p38 M A P K in bone marrow-derived mast cells. Cells were incubated at 37 °C in RPMI 1640 for 1 hr and then left untreated (CON), or treated with saturating doses of IL-3 (IL-3), IL-4 (IL-4), or SLF (SLF) for the indicated times. Immunoprecipitated p38 M A P kinase was analyzed after SDS-PAGE by immunoblotting (IB), initially with the anti-phosphotyrosine antibody 4G10 (4G10), and subsequently with an anti-p38 M A P kinase antibody (anti-p38) to quantitate loading. The position of p38 M A P K is indicated by an arrow. 79 £p IP: anti-p38 cf ATF -2 IB: 4G10 IB: anti-p38 Figure 3.3 - Activation of p38 M A P K by hematopoietic growth factors in M C / 9 cells. Cells were incubated in RPMI 1640 at 37 °C for 1 hr. Equal numbers of cells were then left untreated (CON), or stimulated with saturating doses of IL-4 (IL-4), murine GM-CSF (GM) or SLF (SLF) or 0.2 M NaCl (NaCl) for the indicated times. The cells were lysed and p38 MAP kinase was immunoprecipitated with anti-p38 MAP kinase anti-serum. The relative kinase activity was assessed by an immune complex kinase assay using a truncated form of ATF-2 as substrate. SB 203580 (10 uM) was included in one sample (SB) to inhibit the in vitro kinase activity of p38 MAP kinase. The reaction products were resolved by SDS-PAGE and transferred onto nitrocellulose. Phosphorylation of ATF-2 was assessed by autoradiography (Top). The membrane was immunoblotted (IB) initially with the anti-phosphotyrosine antibody 4G10 (4G10) to assess tyrosine phosphorylation of p38 MAP kinase (Middle) and with an anti-p38 MAP kinase antibody (anti-p38) to assess loading (Bottom). 80 from cells stimulated with GM-CSF or SLF exhibited increased levels of kinase activity, which correlated with the levels of tyrosine phosphorylation of the enzyme. The activity of p38 MAP kinase was completely abolished by the inclusion of SB 203580 during the in vitro assay (Fig. 3.3). To examine the kinetics of SLF and GM-CSF induced activation of p38 M A P kinase in MC/9 cells, we used an antibody specific for the activated form of p38 M A P kinase. This antibody recognizes p38 M A P kinase when phosphorylated on the tyrosine of the T G Y activation motif. The activation of p38 M A P kinase is dependent on dual phosphorylation on both threonine and tyrosine residues as previous demonstrated (Doza et al., 1995; Raingeaud et al., 1995). Treatment with growth factors resulted in a rapid increase in the levels of tyrosine phosphorylation of p38 M A P kinase (Fig. 3.4). Cells treated with SLF exhibited detectable phosphorylation of p38 M A P kinase as early as 2 minutes and maximal phosphorylation of p38 M A P kinase between 5 and 10 minutes. Cells treated with GM-CSF had maximal phosphorylation of p38 M A P kinase at around 10 minutes. In both instances the phosphorylation of p38 M A P kinase was transient and returned to almost basal levels by 30 minutes. It was of particular interest to investigate the effect of IL-4 as we had previously shown that it failed to activate E R K M A P kinases (Welham et al., 1992). IL-4 failed to induce tyrosine phosphorylation of p38 M A P kinase in primary mast cells (Fig. 3.2), in MC/9 (Fig. 3.3) or in FD-5/13R (Fig. 3.5). As predicted from these results, IL-4 also failed to stimulate enzymatic activity of p38 M A P kinase (Fig. 3.3). Interestingly, treatment with IL-4 resulted in a small but reproducible reduction in both the levels of tyrosine phosphorylation and the enzymatic activity of p38 M A P kinase (Fig. 3.3) compared with untreated cells. The related cytokine IL-13 also failed to induce tyrosine phosphorylation of p38 M A P K (Fig. 3.5). Thus, both IL-4 and IL-13 fail to activate either p38 M A P K or E R K M A P K (Welham et a l , 1995) 3.2.3 Activation of p38 MAPK by CSF-1 in FD-MACII cells. To test the effects of CSF-1 or GM-CSF on the activity of p38 M A P kinase, we used murine factor-dependent cell lines that respond to these factors (Welham et al., 1992; Welham et al., 1994). Stimulation of the 8 1 M i n : 0 2 5 10 30 60 G M - C S F S L F IB: anti-phospho-p38 o 73 c o GM-CSF • SLF 0 2 5 10 30 60 Minutes Figure 3.4 - Kinetics of tyrosine phosphorylation of p38 M A P kinase by G M - C S F or S L F . After 1 hr in RPMI 1640 at 37 °C, MC/9 cells were left untreated, or were stimulated with saturating doses of GM-CSF or SLF for the indicated times. The cells were lysed and the NP-40 soluble fraction was analysed by SDS-PAGE followed by immunoblotting using an antibody specific for activated p38 M A P kinase. Levels of activation of p38 M A P kinase as detected by this antibody were quantitated using densitometry. 8 2 • > p p IB: 4G10 IB: anti-phospho-p38 Figure 3.5 - IL-13 fails to activate p38 M A P Kinase in FD5/IL13R ce l l s . Cells were incubated in RPMI 1640 at 37 °C for 1 hr. Equal numbers of cells were then left untreated (CON), or stimulated with saturating doses of IL-4 (IL-4), IL-13 (IL-13) or 0.2 M NaCl (NaCl) for 10 min. The cells were lysed and the cellular lysate was resolved by SDS-PAGE and transferred onto nitrocellulose. The membrane was immunoblotted (IB) initially with the anti-phospho-p38 antibody (anti-phospho-p38) to assess tyrosine phosphorylation of p38 MAP kinase (right). The membrane was also immunoblotted with 4G10 to demonstrate the cells responded to IL-4 and IL-13 (left). 83 macrophage-like FD-MACII cells with CSF-1 resulted in rapid tyrosine phosphorylation of p38 M A P kinase (Fig. 3.6). This effect was seen following treatment with either recombinant murine CSF-1 or a source of natural murine CSF-1, L-cell conditioned medium. The tyrosine phosphorylation of p38 M A P kinase was abrogated by the presence of a monoclonal antibody that neutralizes CSF-1 activity (Fig. 3.6), demonstrating that the induced phosphorylation of p38 M A P kinase was due to CSF-1 and not, for example, to contamination by endotoxin or to osmotic stress. 3.2.4 Activation of p38 MAPK by cross-linking of the Fc Receptor for Immunoglobulin G. Besides hematopoietic growth factors, MC/9 mast cells also respond to ligation of the Fc receptor for Immunoglobulin G (FcR). We used two approaches to determine if p38 M A P K was tyrosine phosphorylated after cross-linking of the FcR. The first involved the use of antigen»antibody complexes, and the second involved cross-linking an anti-FcR antibody bound to FcR on the cell-surface with a secondary antibody against a-FcR (a-Ig). We found that treating MC/9 cells with either K L H (Ag) or an antibody to K L H (Ab) failed to induce tyrosine phosphorylation of p38 M A P K . However, tyrosine phosphorylation of p38 M A P K was observed after the cells were treated with Ag»Ab complexes (Fig. 3.7). Signalling through the FcyRII and FcyRIII can be specifically prevented with 2.4G2 (a-FcR), a monoclonal antibody that binds to these FcR without signalling (Unkeless, 1979). Pretreatment of the MC/9 cells with a-FcR blocked the ability of Ag»Ab complexes to tyrosine phosphorylate p38 M A P K , indicating the complexes were specifically signalling through the FcR to activate p38 M A P K . Importantly, cells treated with either a-FcR or a-Ig alone failed to induce tyrosine phosphorylation of p38 M A P K . However, cells pretreated with a-FcR followed by treatment with a-Ig to cross-link a-FcR bound to the cell surface also induced the tyrosine phosphorylation of p38 M A P K (Fig. 3.7). 3.2.5 Hematopoietic growth factors activate MAPKAP kinase-2. M A P K A P kinase-2 has been reported to be a substrate of p38 M A P kinase (Freshney et al., 1994; Raingeaud et al., 84 CSF-1: - + + IP*- anti-p38 cc-CSF-1: + - + - + LCCM: - - - - + + » — IB: 4G10 IB:anti-p38 Figure 3.6 - Colony Stimulating Factor ( C S F ) - l induces tyrosine phosphorylation of p38 M A P K in F D M A C I I cells . Cells were incubated in RPMI 1640 for 1 hr prior to stimulation, and then treated with CSF-1 (CSF-1) , L-cell conditioned medium ( L C C M ) as a source of natural CSF-1, a neutralizing antibody (5A1) against CSF-1 (oc-CSF-1), or with indicated combinations of these for 5 min. Immunoprecipitated p38 MAP kinase was analyzed after SDS-PAGE by immunoblotting (IB) with the anti-phosphotyrosine antibody 4G10 (4G10), and subsequently with an anti-p38 MAP kinase antibody (anti-p38) to quantitate loading. 85 — — I B : 4G10 I B : anti-p38 Figure 3.7 - Cross-l inking of the FcR for IgG induces tyrosine phosphorylation of p38 M A P K in M C / 9 mast cells . MC/9 mast cells were pre-treated with a-FcR (a-FcR) for 10 min as indicated, and then incubated for 10 min in medium alone, or with a rabbit a-rat immunoglobulin antibody (a-Ig). MC/9 cells were also pre-treated for 10 min with an anti-keyhole limpet hemocyanin (KLH) antibody (Ab), and then incubated with or without K L H (Ag) for 10 min as indicated. Cellular lysates were immunoprecipitated with antibodies recognizing p38 MAPK. Immunoprecipitated p38 MAP kinase was analyzed after SDS-PAGE by immunoblotting (IB) with the anti-phosphotyrosine antibody 4G10 (4G10), and subsequently with an anti-p38 MAP kinase antibody (anti-p38) to quantitate loading. 86 1995), and to be activated in cells stimulated with GM-CSF or IL-3 (Ahlers et al., 1994). To determine whether the activation of M A P K A P kinase-2 by hematopoietic growth factors was due to activation of p38 M A P kinase, we imrnunoprecipitated M A P K A P kinase-2 from MC/9 cells that had been stimulated with IL-4, GM-CSF, SLF or 0.2 M NaCl and assessed its activity in an in vitro kinase assay using recombinant murine Hsp25 as substrate. As shown in the previous report (Ahlers et al., 1994), treatment with GM-CSF resulted in activation of M A P K A P kinase-2 (Fig. 3.8). SLF also induced strong activation of M A P K A P kinase-2 (Fig. 3.8). However, consistent with our finding that IL-4 failed to stimulate the enzymatic activity of p38 M A P kinase, IL-4 failed to induce activation of M A P K A P kinase-2 (Fig. 3.8). Indeed, treatment with IL-4 reduced the activity of M A P K A P kinase-2 to below the levels seen in untreated cells (Fig. 3.8), consistent with the reduction in p38 M A P kinase activity seen in IL-4 treated cells (Fig. 3.3). IL-4 was active on these cells as demonstrated by the induced tyrosine phosphorylation of a protein known to be IRS-2, or pl70 (Fig. 3.8) (Welham et al., 1997). Thus the ability of hematopoietic growth factors to activate M A P K A P kinase-2 correlated with their ability to activate p38 M A P kinase. 3.2.6 GM-CSF or SLF activate MAPKAP kinase-2 via p38 MAPK. M A P K A P kinase-2 has been reported to be activated by members of the E R K M A P kinase family (Stokoe et al., 1992). GM-CSF, IL-3 and SLF induce activation of both E R K (Welham et al., 1992) and p38 M A P kinases (Fig. 3.2 and 3.3). To investigate which of these kinases was responsible for the activation of M A P K A P kinase-2 by these hematopoietic growth factors, we used SB 203580, a specific inhibitor of p38 M A P kinase activity (Cuenda et al., 1995; Lee et al., 1994). Pre-treatment of cells for 20 minutes with 1 uM SB 203580 abrogated the ability of IL-3 (Fig. 3.9), GM-CSF (Fig. 3.10) or SLF (Fig. 3.11) to induce activation of M A P K A P kinase-2. In in vitro experiments, the enzymatic activity of ERK-2 was not inhibited by concentrations of SB 203580 that abolished the enzymatic activity of p38 M A P kinase (Cuenda et al., 1995). To confirm that SB 203580 did not affect the activity of the E R K M A P kinases in vivo, we stimulated cells with GM-CSF or SLF and investigated the effects of the compound on the activation of p90 r sk, which 87 4 Hsp25 Mil IB: 4G10 IP: a n t i - M A P K A P K 2 Figure 3.8 - Activation of M A P K A P kinase-2 by hemopoietic growth factors. MC/9 cells were incubated at 37°C in RPMI 1640 for 1 hr prior to simulation. Cells were then left untreated (CON), or treated with maximal doses of IL-4 (IL-4), GM-CSF ( G M - C S F ) , SLF (SLF) or 0.2 M NaCl (NaCl) for the indicated times. Cells were lysed and the majority of the lysate was used for a M A P K A P kinase-2 immune complex kinase assay with Hsp25 as a substrate. Autoradiograph of Hsp25 phosphorylation after SDS-PAGE (right). Immunoblotting of the proteins in the remaining cell lysate with an anti-phosphotyrosine antibody (4G10) after SDS-PAGE (left). 88 Untreated 1 uM IP: anti-MAPKAPK2 SB203580 IL-3: - 15 30 60 - 15 30 60 Hsp25 Figure 3.9 - IL-3 activates M A P K A P kinase-2 via p38 M A P K . Ba/F3 cells were incubated at 37°C in RPMI 1640 with or without 1 uM SB 203580 (SB 203580) for 20 minutes prior to stimulation. The cells were then left untreated, or were stimulated with saturating doses of IL-3 (IL-3) for the indicated times. M A P K A P kinase-2 was immunoprecipitated and its activity was assessed in immune complex kinase assays using recombinant murine Hsp25 as substrate. Reaction products were resolved by SDS-PAGE and the levels of phosphorylation of Hsp25 was assessed by autoradiography. 8 9 anti-p90rsk anti-MAPKAPK2 GM-CSF + + + + SB203580 - - + - - + Hsp25 MBP Figure 3.10 - G M - C S F activates M A P K A P kinase-2 via p38 M A P K . MC/9 cells were incubated at 37°C in RPMI 1640 with or without 1 uM SB 203580 (SB 203580) for 20 min prior to stimulation. The cells were then left untreated, or were stimulated with a saturating dose of GM-CSF ( G M - C S F ) for 10 min. M A P K A P kinase-2 or p90 r s k was immunoprecipitated from aliquots of cell lysates and their activities assessed in immune complex kinase assays using recombinant murine Ffsp25 or M B P as the respective substrates. Reaction products were resolved by SDS-PAGE and the levels of phosphorylation of Hsp25 or M B P were assessed by autoradiography. 9 0 anti-p90rsk anti-MAPKAPK2 SLF - - + + - - + + SB203580 - + - + - + - + MBP Hsp25 Figure 3.11 - S L F activates M A P K A P kinase-2 via p38 M A P K . MC/9 cells were incubated at 37°C in RPMI 1640 with or without 1 uM SB 203580 (SB 203580) for 20 min prior to stimulation. The cells were then left untreated, or were stimulated with a saturating dose of SLF (SLF) for 5 min. MAPKAP kinase-2 or p90 r s k was immunoprecipitated from aliquots of cell lysates and their activities assessed in immune complex kinase assays using recombinant murine Hsp25 or MBP as the respective substrates. Reaction products were resolved by SDS-PAGE and the levels of phosphorylation of Hsp25 or MBP were assessed by autoradiography. 9 1 is known to be a downstream target of the E R K M A P kinases (Blenis, 1993). As shown in Fig. 3.10 and 3.11, pretreatment of cells with SB 203580 did not affect the activation of p 9 0 r s k by GM-CSF or SLF, implying that the E R K M A P kinase pathway was not affected by SB 203580 and that p38 M A P kinase did not activate p90 r s k . 3.2.7 p38a is sufficient for activation of MAPKAP kinase-2. Two different p38 M A P K genes, p38a and p38(3, encode protein kinases that are sensitive to inhibition by SB 203580 (Gum et al., 1998; Jiang et al., 1997; Kumar et al., 1997; Lee et al., 1994; L i et al., 1996). A mutant of p38cc M A P K was produced that was resistant to inhibition by SB 203580 (SBR-p38) (Gum et al., 1998). We made a stable line of Ba/F3 cells that expressed about 1/3 as much mutant p38cc M A P K compared to endogenous p38oc M A P K (Fig. 3.12, bottom). To test if SB 203580-resistant p38a M A P K was sufficient to restore M A P K A P kinase-2 activity in vivo, we stimulated both wild-type and SB 203580-resistant p38a MAPK-expressing Ba/F3 cells with 0.2 M NaCl for 15 minutes. This expression of p38a M A P K was sufficient to completely restore the activation of M A P K A P kinase-2 after hyperosmotic shock (Fig. 3.12, top). 3.2.8 Role of p38 MAPK in DNA synthesis. To evaluate the physiological significance of the activation of p38 by these growth factors, we utilized SB 203580 to inhibit p38 M A P K activity. A series of experiments employing two factor-dependent hematopoietic cell lines, MC/9 and Ba/F3, indicated that SB 203580 inhibited cell growth. As shown in Fig. 3.13, MC/9 cells that were cultured with titrated amounts of SB 203580 in the presence of IL-3 exhibited a dose-dependent inhibition of ^H-thymidine incorporation with an I C 5 0 °f 3-5 uM. Experiments using the IL-3 dependent myeloid cell-line Ba/F3, grown in the presence of IL-3, showed a similar inhibition of D N A synthesis (Fig. 3.14). We then examined the proliferation of Ba/F3 cells stably over-expressing the SB 203580-resistant form of p38a M A P K . However, the SB 203580-resistant mutant of p38a M A P K was unable to rescue DNA synthesis in the presence of SB 203580 (Fig. 3.14). 92 WT-p38 SBR-p38 NaCl : - - + + - - + + SB203580: - + - + - + - + Hsp25 SBR-p38 WT-p38 Figure 3.12 - p38a M A P K is sufficient for activation of M A P K A P kinase-2. Ba/F3 cells stably transfected with either the empty vector (WT-p38) or a mutant isoform of p38a (SBR-p38) were incubated at 37°C in RPMI 1640 with or without 1 uM SB 203580 (SB 203580) for 20 min prior to stimulation. The cells were then left untreated, or were stimulated with 0.2 M NaCl (NaCl) for 15 min as indicated. M A P K A P kinase-2 was immunoprecipitated and its activity was assessed in immune complex kinase assays using recombinant murine Hsp25 as substrate. (Top) The phosphorylation of Hsp25 was assessed by autoradiography. (Bottom) The relative level of expression of SBR-p38 and endogenous p38 are indicated. 9 3 Figure 3.13 - p38 MAPK activity is required for DNA synthesis in hematopoietic cells. ^.Thymidine incorporation in MC/9 cells was measured at 48 hours, and is shown plotted against the concentrations of SB 203580. Triplicate 15 uL cultures contained 103 cells in medium with or without saturating IL-3, 10% FCS and the indicated concentrations of SB 203580 diluted in culture media. Mean values of cpm in cultures lacking SB 203580 were 2,969 cpm in the absence of IL-3 and 19,750 cpm in the presence of IL-3. 94 Figure 3.14 - p38a MAPK fails to rescue the inhibition of DNA synthesis in hematopoietic cells. ^.Thymidine incorporation in Ba/F3 cells was measured at 24 hours, and is shown plotted against the concentrations of SB 203580. Triplicate 15 uL cultures contained 103 cells in medium with or without saturating IL-3, 10% FCS and the indicated concentrations of SB 203580. 95 3.3 Discussion These experiments demonstrate that a series of hematopoietic growth factors which regulate the normal development and function of cells of the immune system stimulate tyrosine phosphorylation and enzymatic activation of p38 MAP kinase. This was the case for both those hematopoietic growth factors that signal through tyrosine kinase receptors, namely SLF (Fig. 3.2 and 3.3) and CSF-1 (Fig. 3.6), as well as those that signal through receptors of the hematopoietin receptor superfamily, namely IL-3 (Fig. 3.2) and GM-CSF (Fig. 3.3). Ligation of the Fc receptor for Immunoglobulin G, a receptor containing immunotyrosine activation motifs, also induces the tyrosine phosphorylation of p38 MAPK (Fig. 3.7). The observed correlation of tyrosine phosphorylation and activation of p38 MAP kinase (Fig. 3.3) is consistent with other evidence that phosphorylation of the tyrosine of the TGY activation motif of p38 MAP kinase is required for enzymatic activity (Doza et al., 1995; Raingeaud et al., 1995). The activation of p38 MAP kinase by GM-CSF and SLF in hematopoietic cells was rapid and transient (Fig. 3.4), and of the same order as that induced by hyperosmotic stress (Fig. 3.3). This contrasts with reports that stimulation of HeLa cells with epidermal growth factor activated p38 MAP kinase only weakly (Raingeaud et al., 1995) and that nerve growth factor (NGF) failed completely to activate p38 MAP kinase (RK) in PC-12 cells (Rouse et al., 1994). This may reflect differences in the cell types used in these studies. It should be noted that p38 MAP kinase was activated by hematopoietic growth factors in both cell lines and in mast cells from primary cultures. Given that MAPKAP kinase-2 is known to be an in vivo substrate of p38 MAP kinase (Freshney et al., 1994; Rouse et al., 1994), our observations that p38 MAP kinase is activated in response to hematopoietic growth factors are consistent with the earlier report that IL-3 and GM-CSF activate MAPKAP kinase 2 (Ahlers et al., 1994). Our results confirm this observation and extend it by demonstrating that treatment of cells with SLF also induces MAPKAP kinase-2 activity (Fig. 3.8). In that the specific inhibitor of p38 MAP kinase, SB 203580, completely abrogated MAPKAP kinase-2 activation by IL-3 (Fig. 3.9), GM-CSF (Fig. 3.10) and SLF (Fig. 9 6 3.11), our data also extend current knowledge by indicating that the activation of M A P K A P kinase-2 by GM-CSF or SLF depended on p38 M A P kinase activity. Thus, despite the fact that treatment of cells with GM-CSF or SLF activates both E R K (Welham et al., 1992) and p38 MAP kinases (Fig. 3.3), SB 203580 specifically inhibited the in vivo activation of M A P K A P kinase-2 but not of p 9 0 r s k (Fig. 3.10 and 3.11). These data demonstrate that SB 203580 fails to inhibit the activity of E R K M A P kinases in vivo, consistent with in vitro data that SB 203580 fails to inhibit ERK-2 or p 9 0 r s k (Cuenda et al., 1995). These results are also consistent with experiments in which in vivo activation of E R K M A P kinases did not result in in vivo activation of M A P K A P kinase-2 (Rouse et al., 1994). Despite the fact that both E R K and p38 M A P kinases can activate M A P K A P kinase-2 in vitro (Freshney et al., 1994; Rouse et al., 1994; Stokoe et al., 1992), and both are activated in cells treated with GM-CSF or SLF, p38 M A P kinase activity was essential for activation of M A P K A P kinase-2 in vivo (Fig. 3.10 and 3.11). This evidence that M A P K A P kinase-2 is activated by p38 M A P kinase and not by E R K M A P kinase, suggests differences in molecular localization of these enzymes. Another possibility is that inactive p38 M A P kinase has a greater affinity for M A P K A P kinase-2 than the E R K M A P kinases, and that, in vivo, M A P K A P kinase-2 is complexed with inactive p38 M A P kinase, and not with E R K M A P kinases. In keeping with this notion, p38 MAP kinase has been shown to form a complex in vivo with human M A P K A P kinase-3, a close homologue of M A P K A P kinase-2 (70% amino acid identity) (McLaughlin et al., 1996). The failure of SB 203580 to inhibit the in vivo activation of p 9 0 r s k in the same cells in which it inhibited the in vivo activation of M A P K A P kinase-2 (Fig. 3.10 and 3.11) is consistent with the notion that p 9 0 r s k is activated only by the E R K M A P kinases and not by p38 MAP kinase. These results confirm the suggestion from unpublished in vitro experiments that p38 MAP kinase (RK) was unable to activate p 9 0 r s k (Rouse et al., 1994). It should be noted that the anti-MAPKAP kinase-2 antibody that we used was raised against a 16 amino-acid peptide from mouse M A P K A P kinase-2. This peptide differs by only two closely spaced, conservative amino acid substitutions from the corresponding peptide of human 97 MAPKAP kinase-3. If there is a murine homologue of human MAPKAP kinase-3, we would probably have precipitated it with this antibody. In the human, both MAPKAP kinase-2 and MAPKAP kinase-3 are activated by p38 MAP kinase (CSBP) (McLaughlin et al., 1996). Moreover both enzymes act as Hsp25 kinases in vitro, although whether both phosphorylate Hsp25 in vivo is unclear. The functional significance of activation of the p38 MAP kinase pathway by growth factors and its relationship to actions of growth factors such as promotion of cell-cycle progression or suppression of apoptosis is unclear. Two possible roles for p38 MAP kinase relate to the regulation of actin polymerization and the activation of transcription factors. Actin polymerization appears to be regulated by phosphorylation of Hsp25, which lowers the affinity of its interaction with the barbed ends of filamentous (F) actin, thus allowing polymerization and the accumulation of F-actin (Lavoie et al., 1995). Growth factors and serum induce phosphorylation of Hsp25 on the same residues that are phosphorylated in response to stress (Landry et al., 1992; Saklatvala et al., 1991), consistent with the involvement of the same enzyme, most likely MAPKAP kinase-2 or MAPKAP kinase-3. Our results with hematopoietic growth factors demonstrate that inhibition of p38 MAP kinase completely blocks activation of MAPKAP kinase-2 (Fig. 3.10 and 3.11), suggesting that growth factor stimulated phosphorylation of Hsp25 reflects activation of p38 MAP kinase. Furthermore, the over-expression of an SB 203580-resistant isoform of p38a MAPK was sufficient to restore MAPKAP kinase-2 activation (Fig. 3.12). The role of actin polymerization in growth factor action is unclear although cell growth is inhibited by cytochalasin D (Lavoie et al., 1995) which, like unphosphorylated Hsp25, prevents elongation of F-actin (Sampath and Pollard, 1991). Over-expression of wild-type Hsp25, but not mutant Hsp25 that is unable to become phosphorylated, renders cells relatively resistant to the growth inhibitory effect of cytochalasin D (Lavoie et al., 1995). This implies a requirement for Hsp25 phosphorylation and F-actin accumulation for cell cycle progression. Our observations that cells treated with SB 203580 underwent a rapid and reversible change in cellular morphology (F. Lee, unpublished data) were also consistent with the notion that p38 MAP kinase regulated actin polymerization. 98 Transcription factors regulating cell growth may be regulated by the p38 M A P kinase pathway through several mechanisms. In vivo activation of p38 M A P kinase increases the transcriptional activity of Elk-1 (Raingeaud et al., 1996), implying a role for p38 M A P kinase in the transcriptional regulation of proteins such as c-Fos. Interestingly p38p, but not p38a MAP kinase, has been shown to phosphorylate and activate ATF-2 in vivo (Jiang et al., 1996), suggesting differential regulation of transcription factors by the p38 M A P kinase family members. Further experiments will be required to determine whether all family members are activated by hematopoietic growth factors. Recently p38 M A P kinase has also been shown to regulate the phosphorylation of CREB and ATF-1 through M A P K A P kinase 2 (Tan et al., 1996). In addition, p38 M A P kinase has been shown to regulate the production of IL-1, TNFa , IL-6 and GM-CSF (Beyaert et al., 1996; Lee et al., 1994), which are all encoded by mRNAs containing AU-rich motifs. It is possible that the production of the transcription factors c-Myc, c-Fos and c-Jun, which are translated from mRNA containing the same AU-rich motifs characteristic of cytokine mRNA (Chen and Shyu, 1995), may also be regulated in a p38 M A P kinase-dependent fashion. The notion that activation of the p38 M A P kinase pathway has a critical role in cell cycle progression is suggested by our observations that the specific p38 M A P kinase inhibitor SB 203580 inhibited DNA synthesis (Fig. 3.13 and 3.14). However the over-expression of an SB 203580-resistant isoform of p38a M A P K was unable to rescue D N A synthesis in Ba/F3 cells (Fig. 3.14), even though M A P K A P kinase-2 activation was restored, suggesting that M A P K A P kinase activity was not sufficient to rescue DNA synthesis. However it is possible that M A P K A P K 2 activity is not completely restored under normal cell culture conditions, and such possibilities require further investigation. These findings also suggest that there may be differences in the substrate specificity of p38a and p38p in vivo, or that the inhibition of DNA synthesis was due to a non-specific effect of SB 203580. The correct interpretation of these data requires the analysis of an identical SB 203580-resistant mutant of p38p M A P K . It will be important to develop precise genetic tools to explore the role of p38 M A P kinase in growth factor action and cell cycle progression in mammalian cells. 99 A recent report implicated p38 M A P kinase in the induction of apoptosis (Xia et al., 1995). Withdrawal of nerve growth factor from PC-12 cells led to activation of p38 M A P kinase and the c-Jun N-terminal kinases (JNK), followed by apoptosis. Over-expression of a constitutively active M K K 3 , which at least in some cells activates p38 M A P kinase (Raingeaud et al., 1995; Xia et al., 1995), promoted apoptosis, whereas over-expression of a dominant negative M K K 3 inhibited apoptosis (Xia et al., 1995). However, these results do not discriminate between roles for p38 M A P kinase and JNK, as over-expression of a dominant negative M K K 3 appears to block activation of both of these kinases (Raingeaud et al., 1995). In contrast, our results show that p38 M A P kinase is activated by stimulation by growth factors (Fig. 3.2 and 3.3), not by their withdrawal. Moreover, in that hematopoietic growth factors suppress apoptosis (Williams et al., 1990), our findings indicate that, at least in hematopoietic cells, activation of p38 M A P kinase correlates with the suppression of apoptosis rather than its induction. Finally, treatment of cells with concentrations of SB 203580 that we have shown to inhibit p38 M A P kinase activity in vivo (Fig. 3.8) failed to inhibit the apoptosis induced by withdrawal of hematopoietic growth factors (I. Foltz, unpublished data). Interleukin-4 and IL-l3 are notable in being the only growth factors we investigated that failed to induce tyrosine phosphorylation (Fig. 3.2, 3.3 and 3.4) or activation of p38 M A P kinase (Fig. 3.3). The inability of IL-4 to activate p38 M A P kinase accounts for its failure to activate M A P K A P kinase-2 (Fig. 3.8) and correlates with its failure to activate Ras (Duronio et al., 1992; Satoh et al., 1991), and E R K M A P kinase (Welham et al., 1992; Welham et al., 1994). Further experiments will be required to determine whether the activation of p38 M A P kinase by growth factors in hematopoietic cells is Ras dependent. Interestingly, IL-4 consistently reduced the state of tyrosine phosphorylation and enzymatic activity of p38 M A P kinase to levels below those observed in control cells (Fig. 3.3). Likewise, treatment of cells with IL-4 reduced the in vivo activity of M A P K A P kinase-2 to levels below those in control cells (Fig. 3.8). The basis for the inhibition of the p38 M A P kinase pathway by IL-4 is under further investigation. 100 In conclusion, these experiments demonstrate that p38 M A P kinase participates in the responses to hematopoietic growth factors that interact with two structurally distinct classes of receptor. They demonstrate that p38 M A P kinase is not only involved in responses to stresses, but also in the action of growth factors that regulate the development and function of hematopoietic cells. 101 CHAPTER 4 - Activation of JNK in Hematopoietic Cells. 4.1 Introduction While working to characterize the hematopoietic growth factors IL-3, GM-CSF and SLF as activators of the p38 M A P K pathway, it became apparent that the activation of p38 M A P K strongly correlated with the activation of the JNK pathway. Stimuli such as IL-1, TNFa , and U V light were characterized as activators of both p38 M A P K and JNK. In fact, a stimulus capable of separating the activation of p38 M A P K and JNK had not been described as yet. Previous unpublished data in our laboratory conducted by James Wieler had established that IL-3. GM-CSF and SLF induced mRNAs encoding c-Jun. However, IL-4 was unable to induce the expression of c-Jun. JNK had been identified as the relevant kinase for increasing c-Jun transactivation through N-terminal phosphorylation, thereby increasing AP-1 activity. The promoter of c-Jun contains AP-1 sites and therefore the literature suggested the induction of c-Jun was indicative of J N K activity. Hypothesis: (1) IL-3, GM-CSF and SLF would increase the activity of JNK1 and JNK2. (2) These cytokines would induce the phosphorylation of M K K 4 . (3) IL-4 would fail to activate either isoform of JNK or induce the phosphorylation of M X K 4 . Objectives: (1) The transcription factor c-Jun had been described as having affinity for JNK through the delta peptide. We used GST-c-Jun to affinity precipitate JNK and perform in vitro kinase assays to determine the effect of cytokine stimulation on the activation of JNK. We also intended to identify commercial antibodies that could be used for immunoprecipitation of JNK for use in gel kinase assays, or immune-complex kinase assays. 102 (2) At this time the only known activator of JNK was the dual specificity kinase, M K K 4 . There were no antibodies commercially available to immunoprecipitate M K K 4 for direct analysis of kinase activity. However, New England Biolabs had just started producing antibodies that specifically recognized M K K 4 when phosphorylated on an activating threonine residue. We decided to use this approach to correlate the activation of JNK with the activation state of M K K 4 . (3) Stimuli capable of separating the activation of p38 M A P K and JNK had not yet been described, and as such we did not expect IL-4 to be able to induce JNK activation. Both in gel kinase assays and immune complex kinase assays of cell extracts from cells treated with IL-4 would be conducted to determine i f IL-4 could activate JNK or its activator M K K 4 . 4.2 Results 4.2.1 Activation of JNKl and JNK2 by Hematopoietic Growth Factors with the Exception of IL-4 or IL-13. To determine whether JNK was involved in responses to IL-3 , GM-CSF or SLF, we stimulated MC/9 mast cells with these factors and immunoprecipitated J N K , using antibodies that precipitated both the 46 and 55 kDa isoforms of either J N K l or JNK2. The precipitate was subjected to SDS-PAGE followed by renaturation and JNK activity was assessed using an "in gel" kinase assay. We found that stimulation with GM-CSF, IL-3 or SLF induced activation of both the 46 and 55 kDa isoforms of J N K l (Fig. 4.1). It was of particular interest to investigate the effect of IL-4 on J N K l activity, as our laboratory had previously shown that IL-4 failed to activate Ras (Duronio et al., 1992; Welham et al., 1994), ERK-1/2 M A P kinases (Welham et al., 1992; Welham et al., 1994) or p38 MAP kinase (Foltz et al., 1997). Unlike the other hematopoietins, IL-4 and IL-13 failed to activate either J N K l (Fig. 4.1 and 4.2) or JNK2 (Fig. 4.2 and 4.3). We also examined the activation of JNK2, and found that GM-CSF or SLF induced the activation of both 46 and 55 kDa isoforms of JNK2 (Fig. 4.3). To examine the kinetics of the activation of JNK induced by treatment of MC/9 cells with SLF, IL-3 and GM-CSF, we used GST-c-Jun to preferentially immunoprecipitate activated JNK 103 A IP: anti-JNKl < f w- N c^V i f 10 10 10 5 15 f^lfl J • - «» m P 4 6 Figure 4.1 - Activation of JNK1 by hematopoietic growth factors with the exception of IL-4 in MC/9 cells. Cells were incubated at 37°C in RPMI 1640 for 1 hr and then left untreated (CON), or treated with saturating doses of IL-3 (IL-3), IL-4 (IL-4), GM-CSF (GM), SLF (SLF) or 0.2 M NaCl (NaCl) for the indicated times. Immunoprecipitated JNK1 was analyzed using an in gel kinase assay after SDS-PAGE with a separating gel containing GST-c-jun. The phosphorylation of c-Jun was assessed by autoradiography and the presence of 46 and 55 kDa splice variants of JNK1 are indicated. 1 0 4 AP:GST-c-Jun GST-c-Jun Figure 4.2 - IL-13 fails to activate J N K l in FD5/IL13R cells. FD5 cells stably expressing the IL-13Ra chain (FD5/IL13R) were incubated at 37°C in RPMI 1640 for 1 hr and then left untreated (CON), or treated with saturating doses of IL-4 (IL-4), IL-13 (IL-13) or 0.2 M NaCl (NaCl) for 10 min. JNK was affinity precipitated (AP) using GST-c-Jun, and J N K activity was analyzed in a kinase assay with GST-c-Jun as substrate. The phosphorylation of c-Jun was assessed by autoradiography. 105 A IP: anti-JNK2 p55 p46 Figure 4.3 - Activation of J N K 2 by G M - C S F and S L F , but not IL-4, in M C / 9 cells. Cells were incubated at 37°C in RPMI 1640 for 1 hr and then left untreated (CON), or treated with saturating doses of IL-4 (IL-4), GM-CSF (GM), SLF (SLF) or 0.2 M NaCl (NaCl) for the indicated times. Immunoprecipitated JNK2 was analyzed after SDS-PAGE with a separating gel containing GST-c-Jun, using an in gel kinase assay. The phosphorylation of c-Jun was assessed by autoradiography and the presence of 46 and 55 kDa splice variants of JNK2 are indicated. 106 (Adler et al., 1994; Dai et al., 1995; Kallunki et al., 1996). Treatment with SLF or GM-CSF resulted in a rapid increase in the activation of JNK (Fig. 4.4). Cells treated with SLF exhibited detectable activation of JNK after 2 minutes, with maximal activation of JNK occurring around 10 minutes (Fig. 4.4). In cells treated with GM-CSF the activation of JNK again peaked at around 10 minutes (Fig. 4.4). In both instances the activation of JNK was transient and was returning to basal levels by 30 minutes (Fig. 4.4). 4.2.2 Threonine Phosphorylation of MKK4 is induced by GM-CSF or SLF, but not IL-4. At the time these experiments were done, M K K 4 was the only established upstream activator of JNK (Derijard et al., 1995; Lin et al., 1995; Sanchez et al., 1994; Yan et al., 1994). Therefore we determined whether there was a correlation between activation of JNK by hematopoietic growth factors and activation of M K K 4 . We immunoblotted cell extracts from MC/9 cells that had been stimulated with GM-CSF, IL-4 or SLF (Fig. 4.5, top), or GM-CSF, SLF or 0.2 M NaCl (Fig. 4.5, bottom) with an antibody that specifically recognized the activated forms of human and murine M K K 4 . It was evident (Fig. 4.5, bottom) that stimulation of cells with GM-CSF, SLF or NaCl induced rapid phosphorylation of M K K 4 . This phosphorylation was transient and was returning to basal levels by 10 minutes. On shorter exposures, it was evident that GM-CSF was a weaker activator of M K K 4 than SLF (Fig. 4.5, top). These data indicate the activation of M K K 4 preceded the activation of JNK, consistent with a role in activation of JNK by these stimuli. In keeping with our finding that IL-4 failed to stimulate the enzymatic activity of JNK, IL-4 failed to induce phosphorylation of M K K 4 (Fig. 4.5). The activation of JNK in all cases correlated with activation of M K K 4 . 4.3 Discussion These experiments utilizing MC/9 mast cells demonstrate that a series of hematopoietic growth factors which regulate the normal development and function of cells of the immune system 1 0 7 Figure 4.4 - Kinetics of J N K activation by G M - C S F or S L F in MC/9 cells. After 1 hr in RPMI 1640 at 37°C, MC/9 cells were left untreated, or treated with saturating doses of G M -CSF (GM-CSF) , SLF (SLF), or 0.2 M NaCl (NaCl) as a positive control for the indicated times in minutes. Cell extracts were prepared and GST-c-Jun was used as both affinity reagent and substrate for an in vitro kinase assay. The phosphorylation of c-Jun was assessed by autoradiography (left). The fold activation of JNK in cells treated with SLF or GM-CSF compared to untreated cells was quantitated using densitometry (right). 108 c f ^ & & - 10 10 5 15 — phospho-MKK4 fl SLF NaCl GM-CSF 6 5 10 5 10 5 10 Figure 4.5 - Threonine phosphorylation of M K K 4 by SLF or GM-CSF, but not IL-4. MC / 9 cells were incubated at 37°C in RPMI 1640 for 1 hr prior to simulation. Cells were then left untreated (CON), or (Top) treated with maximal doses of IL-4 (IL-4), GM-CSF (GM-CSF) or SLF (SLF) or (Bottom) treated with maximal doses of GM-CSF (GM-CSF), SLF (SLF) or 0.2 M NaCl (NaCl) for the indicated times. After lysis, cell extracts were analysed by SDS-PAGE, and were immunoblotted with an antibody that specifically recognizes the activated form of SEK1/MKK4. 109 activate both JNK1 (Fig. 4.1) and JNK2 (Fig. 4.3). One of these hematopoietic growth factors, SLF, signals through a tyrosine kinase receptor, while the others, IL-3 and GM-CSF, signal through receptors of the hematopoietin receptor superfamily (Fantl etal., 1993; Taniguchi, 1995). Cells treated with GM-CSF, IL-3 or SLF exhibited increased activity of 46 and 55 kDa proteins corresponding to different splice variants of JNK1 (Fig. 4.1). Similarly, cells treated with G M -CSF or SLF activated both 46 and 55 kDa splice variants of JNK2 (Fig. 4.3). Together our results indicate that IL-3 (Fig. 4.1), GM-CSF (Fig. 4.1 and 4.3) and SLF (Fig. 4.1 and 4.3) stimulate the activation of 46 and 55 kDa isoforms of both JNK1 and JNK2 in MC/9 cells. The kinetics of JNK activation in MC/9 cells treated with GM-CSF or SLF resembled that observed for activation of E R K (Welham et al., 1992; Welham et al., 1994) and p38 M A P K (Foltz et al., 1997) by these factors. The activation of JNK by SLF showed similar kinetics to that induced by GM-CSF (Fig. 4.4). In all cases the activation of JNK following stimulation with these factors was rapid and transient (Fig. 4.4). We also used an antibody specific for the activated form of M K K 4 to analyze the ability of these growth stimuli to activate M K K 4 , the only known upstream activator of JNK (Derijard et al., 1995; Lin et al., 1995; Sanchez et al., 1994; Yan et al., 1994). The phosphorylation of M K K 4 induced by treatment of cells with SLF, G M -CSF or 0.2 M NaCl was evident at 5 minutes and was declining by 10 minutes when levels of JNK activity were peaking. However, the phosphorylation of M K K 4 by SLF was greater than that observed when cells were treated with GM-CSF. The earlier kinetics of phosphorylation of M K K 4 , compared with activation of JNK, are consistent with M K K 4 being upstream of JNK in the cellular response to these stimuli. These results suggest that M K K 4 is involved in the activation of JNK in response to SLF and GM-CSF. There have been reports of other activators of JNK (Moriguchi et al., 1995), but these proteins had not been characterized at the time these experiments were done. Interleukin-4 and IL-13 were unique among the hematopoietins that we examined in this study in failing to increase the activation of JNK1 (Fig. 4.1) or JNK2 (Fig. 4.3). As noted, the inability of IL-4 to activate JNK correlated with its inability to induce phosphorylation of M K K 4 110 (Fig. 4.5). This failure of IL-4 to activate JNK correlates with its inability to activate either the E R K (Welham et al., 1992; Welham et al., 1994) or the p38 M A P kinase pathway (Foltz et al., 1997). Interleukin-4 is also notable for its inability to activate Ras (Duronio et al., 1992; Satoh et al., 1991). We hypothesize that hematopoietic growth factors activate p38 M A P K and JNK in a Ras-dependent fashion. Indeed, other growth factors activate JNK through Ras-dependent mechanisms as dominant inhibitory mutants of Ras have been shown to prevent the activation of JNK in response to EGF and NGF (Minden et al., 1994). Furthermore, JNK activity was increased by the expression of constitutively active mutants of Ras (Minden et al., 1995). However, dominant inhibitory mutants of Cdc42 or Racl prevent JNK activation by Ras (Minden et al., 1995), and constitutively active mutants of Cdc42 or Racl increased JNK activity (Bagrodia et al., 1995; Coso et al., 1995; Minden et al., 1995). These findings suggest that Ras was mediating JNK activation through either Cdc42 or Racl . Observations that the activation of J N K by TNFa is not inhibited by over-expression of dominant-negative Ras however suggest that there may also be other Ras-independent pathways of JNK activation (Minden et al., 1995). Further studies using dominant inhibitory mutants of Ras and the Rho family of small GTPases will be required to elucidate the hierarchy of proteins leading to JNK activation by hematopoietic growth factors. Hematopoietic growth factors promote cell cycle progression in cells of the immune system. The functional significance of the activation of J N K l and JNK2 by hematopoietic growth factors, for example in the promotion of cell cycle progression, is unknown. Clearly the best argument for a role of JNK in cell cycle progression is their ability to phosphorylate and activate the transcription factors including c-Jun, ATF-2 and Elk-1 (Cavigelli et al., 1995; Derijard et al., 1994; Gupta et al., 1995; Kyriakis et al., 1995; Minden et al., 1994). JNK may have an obligate role in cell cycle regulation as JNK are the only known kinases capable of phosphorylating c-Jun on serines 63 and 73 and thereby increasing its transcriptional activity (Derijard et al., 1994; Kyriakis et al., 1995; Minden et al., 1994). The observation that c-Jun is required for the 1 1 1 transformation of fibroblasts by activated Ras implies a role for JNK in the Ras-mediated control of growth (Johnson et al., 1996). Hematopoietic growth factors not only provide growth signals that promote growth of hematopoietic cells, but also signals that suppress apoptosis. In some cells activation of J N K correlates not with suppression of apoptosis but with its induction (Chen et al., 1996; Xia et al., 1995). It is possible that activation of the JNK pathway in the absence of other signals such as E R K activity results in a pro-apoptotic signal, whereas the integrated activation of ERK, JNK and p38 M A P K pathways, stimulated by factors such as GM-CSF, IL-3 and SLF, delivers a signal for the suppression of apoptosis. However, our data indicates that the activation of ERK, JNK or p38 M A P K pathways is not essential for the suppression of apoptosis as IL-4, which we have shown fails to activate any of these kinases, nevertheless suppresses the apoptosis of many hematopoietic cells, including the factor dependent cell line MC/9 used in these experiments. In conclusion, we have demonstrated that JNK1 and JNK2 are activated in response to IL-3, GM-CSF and SLF, but not IL-4. Furthermore our results suggest that SAPK activation is mediated by M K K 4 , as both SLF and GM-CSF, but not IL-4, activated M K K 4 prior to activation of JNK. Importantly, our results demonstrate that JNK activity is involved not only in response to stress, but also in signaling by hematopoietic growth factors that in some cases, such as SLF, regulate the normal growth and development of the hematopoietic system. 112 CHAPTER 5 - MKK7 - A Specific Activator of JNK. 5.1 Introduction The activation of JNK by GM-CSF or SLF followed very similar kinetics. However, the induction of threonine phosphorylation of M K K 4 by these cytokines was much more pronounced with SLF than with GM-CSF or NaCl. These findings suggested the existence of another J N K kinase that was regulated by GM-CSF. Several other lines of evidence pointed to the existence of another M K K capable of activating the JNK family. Moriguchi et al. identified at least one J N K kinase activity in addition to the activity of M K K 4 in rat fibroblastic 3Y1 cells stimulated by hyperosmotic shock (Moriguchi et al., 1995). Similarly Meier et al. identified two JNK kinase activities operationally termed SKK4 and SKK5 in K B cells, a human oral carcinoma cell line, following treatment with hyperosmolarity or anisomycin (Meier et al., 1996). The generation of embryonic stem (ES) cells in which the M K K 4 gene was disrupted provided conclusive evidence for the existence of another JNK kinase. ES cells lacking M K K 4 still exhibited increased J N K activity following hyperosmotic shock or U V irradiation, but failed to exhibit JNK activation after treatment with anisomycin or interleukin-1 (Meier et al., 1996; Nishina et al., 1997; Yang et al., 1997). Together these studies suggested the existence of another JNK kinase. Hypothesis: (1) MC/9 cells express a novel M K K that acts as a direct activator of JNK. (2) The unknown M K K is activated by GM-CSF, IL-3, and NaCl. Objectives: (1) Screen the expressed sequence tags (EST) database to identify a novel M K K . The sequences of the known M K K were aligned, and a consensus sequence at the C-terminus of these enzymes 1 1 3 was determined (Fig. 5.1). The resulting peptide was converted to a degenerate oligonucleotide (Fig. 5.2), and the oligonucleotide was used to screen the EST database. (2) Determine the expression pattern of any unknown M K K that was identified. (3) Characterize the enzymes for their substrate specificity and upstream activators. 5.2 Results 5.2.1 Cloning strategy to discover unknown MKK. We screened the EST (expressed sequence tags) database using a degenerate oligonucleotide sequence based on the amino acids conserved in the C-terminus of all previously characterized mitogen-activated protein kinase kinases (Fig. 5.1) (Lin et al., 1995). We identified 7 clones that encoded known M K K family members and one clone (aa019720) that contained a novel human sequence (Table 5.2). This clone contained 464 base pairs (bp) of coding region and approximately 1200 bp of 3' untranslated region. We used 5 ' -RACE (Rapid Amplification of cDNA Ends) to isolate overlapping 5'-fragments of this novel cDNA from a human fetal kidney R A C E cDNA library (Fig. 5.2). We amplified several over-lapping clones of M K K 7 using a fully nested PCR with the adaptor primers AP1/AP2 and primers IF113/IF111 or IF117/IF115 for the first or second/third screens respectively (Fig. 5.3). Using this technique we cloned a further 861 bp of human M K K 7 (hMKK7a) that contained an in-frame stop codon. When theoretical translation was initiated at the first methionine, this cDNA encoded a 419 amino acid protein (Fig. 5.4) that contained a putative protein kinase domain. We also isolated a splice variant of human M K K 7 (MKK7(3) that contained an additional 126 base pairs encoding an insert of 42 amino acids (Fig. 5.5). We screened the yeast and invertebrate databases for other M K K related to M K K 7 and although we failed to identify a yeast homolog, we did find an ortholog of M K K 7 in D. melanogaster (dHep - 69% identity) (Glise et al., 1995). In C. elegans we identified an ortholog with 54% identity we termed cMKK7 (Wilson et al., 1994). We compared the catalytic domains of M K K 7 with dHep and cMKK7 (Fig. 5.6) or all other known human M K K (Fig. 5.7). M K K 7 was orthologous with 114 Alignment of M K K : MKK4: FSPSFINFVNLCLTKDESKRPKYKELLKHPF MKK3: . . . E . V D . T A Q . . R . N P A E . M S . L . . M E . . . MEK2: . T . D . Q E . . . K . . I . N P A E . A D L . M . T N . T . MEK1: . . L E . Q D . . . K . . I . N P A E . A D L . Q . M V . A . PBS2: . . S D A Q D . . S . . . Q . I . E R . . T . A A . T . . . W C o n s e n s u s : F F . . . C L . K . . . .R L . . H . . Nucleotide Sequence: TT[TC].(.)16TT[TC].(.)7.TG[TC].T....AA[AG].(.)11.G.(.)15.T.(.)5.CA Figure 5.1 - Designing a probe to screen the EST database. The C-terminii of the primary structures of human M K K 4 , M K K 3 , M E K 2 , MEK1 and 5. cerevisiae PBS2 were aligned to determine a consensus motif to identify novel M K K . The amino acid motif was back-translated into degenerate nucleotides according to the human genetic code (Table 5.1). The resulting nucleotide sequence was used to screen the EST database. Table 5.1 - The Genetic Code. T C A G T TTT - F TTC - F TTA - L TTG - L TCT - S TCC - S TCA - S TCG - S TAT - Y TAC - Y TAA - * TAG - * TGT - C TGC - C TGA - * TGG - W C CTT - L CTC - L CTA - L CTG - L CCT - P CCC - P CCA - P CCG - P CAT - H CAC - H CAA - Q CAG - Q CGT - R CGC - R CGA - R CGG - R A ATT - I ATC - I ATA - I ATG - M ACT - T ACC - T ACA - T ACG - T AAT - N AAC - N AAA - K AAG - K AGT - S AGC - S AGA - R AGG - R G GTT - V GTC - V GTA - V GTG - V GCT - A GCC - A GCA - A GCG - A GAT - D GAC - D GAA - E GAG - E GGT - G GGC - G GGA - G GGG - G 115 Table 5.2 - Analysis of Clones derived from the EST database. EST Clone Protein Species aa008333 MKK3 Human aa019720 Novel - MKK7 Human n98702 MKK4 Human rll022 MEK2 Human r51239 - -ricr2291a Novel Rice t23364 Novel GCK-like Human wl6504 Novel Grain W29331 MKK5 Human W78467 MKK3 Human aa050011 MKK3 Mouse aa026071 - -aa071840 MKK4 Mouse ATG Step 1: Nested PCR A P I AP2 I 1 Adapter i Step 2: Blunt end sub-cloning of PCR products into pBSKS 1 Step 3: Blue/White Colony Selection 1 Step 4: Colony Blot with Specific Probe from EST Clone I Step 5: Sequence Putative Positive Clones Figure 5.2- Strategy for cloning full length MKK7. EST clone: aa019720 TAG Probe 116 EST clone: aa019720 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 0 100 200 300 400 I l I I I I I I I L i — — — ATC IF115IF117 IF111 IF113 TAG Primary Screen: Clone 10 Clone 24 Secondary Screen: Clone 7 Clone 17 Tertiary Screen: ^ — ^ — ^ C l o n e FI Figure 5.3- Amplification of fragments of MKK7 using 5'-RACE PCR. A human fetal kidney cDNA library was used to amplify M K K 7 . The adapter primers API and AP2 were used for all PCR amplifications as depicted in Figure 5.2. A fully nested PCR using either the primers IF113 and IF 111, or the primers IF117 and IF115, was used for the primary screen, or the secondary and tertiary screens respectively. The positive clones found in these screens are depicted to scale with respect to the full length human M K K 7 a , and the EST clone aaO 19720. 117 GGCGGTGTTTGTCTGCCGGACTGACGGGCGGCCGGGCGGTGCGCGGCGGCGGTGGCGGCCGGGGAAA ATGGCGGCGTCCTCCCTGGAACAGAAGCTGTCCCGCCTGGAAGCAAAGCTGAAGCAGGAGAACCGGGA M A A S S L E Q K L S R L E A K L K Q E N R E GGCCCGGCGGAGGATCGACCTCAACCTGGATATCAGCCCCCAGCGGCCCAGGCCCACCCTGCAGCTCC A R R R I D L N L D I S P Q R P R P T L Q L CGCTGGCCAACGATGGGGGCAGCCGCTCGCCATCCTCAGAGAGCTCCCCGCAGCACCCCACGCCCCCC P L A N D G G S R S P S S E S S P Q H P T P P GCCCGGCCCCGCCACATGCTGGGGCTCCCGTCAACCCTGTTCACACCCCGCAGCATGGAGAGCATTGA A R P R H M L G L P S T L F T P R S M E S I E GATTGACCAGAAGCTGCAGGAGATCATGAAGCAGACGGGCTACCTGACCATCGGGGGCCAGCGCTACC I D Q K L Q E I M K Q T G Y L T I G G Q R Y AGGCAGAAATCAACGACCTGGAGAACTTGGGCGAGATGGGCAGCGGCACCTGCGGCCAGGTGTGGAAG Q A E I N D L E N L G E M G S G T C G Q V W K ATGCGCTTCCGGAAGACCGGCCACGTCATTGCCGTTAAGCAAATGCGGCGCTCCGGGAACAAGGAGGA M R F R K T G H V I A V K Q M R R S G N K E E GAACAAGCGCATCCTCATGGACCTGGATGTGGTGCTGAAGAGCCACGACTGCCCCTACATCGTGCAGT N K R I L M D L D V V L K S H D C P Y I V Q GCTTTGGGACGTTCATCACCAACACGGACGTCTTCATCGCCATGGAGCTCATGGGCACCTGCGCTGAG C F G T F I T N T D V F I A M E L M G T C A E AAGCTCAAGAAGCGGATGCAGGGCCCCATCCCCGAGCGCATTCTGGGCAAGATGACAGTGGCGATTGT K L K K R M Q G P I P E R I L G K M T V A I V GAAGGCGCTGTACTACCTGAAGGAGAAGCACGGTGTCATCCACCGCGACGTCAAGCCCTCCAACATCC K A L Y Y L K E K H G V I H R D V K P S N I TGCTGGACGAGCGGGGCCAGATCAAGTTCTGCGACTTCGGCATCAGCGGCCGCCTGGTGGACTCCAAA L L D E R G Q I K F C D F G I S G R L V D S K GCCAAGACGCGGAGCGCCGGCTGTGCCGCCTACATGGCACCCGAGCGCATTGACCCCCCAGACCCCAC A K T R S A G C A A Y M A P E R I D P P D P T CAAGCCGGACTATGACATCCGGGCCGACGTATGGAGCCTGGGCATCTCGCTGGTGGAGCTGGCAACAG K P D Y D I R A D V W S L G I S L V E L A T GACAGTTTCCCTACAAGAACTGCAAGACGGACTTTGAGGTCCTCACCAAAGTCCTACAGGAAGAGCCC G Q F P Y K N C K T D F E V L T K V L Q E E P CCGCTTCTGCCCGGACACATGGGCTTCTCGGGGGACTTCCAGTCCTTCGTCAAAGACTGCCTTACTAA P L L P G H M G F S G D F Q S F V K D C L T K AGATCACAGGAAGAGACCAAAGTATAATAAGCTACTTGAACACAGCTTCATCAAGCGCTACGAGACGC D H R K R P K Y N K L L E H S F I K R Y E T TGGAGGTGGACGTGGCGTCCTGGTTCAAGGATGTCATGGCGAAGACTGAGTCACCGCGGACTAGCGGC L E V D V A S W F K D V M A K T E S P R T S G GTCCTGAGCCAGCCCCACCTGCCCTTCTTCAGGTAGCTGCTTGGCGGCGGCCAGCCCCACAGGGGGCC V L S Q P H L P F F R * AGGGGCATGGCCACAGGCCCCCCTCCCCACTTGGCCACCCAGCTGCCTGCCAGGGGAGACCTGGGACC TGGACGGCCACTAGGACTGAGGACAGAGAGT Figure 5.4 - Nucleotide and primary amino acid sequence of human M K K 7 . The nucleotides of the cDNA encoding human MKK7a. The initiating methionine is underlined and stop codons are shown in bold. The primary amino acid sequence is shown below the nucleotide sequence as predicted from the human codon usage chart (Table 5.1). 118 hMKK7 a MAAS SLEQKLSRLEAKLKQENREARRRIDLNLDIS PQRPRPT mMKK7a mMKK7y IIVITLSPAPAPSQRAA LQ hMKK7a LPLANDGGSRSPSSESSPQHPTPPARPRHMLGLPSTLFTPRSMESIEIDQKLQEIMKQTG mMKK7a T hMKK7a YLTIGGQ RYQAEINDLE hMKK7(3 VPPSLWRGEGGGPARLDPSWERQWGAGGGGRAPGTLQPSLSSQ mMKK7a I II I I I IV hMKK7a NLGEMGSGTCGQVWKMRFRKTGHVIAVKQMRRSGNKEENKRILMDLDWLKSHDCPYIVQ mMKK7a I hMKK7 a CFGTFITNTDVFIAMELMGTCAEKLKKRMQGPIPERILGKMTVAIVKALYYLKEKHGVIH mMKK7a .V. VI VII * * V I I I hMKK7a RDVKPSNILLDERGQIKLCDFGISGRLVDSKAKTRSAGCAAYMAPERIDPPDPTKPDYDI mMKK7a IX X hMKK7a RADVWSLGISLVELATGQFPYKNCKTDFEVLTKVLQEEPPLLPGHMGFSGDFQSFVKDCL XI hMKK7a TKDHRKRPKYNKLLEHSFIKRYETLEVDVASWFKDVMAKTESPRTSGVLSQPHLPFFR Figure 5.5 - Alignment of primary structures of human and murine isoforms of M K K 7 . Predicted amino acid sequence of human MKK7oc (hMKK7a) and MKK7(3 (hMKK7(3) and of the N-termini of murine M K K 7 a (mMKK7a) and M K K 7 y (mMKK7Y). Where residues are identical with those of hMKK7cc, they are indicated by periods. hMKK7(3 and mMKK7y are shown only where they differ from M K K 7 a . Gaps were introduced to optimize the alignment and are indicated by dashes. 119 dHep MSTIEFETIGSRLQSLEAKLQAQNESHDQIVLSGARGPWSGSVPSARVPPLATSASAA hMKK7 MAAS SLEQKLSRLEAKLKQENREARRRIDLNLDIS PQRPRPTLQ dHep TSATHAPSLGASSVSGSGI.IAQRPAPPVPHATLRS PSAS S S S S SRSAFR.AAPATGLRW hMKK7 LPLANDGDSRSPSSESSPQHPTPPARPRHMLGLPSTLFTPRSMESIEIDQKLQEIMKQTG dHep TYTPPTTRVSRATPTLPMLSSG.GGDVECTRPVILP.P..PHPPVS.T.M..KI..E... CMKK7 MER.F.LGMGRPGGLGGLGGE.IMQQMPQPA.HHPSRSSNDHNVKNLM.QA.—ENS. I II hMKK7 YLTIGGQRYQAEINDLENLGEMGSGTCGQVWKMRFRKTGHVIAVKQMRRSGNKEENKRIL dHep K. N . N. RQ . PTD .... KH .. DL . N.. S . N... . MHLSSNTI T . . A CMKK7 ....L.N.RK.DLKE.QFVEDI.H.S..T.T.C.YKSV--IM...T.P.TS.SY.MS... I l l VI V hMKK7 MDLDWLKSHDC PYIVQCFGTFITNTDVFIAMELMGTCAEKLKKRMQGPIPERILGKMTV dHep K...K.L.C. VRDP . .W.C. . . . SM. FD . . L . LSKK .V. .Q. . . .V. . CMKK7 ICL.F R...Y....F.. RVC . . C . A. . LDR. LI . IKQ I. . LS . VI VII * * hMKK7 AIVKALYYLKEKHGVIHRDVKPSNILLDERGQIKLCDFGISGRLVDSKAKTRSAGCAAYM dHep .T.N..S...D..G I....N N CMKK7 S.I...H...T..QIM WS.V A...IE.R. HSKQ . . . PL. . VIII IX X hMKK7 APERIDPPDPTKPDYDIRADVWSLGISLVELATGQFPYKNCKTDFEVLTKVLQEEPPLLP dHep .K..K T ARS . . EG . N DS . . . C . . CMKK7 G. . .L. .NNFDS— ....S....F.VT QYP.AG--.E.DMMS.I.ND. . .R.D XI hMKK7 GHMG--FSGDFQSFVKDCLTKDHRKRPKYNKLLEHSFIKRYETLEVDVASWFKDVMAKTE dHep YGE.YN..QQ.RD..IK....N.QD....PE..AQP..RI..SAK...PN..QSIKDNDC CMKK7 PA K. . P . . CQL . ES . . QR. PTM. . N. DM. . Q . P . WHH . KI. T . . EE . . A. . . G-EC hMKK7 SPRTSGVLSQPHLPFFR dHep GQWRSNAPEVT CMKK7 G Figure 5.6 - Alignment of human M K K 7 with its orthologs in C. elegans and D. melanogaster. Predicted amino acid sequence of human M K K 7 a (hMKK7), D. melanogaster M K K 7 (dHep) and C. elegans M K K 7 (cMKK7). Where residues are identical with those of hMKK7, they are indicated by periods. Gaps were introduced to optimize the alignment and are indicated by dashes. 120 I II I I I MKK7 DLENLGEMGSGTCGQVWKMRFRKTGHVIAVKQMRRSGNKEENKRILMDLDWLKSHDCPY MKK4 . .KD. . .I.R.AY.S.N. .VHKPS.QIM. . .RI.STVDEK.Q.QL ..MR.S. . . . MKK6 ...PIM.L.R.AY.V.E...HVPS.QIM...RI.ATV.SQ.Q..L ISMRTV...F MKK 3 . .VTIS.L.R.AY.V.E.V.HAQS.TIM. . . RI . ATV. SQ . Q . . L INMRTV. . . . MEK5 .IRYRDTL.H.NG.T.Y.AYHVPS.KIL...VILLDITL.LQ.Q.MSE.EI-.YKC.SS. MEK2 .F.RIS.L.A.NG.V.T.VQH.PS.LIM R.LIHLEIKPAIRNQ.IRE.Q.-.HECNS.. MEKl .F.KIS.L.A.NG.V.F.VSHKPS.L.M.R.LIHLEIKPAIRNQ.IRE.Q.-.HECNS.. VI V MKK7 IVQCFGTFITNTDVFIAMELMGT-CAEKLKK RMQGPIPERILGKMTVAIVKALYYL MKK 4 . . .FY ALFREG.CW c. S.-SFD.FY YVYSVLDDV. . .E. . . . I . L . T . . . NH MKK 6 T.TFY ALFREG..W c. D.-SLD.FY QVIDK -GQT. . .D. . ..IA.S.... EH MKK 3 T.TFY ALFREG..W c. D.-SLD.FYRKVLDK -NMT. . .D. . ..IA.S..R. EH MEK5 . IGFY A.FVENRIS CT F DGGSLDVY-- --RKM . .HV. .RIA..V..G T. MEK2 . . GFY A.YSDGEIS C. H DGGSLDQVL E -AKR. . .E. . ..VSI.VLRG A. MEKl . .GFY A.YSDGEIS C. H DGGSLDQVL K -A.R. . .Q. . ..VSI.VI.G T. VI VII * * VIII MKK7 KEKHGVIHRDVKPSNILLDERGQIKLCDFGISGRLVDSKAKTRSAGCAAYMAPERIDPPD MKK 4 ..NLKI . I . . ,RS N • Q T . . .D. RP. . . _g MKK 6 HSKLS V INAL V M. . Y V ID. .KP... ....N.-E MKK 3 HSKLS.,, . .V INKE HV M. . . Y V . .MD. .KP... ....N.-E MEK5 WS-LKIL M VNTR . V . .V TQ .N I . . Y-V TN.... ....SGEQ MEK2 R...QIM VNSR E , . ,V • Q T . M NSF-V TRS... ...LQGTH MEKl R...KIM VNSR E. . .V • Q I . M NSF-V TRS... ...LQGTH IX MKK7 PTKPDYDIRADVWSLGISLVELATGQFPYKNCKT MKK 4 ASRQG ..V.S T.Y R... PKWNS MKK6 LNQKG.SVKS.1 TMI. . .ILR. . .DSWG. MKK3 LNQKG.NVKS.V TMI.M.ILR. . .ESWG. MEK5 .GIHS FM.IQ MEK2 . SVQS . I . . M. L V. RY . IPPPDAKELEAIFGRPWDGEEGEPHSISPRF MEKl .SVQS.I..M.L....M.V.RY.IPPPDAKELELMFGCQV EGDAAETPPRF MKK 7 MKK 4 MKK 6 MKK 3 MEK5 MEK2 MEKl DFEVLTKVLQEEPPLLPGHMG-- FSGDFQSFVKDCLTKDHR _ v DQ • Q VKGD. Q. SNSEERE . .PS IN .NL .ES p QQ KQ VE PS Q . ADK . .AE VD TSQ .K NSK p QQ KQ VE PS Q .ADR . . PE VD TAQ .R NPA KNQGSLMPLQL QCIVD DS .V . VGE . .EP VH ITQ MR QPK RPPGRPVSGHGMDSRPAMAI . L DYIVN P. K . NGV .TP. .E .NK . I NPA RTPGRPLSSYGMDSRPPMAI .L DYIVN P. K . SGV . .LE .D .NK . I NPA XI MKK7 KRPKYNKLLEHSF MKK4 KE . . K . P . MKK6 E..T.PE.MQ.P. MKK3 E.MS.LE.M..P. MEK5 E..APEE.MG.P. MEK2 E.ADLKM.TN.T. MEKl E.ADLKQ.MV.A. Figure 5.7 - Alignment of the catalytic domains of known human M K K . The predicted amino acid sequence of known human M K K . Where residues are identical with those of M K K 7 , they are indicated by periods. Gaps were introduced to optimize the alignment and are indicated by dashes. 121 dHep and cMKK7 and was more closely related to these than to the known human M K K (Fig. 5.8). We used primers derived from the human sequence of M K K 7 to amplify a 869 bp fragment of cDNA from the murine Ba/F3 hematopoietic cell line. Sequencing indicated that this cDNA encoded the N-terminus and most of the kinase domain of a murine homologue of M K K 7 (mMKK7oc). We screened the EST database for cDNA encoding the N-terminus and identified another splice variant of mMKK7 (mMKK7Y) that contained an additional 48 base pair exon or in-frame intron (Fig. 5.5). We have not yet identified a human homologue of mMKKTy. These splice variants may encode alternative forms of M K K 7 or may represent incompletely processed mRNA. By far the most highly represented form of M K K 7 in a human fetal kidney cDNA library was M K K 7 a and therefore we chose this isoform for continued analysis of M K K 7 function. 5.2.2 Expression of MKK7 in human and murine cells. To investigate expression of endogenous and exogenous M K K 7 we immunoblotted lysates of MC/9 mast cells, Ba/F3 cells and a clone of Ba/F3 cells that we generated stably expressing M K K 7 (Ba/F3-MKK7) with an antiserum raised against the N-terminus of M K K 7 . We identified an immunoreactive protein of 47 kDa, consistent with the size predicted from the cDNA of MKK7cc or M K K 7 y (Fig. 5.9). Thus Ba/F3 and MC/9 cells expressed the N-terminus predicted by translation of the cDNA encoding mMKK7a or mMKK7Y . We were unable to detect an immunoreactive protein at the size (53 kDa) predicted to correspond to the hMKK7p splice variant in murine cells. These experiments indicated that the levels of exogenous M K K 7 expressed in Ba/F3-MKK7 cells were at least 30-fold greater than endogenous M K K 7 (Fig. 5.9). This was interesting as the Ba/F3-MKK7 cells exhibited no obvious abnormalities in growth and remained dependent on IL-3 for growth and survival. Moreover, over-expression of human M K K 7 did not appear to affect the expression of endogenous murine M K K 7 , as 5 x 105 cells of both untransfected and Ba/F3-MKK7 cells expressed comparable levels of endogenous M K K 7 (Fig. 5.9). 122 M K K 5 Figure 5.8 - Dendrogram of known human M K K and the orthologs of MKK7. A radial dendrogram (Fitsch method) depicting the phylogeny of the catalytic domains of M K K 7 , dHep, c M K K 7 and other known human M K K . The percent identities within the kinase domain of other M K K with human M K K 7 a were: dHep - 69%; M K K 4 - 56%; c M K K 7 - 54%; M K K 6 -49%; M K K 3 - 47%; MEK1/2 - 35% and MEK5 - 30%. 123 Ba/F3 M K K 7 e e tf) N Ift M 50 kDa _ Cells x IO-4 m y c - M K K 7 M K K 7 Figure 5.9 - Expression of M K K 7 in Ba/F3 and M C / 9 cells. Immunoblot of endogenous M K K 7 in lysates of Ba/F3 and MC/9 hematopoietic cells and endogenous and exogenous M K K 7 in lysates of a clone of Ba/F3 cells that expressed human myc-tagged M K K 7 . The indicated numbers of cells were lysed and proteins were separated by SDS-PAGE. An antiserum raised against the N-terminus of M K K 7 (a.a.4-26) was used for immunoblotting. The position of endogenous M K K 7 and myc-tagged exogenous M K K 7 are indicated by arrowheads. 124 We used Northern blotting to investigate the expression of M K K 7 in multiple human tissues. The probe contained the unique N-terminus of M K K 7 and the 5'-end of the kinase domain which exhibits little sequence homology with other known M K K family members. The probe bound to a single transcript of around 4 kbp in all tissues tested, with highest levels of hybridization occurring with mRNA from skeletal muscle (Fig. 5.10). We also used RT-PCR to examine expression of M K K 7 in a number of human and murine cells and confirmed the PCR products as being derived from M K K 7 by sequencing and restriction analysis. M K K 7 mRNA was present in all tested cell lines corresponding to a number of cell lineages (Table 5.3). Thus, M K K 7 is widely expressed. 5.2.3 MKK7 specifically activates JNK, but not p38 MAPK. It has been previously shown that the transient co-expression of mitogen-activated protein kinases with the appropriate upstream M K K leads to in vivo activation of the M A P K . We investigated the substrate specificity of M K K 7 by co-expression of M K K 7 with either J N K l or p38a M A P kinase. Co-expression of M K K 7 with J N K l in HeLa epithelial cells (Fig. 5.11) or Ba/F3 hematopoietic cells (data not shown) without any deliberate stimulation of the cells, resulted in easily detectable activation of J N K l . Stimulation of these co-transfected cells with hyperosmotic shock (0.2 M NaCl) or U V light resulted in even greater J N K l activation (Fig. 5.11). These results suggested that M K K 7 was upstream of J N K l and was activated by stimulation of cells by U V light or hyperosmotic shock. In contrast, co-expression of p38a M A P K with M K K 7 had no effect on activation of p38oc M A P K (Fig. 5.12). Nor did co-expression of M K K 7 with p38 M A P K result in increased activation of p38oc M A P K when the transfected cells were stimulated by hyperosmotic shock or U V light (Fig. 5.12). Thus M K K 7 , unlike M K K 4 , specifically activated J N K l , but not p38a M A P kinase. 5.2.4 MKK7 is activated by TNF or multiple stress stimuli. The c-Jun N-terminal kinases have been shown to be activated in response to a number of different stimuli including U V 125 in 3 g CU fl J i CJ cm cu fl "C • r u S fl <•> °5 8 cu eg .= a .5 s .3 C cu kbp - 1.85 Figure 5.10 - Tissue Distribution of m R N A encoding M K K 7 . Northern blot of M K K 7 mRNA using poly-(A)+ mRNA isolated from multiple human tissues. A probe was prepared by random priming a 600 bp fragment from the 5'-end of M K K 7 . Table 5.3 - Expression of m R N A encoding M K K 7 in various cell lines as determined by R T - P C R . Cell Line Species Cel l Lineage H e L a Human Epithelial (Cervical carcinoma) HepG2 Human Hepatocarcinoma Jurkat Human Acute T cell leukemia Daudi Human B lymphoma H E K 293 Human Embryonic kidney cell C937 Human Monocyt ic 129/J ES cells Mouse Embryonic stem cell Ba /F3 Mouse IL-3 dependent hematopoietic cell M C / 9 Mouse IL-3 dependent mast cell 126 pEFBOS M K K 7 J N K 1 J N K 1 c-Jun Figure 5.11 - Co-expression of M K K 7 with J N K 1 activates J N K 1 . HeLa cells were transfected with constructs encoding GST-JNK 1 and either myc-tagged M K K 7 or the empty vector pEF-BOS (pEF). Transfected cells were split into 3 plates and either left untreated (Con) or stimulated with 0.2 M NaCl (Na) or U V irradiation (UV) for 20 min. GST-JNK1 was affinity purified with glutathione-Sepharose and its kinase activity was determined using GST-c-Jun as substrate. The phosphorylation of c-Jun was visualized after SDS-PAGE using autoradiography. 127 pEFBOS p38 M K K 7 p38 IP: anti-Flag U Z P y z p ATF-2 — «— — — — IB:anti-p38 Figure 5.12 - Co-expression of M K K 7 with p38a M A P K activates p38a M A P K . HeLa cells were transfected with constructs encoding Flag-tagged CSBP2/p38a and either myc-tagged M K K 7 or the empty vector pEF-BOS (pEF). Transfected cells were split into 3 plates and either left untreated (Con) or stimulated with 0.2 M NaCl (Na) or U V irradiation (UV) for 20 minutes. Flag-tagged p38oc M A P K was immunoprecipitated with the M2 antibody and its activity determined using ATF-2 as substrate. The phosphorylation of ATF-2 was visualized after SDS-PAGE using autoradiography. 128 light, hyperosmotic shock, protein synthesis inhibitors, heat shock, the pro-inflammatory cytokines IL-1 and TNFa and hematopoietic growth factors (Foltz and Schrader, 1997; Raingeaud et al., 1995). We tested the ability of these stimuli to activate GST-MKK7 that had been transiently over-expressed in HeLa or Ba/F3 cells. GST or G S T - M K K 7 was purified from extracts of cells subjected to various stimuli, and its ability to phosphorylate GST-JNK1 was assessed in an in vitro kinase assay. The activity of M K K 7 was increased by treatment of cells with hyperosmotic shock (11 fold), heat shock (7.5 fold), U V light (5.5 fold), or TNFa (3.5 fold), but not by EGF (Fig. 5.13, top). The ability of these stimuli to activate M K K 7 and to in turn activate JNK1 in vitro was also assessed. We observed that the ability of M K K 7 to activate JNK1 was increased in transfected cells treated with TNFa (4 fold), hyperosmotic shock (5 fold) or anisomycin (5 fold - 30 min; 6.5 fold - 45 min) (Fig. 5.13, bottom). Thus M K K 7 was activated by all these known activators of JNK. 5.2.5 Activation of MKK7 by IL-3, but not IL-4, in Ba/F3 cells. We and others have previously demonstrated the activation of JNK in response to stimulation by the hematopoietic growth factor IL-3 in both Ba/F3 hematopoietic cells and MC/9 mast cells (Foltz and Schrader, 1997; Nagata et al., 1997; Terada et al., 1997). In contrast, the related cytokines IL-4 and IL-13 failed to activate JNK (Foltz and Schrader, 1997). We transiently expressed GST-M K K 7 in Ba/F3 cells, subjected them to various stimuli and assayed the ability of M K K 7 , that was activated in vivo, to phosphorylate GST-JNK1 in vitro. As seen in Fig. 5.14, M K K 7 was strongly activated in cells treated with IL-3 (5.5 fold), 0.2 M NaCl (4.5 fold) or U V light (3.5 fold). In contrast, M K K 7 was not activated in cells treated with IL-4 (Fig. 5.14), correlating with its inability to activate JNK (Foltz and Schrader, 1997). 5.2.6 Activation of endogenous MKK4 and MKK7 by IL-3, but not IL-4. We next investigated the activation of endogenous M K K 7 in response to physiological stimuli. In parallel we also investigated the activation of the known JNK activator M K K 4 in order to identify 129 GST G S T - M K K 7 GST-JNK1 IB: ant i-MKK7 B GST G S T - M K K 7 r © § B s cj »H «a o « o Z s S cs U Z U H «< Z c-Jun IB: ant i -MKK7 Figure 5.13 - Activation of M K K 7 by various stimuli. Cells were transiently transfected with vectors encoding GST or GST-MKK7 and aliquots were stimulated as follows. (A) HeLa cells were either left untreated (Con), or stimulated with 100 ng/mL EGF (EGF) for 5 min, 0.2 M NaCl (Na) for 20 min, U V irradiation (UV) for 20 min, 100 ng/mL TNF-a (TNF) for 20 min or heat shocked (Heat) at 42 °C for 20 min. (B) HeLa cells were left untreated (Con), or stimulated with 100 ng/mL TNF-a (TNF) for 15 min, 50 ug/mL anisomycin (Aniso) for 30 or 45 min, or 0.2 M NaCl (Na) for 20 min. In all cases, transiently expressed proteins were affinity precipitated using glutathione Sepharose from samples of lysates that had been normalized for total protein using the Pierce assay. M K K 7 activity was determined by assaying its ability to phosphorylate 1 ug of GST-JNKl , or by measuring its ability to activate J N K l in vitro by incubating it with 1 ug of GST-JNKl and 50 mM unlabelled ATP for 30 min and determining the ability of an aliquot of this reaction mixture to phosphorylate GST-c-Jun. Phosphorylated substrates were visualized after SDS-PAGE by autoradiography. The precipitated proteins were immunoblotted after SDS-PAGE with an anti-MKK7 antibody (a.a.4-26) to quantitate loading. The position of M K K 7 is indicated by an arrowhead. 130 GST G S T - M K K 7 Z U P P Z P c-Jun IB: a n t i - M K K 7 Figure 5.14 - Activation of MKK7 by IL-3, but not I L - 4 , in BaF3 cells. Cells were transiently transfected with vectors encoding GST or GST-MKK7 and aliquots were stimulated as follows. Ba/F3 cells were left untreated (Con), or stimulated with 10 ug/mL synthetic IL-3 ( IL-3) for 5 min, 10 ug/mL synthetic IL-4 (IL-4) for 10 min, 0.2 M NaCl (Na) for 20 min or U V irradiation (UV) for 20 min. In all cases, transiently expressed proteins were affinity precipitated using glutathione Sepharose from samples of lysates that had been normalized for total protein using the Pierce assay. M K K 7 activity was determined by assaying its ability to activate J N K l in vitro by incubating it with 1 ug of GST-JNKl and 50 uM unlabelled ATP for 30 min and determining the ability of an aliquot of this reaction mixture to phosphorylate GST-c-Jun. Phosphorylated substrates were visualized after SDS-PAGE by autoradiography. The precipitated proteins were immunoblotted after SDS-PAGE with an anti-MKK7 antibody (a.a.4-26) to quantitate loading. 131 potential functional differences. The antibodies used for immune complex kinase assays specifically recognized M K K 4 or M K K 7 (Fig. 5.15). We then examined the ability of IL-3, IL-4, anisomycin and 0.2 M NaCl to activate M K K 7 in Ba/F3 cells. Consistent with the results of experiments with transiently expressed M K K 7 , cells treated with IL-3 exhibited an increased activation (8 fold) of endogenous M K K 7 (Fig. 5.16). Cells treated with IL-3 also exhibited an increase in M K K 4 activity (3 fold). In contrast, cells treated with IL-4 failed to activate M K K 7 (Fig. 5.16). Anisomycin and hyperosmotic shock also activated endogenous M K K 7 (4 and 14 fold). 5.2.7 Activation of endogenous MKK4 and MKK7 by TNF. The pro-inflammatory cytokine TNFa is a well described activator of the JNK pathway. We examined the ability of TNFa to activate M K K 4 and M K K 7 in HeLa cells. As seen in Fig. 5.17, we observed an increase in the activity of both M K K 4 (5 fold) and M K K 7 (2.5 fold). 5.2.8 Activation of endogenous MKK4 and MKK7 by cross-linking the Fc receptor for IgG. Activation of JNK has also been shown to follow cross-linking of the receptor for the Fc fragment of Immunoglobulin G (FcR) on myeloid cells. The monoclonal antibody 2.4G2 (a-FcR) binds to both FcgRII and FcgRIII and when cross-linked by a secondary antibody (a-Ig) induces signalling through the FcR. As seen in Fig. 5.18, cells either left untreated or incubated with either a-FcR or a-Ig alone exhibited baseline activities of M K K 4 or M K K 7 . However, when cells were pre-treated for 10 minutes with a-FcR and then incubated with a-Ig to aggregate the a-FcR/FcR complexes, an increase in both M K K 4 (7 fold) and M K K 7 (8 fold) activity was observed. 5.2.9 Constitutively active GTPases activate MKK4 and MKK7. Expression of constitutively active mutants of the Ras and Rho family of small GTPases activates the JNK family of protein kinases (Bagrodia et al., 1995; Coso et al., 1995; Derijard et al., 1994; Hibi et al., 1993; 132 IP IP Tt h t h> w ^ ^ H |2 bd o a a g y a a g i g H ^ » » « : ^ K 7 MKK4 " *" • Figure 5.15 - Specificity of M K K 4 and M K K 7 antibodies. A lysate of WEHI-231 cells was split and immunoprecipitated with antibodies recognizing either M K K 4 , the N-terminus of M K K 7 , or with an irrelevant Ab (Con). Aliquots of immunoprecipitates were analysed by SDS-PAGE and immunobotted with either an antibody recognizing M K K 4 (left) or the C-terminus of M K K 7 (right). A sample containing 5 x 105 cell equivalents of whole cell extract (WCE) was analysed in parallel. The position of M K K 4 (2 isoforms) and M K K 7 are indicated by arrowheads. 133 M K K 4 M K K 7 c-Jun Figure 5.16 - Activation of endogenous M K K 4 and M K K 7 by IL-3, but not I L - 4 . Ba/F3 cells were left untreated (Con), or stimulated with 10 ug/mL synthetic IL-3 (IL-3) for 5 min, 10 ug/mL synthetic IL-4 (IL-4) for 10 min, 0.2 M NaCl (NaCl) for 20 min or 50 ug/mL anisomycin (Aniso) for 30 min. Cellular lysates were immunoprecipitated with antibodies recognizing M K K 4 or M K K 7 . The activity of M K K 4 or M K K 7 was determined by incubating 1 ug of GST-JNKl with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 ug of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography. 134 M K K 4 M K K 7 fl TNF a TNF 5 5 10 3 5 10 mm « m m c-Jun Figure 5.17 - T N F a activates endogenous M K K 4 and M K K 7 in HeLa cells. HeLa cells were left untreated (Con), or stimulated with 100 ng/mL TNF (TNF) for 5 or 10 min. Cellular lysates were immunoprecipitated with antibodies recognizing M K K 4 or M K K 7 . The activity of M K K 4 or M K K 7 was determined by incubating 1 ug of GST-JNK1 with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 ug of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography. 135 M K K 4 M K K 7 c-Jun Figure 5.18 - Cross-linking of the Fc receptor for IgG activates M K K 4 and M K K 7 in MC/9 mast cells . MC/9 mast cells were pre-treated with a-FcR (a-FcR) for 10 min as indicated, and then incubated for 10 min in medium alone, or with a rabbit a-rat immunoglobulin antibody (a-Ig). Cellular lysates were immunoprecipitated with antibodies recognizing M K K 4 or M K K 7 . The activity of M K K 4 or M K K 7 was determined by incubating 1 ug of GST-JNK1 with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 ug of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography. 136 Minden et al., 1995). We confirmed that co-expression of GST-JNK1 and Ras , Rac or V12 Cdc42 activated the JNK pathway in HeLa cells (data not shown). We then co-expressed GST-MKK7 with each of these mutant GTPases, affinity-precipitated the GST-MKK7 and assessed its ability to activate GST-JNKl in vitro, using phosphorylation of GST-c-Jun as a readout of J N K l activity. We found that co-expression of activated mutants of Ras, Rac or Cdc42 and M K K 7 in HeLa cells resulted in marked activation of M K K 7 , demonstrating that this kinase could be activated by signals downstream of each of these GTPases (Fig. 5.19). Similar results were obtained when GST-MKK4 was co-expressed with these GTPases in HeLa cells (Fig. 5.19). We also performed similar experiments in the murine hematopoietic cell line Ba/F3. Co-expression of G S T - J N K l and the constitutively active mutants of Rac and Cdc42 resulted in activation of J N K l in Ba/F3 cells; however co-expression of activated Ras with J N K l failed to activate J N K l (data not shown). In keeping with these findings, co-expression of M K K 7 and Rac or Cdc42 in Ba/F3 cells activated M K K 7 ; however the co-expression of activated Ras with M K K 7 was not sufficient to activate M K K 7 (Fig. 5.20). 5.3 Discussion Our results indicate that human and murine M K K 7 are highly conserved (99% identity, Fig. 5.5) and are most closely related to dHep, a M K K in Drosophila , exhibiting 69% identity in the kinase domain (Fig. 5.6). M K K 7 was less related to M K K 4 , M K K 6 or M K K 3 (-45-55% identity), the other mammalian M K K known to be activators of JNK and p38 MAPE1, and was even less similar to MEK1 or MEK2 (-30-35% identity), the activators of E R K M A P K (Fig. 5.7 and 5.8). We conducted extensive searches of the yeast and invertebrate databases and although we failed to identify M K K 7 in S. cerevisiae, we did identify a highly related C. elegans M K K (cMKK7) that exhibited 54% identity to M K K 7 within the kinase domain (Fig. 5.6) (Wilson et al., 1994). A more detailed inspection of alignments revealed a series of residues or motifs that were conserved among dHep, M K K 7 and c M K K 7 , that were not present in M K K 3 , M K K 4 and 137 GST G S T - M K K 7 G S T - M K K 4 fl fl C*5 U W fl C « U W C CZ C CS C3 C3 "C O S S S U Z U Z f t J C c J U U Z PJ « u c-Jun Figure 5.19 - Activation of M K K 7 or M K K 4 by the small GTPases Ras, Rac and Cdc42 . HeLa cells were transiently co-transfected with a vector encoding either GST, GST-M K K 7 or GST-MKK4 and pEF-BOS that encoded the constitutively active mutants of Ras (Ras), Rac (Rac), Cdc42 (Cdc42) or an empty vector as a control. In every case, a reporter construct encoding (3-galactosidase in the same pEF-BOS vector was also co-transfected and (3-galactosidase activity was used to normalize the lysates for the expression of transfected genes. Cells transfected with the empty vector and M K K 7 were left unstimulated (Con), or stimulated with 0.2 M NaCl (Na) for 20 min as controls for the kinase assay. The activity of affinity purified M K K 7 or M K K 4 was determined by incubating 1 ug of GST-JNKl with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 ug of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography. 138 G S T G S T - M K K 7 S3 S3 s O 03 O C3 S3 S3 *0 u z o z u c-Jun Figure 5.20 - Activation of M K K 7 by the small GTPases Rac and Cdc42. Ba/F3 cells were transiently co-transfected with a vector encoding either GST or GST-MKK7 and pEF-BOS that encoded the constitutively active mutants of Ras (Ras), Rac (Rac) or Cdc42 (Cdc42) or an empty vector as a control. In every case, a reporter construct encoding p-galactosidase in the same pEF-BOS vector was also co-transfected and P-galactosidase activity was used to normalize the lysates for the expression of transfected genes. Cells transfected with the empty vector and M K K 7 were left unstimulated (Con), or stimulated with 0.2 M NaCl (Na) for 20 minu as controls for the kinase assay. The activity of affinity purified M K K 7 was determined by incubating 1 ug of GST-JNKl with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 pig of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography. 139 M K K 6 , and vice versa. Particularly striking are differences in the activation loops, with M K K 7 , dHep, and cMKK7 being characterized by a basic amino acid (S[K/R]AKT), at the same position where in M K K 3 , M K K 4 , and M K K 6 a hydrophobic residue (S[17V]AKT) is found. Overall these patterns of conservation of specific motifs support the notion that M K K 7 is orthologous with dHep and c M K K 7 , and is more distantly related to M K K 4 , M K K 3 and M K K 6 (Fig. 5.6 and 5.7). It will be interesting to determine the functional significance of the conserved residues that characterize M K K 7 , dHep and cMKK7, and distinguish them from M K K 4 , M K K 3 and M K K 6 . Tournier et al. have recently reported the cloning of two splice variants of murine M K K 7 and the N-terminus of human M K K 7 a (Tournier et al., 1997). The amino acid sequence predicted from their murine MKK7a clone is identical to the sequence that we report here, with the exception that it lacks the N-terminus that was present in all of our predicted human and murine M K K 7 splice variants. We were able to immunoblot endogenous M K K 7 in lysates of Ba/F3 hematopoietic cells and MC/9 mast cells using an antiserum raised against the N-terminus of our human and murine M K K 7 sequence (Fig. 5.9), and therefore we are confident that the form of M K K 7 predicted from our human and murine cDNA is indeed expressed in murine cells. While this work was being completed, a number of articles describing the cloning of M K K 7 were published (Holland et al., 1997; Lawler et al., 1997; Lu et al., 1997; Moriguchi et al., 1997; Wu et al., 1997; Yao et al., 1997). Holland et al. identified two isoforms of murine M K K 7 , one corresponding to murine M K K 7 a and the other having a unique N-terminus (Holland et al., 1997). Moriguchi et al. cloned murine M K K 7 y and identified two M K K 7 isoforms using an antiserum raised against full length murine M K K 7 y (Moriguchi et al., 1997). Based on electrophoretic mobility, we believe that the larger isoform could represent the murine equivalent of our human MKK7(3 (Fig. 5.5). Lu et al. have identified another isoform of human M K K 7 that has the same N-terminus and kinase domain as our M K K 7 isoforms but contains 70 amino acids that are not found in any of the M K K 7 isoforms reported to date (Lu et al., 1997). Based on sequence identities among the known M K K s (Fig. 5.7), we investigated the ability of M K K 7 to activate JNK or p38 M A P K and showed that in co-transfection assays M K K 7 140 acted upstream of J N K l (Fig. 5.11), but not p38 M A P K (Fig. 5.12). The identification of J N K l as an in vitro substrate for M K K 7 permitted us to investigate the ability of a range of stimuli to activate transiently expressed M K K 7 . In HeLa cells, M K K 7 was strongly activated by hyperosmotic shock, UV light, anisomycin, heat shock and to a lesser extent TNFa (Fig. 5.13). We observed no detectable activation of M K K 7 in HeLa cells treated with EGF (Fig. 5.13, top). These results are similar to that seen by Holland et al. with PDGF in NIH 3T3 cells (Holland et al., 1997). In Ba/F3 hematopoietic cells, hyperosmotic shock and U V light also activated M K K 7 (Fig. 5.14). Our observations that stimulation of Ba/F3 cells with IL-3 increased the activity of M K K 7 (Fig. 5.14 and 5.16) correlates with recent observations that JNK was activated by a range of hematopoietic growth factors including IL-3, GM-CSF, G-CSF, EPO or SLF (Foltz and Schrader, 1997; Nagata et al., 1997; Rausch and Marshall, 1997; Terada et al., 1997). Moreover, our observation that IL-4 failed to activate M K K 7 (Fig. 4c, 5c) correlates with the inability of IL-4 to activate the Ras, ERK, p38 or JNK M A P kinase pathways in hematopoietic cells (Duronio et al., 1992; Foltz et al., 1997; Foltz and Schrader, 1997; Satoh et al., 1991; Welham and Schrader, 1992). Co-expression of activated mutants of Ras, Rac or Cdc42 with M K K 7 in HeLa cells resulted in readily detectable activation of M K K 7 and M K K 4 (Fig. 5.19). This is consistent with previous work which demonstrated that the Ras and Rho family of small GTPases are capable of activating JNK (Bagrodia et al., 1995; Coso et al., 1995; Derijard et al., 1994; Hibi et al., 1993; Minden et al., 1995). Slightly different results were obtained in Ba/F3 cells where M K K 7 was activated by co-expression of activated Rac or Cdc42, but not Ras (Fig. 5.20). The inability of Ras to activate M K K 7 was not surprising as a constitutively active Ras was insufficient to activate JNK in Ba/F3 cells, although IL-3-induced JNK activation was blocked by expression of a dominant-negative mutant of Ras (Terada et al., 1997). Taken together, these results support the notion that Ras is necessary but not sufficient for JNK activation. Our evidence that IL-3 increased M K K 7 activity (Fig. 5.14 and 5.16), but that Ras alone failed to increase M K K 7 activity (Fig. 5.20), is also consistent with data on activation of JNK by G-CSF, which is structurally related to 141 IL-3 and acts through a similar receptor. These experiments showed that activation of JNK in cells stimulated with G-CSF depended upon both an intact Ras signalling pathway, as well as a specific tyrosine residue in the G-CSF receptor (Rausch and Marshall, 1997). Together these data suggest that activation of M K K 7 in response to IL-3 involves both activation of the Ras pathway and an as yet unknown signal. The receptor for the Fc fragment of Immunoglobulin G (FcR) has many important roles in the immune system (Takai, 1996). These include the phagocytosis of Ig-coated particles by macrophages and neutrophils, the antibody-dependent cell mediated cytotoxicity by N K cells, the down-regulation of signalling through the BCR, and the release of TNFa and other mediators by macrophages and mast cells. Our observation that ligation of FcR results in the activation of both endogenous M K K 4 and M K K 7 in MC/9 mast cells (Fig. 5.18) supports previous findings that signalling through the FcR activates JNK in bone-marrow derived macrophages (Rose et al., 1997). Recent studies have shown that signalling through the JNK pathway is required for the production of T N F a in MC/9 mast cells (Ishizuka et al., 1997). As signalling through the FcR activates JNK through M K K 4 and M K K 7 , it will be important to determine their individual roles in the production of TNFa in mast cells. The existence of multiple activators of JNK which are responsive to different stimuli was established by the fact that MKK4-deficient ES cells still exhibited activation of JNK in response to hyperosmolarity and U V light, but not heat shock or anisomycin (Derijard et al., 1995; Lin et al., 1995). In that M K K 7 is activated by all of the above stimuli (Fig. 5.13, 5.14 and 5.16), and M K K 7 mRNA is present in ES cells (Table 5.3), the failure of MKK4-deficient ES cells to activate JNK in response to heat shock or anisomycin is paradoxical. It is possible that, although M K K 7 is expressed in ES cells, it is not activated by heat or anisomycin because of cell-specific differences in upstream activators. This notion is supported by a recent report that in K B and U937 cells TNFa activates M K K 7 , but not M K K 4 (Lawler et al., 1997; Moriguchi et al., 1997), whereas in HeLa cells we observed that TNFa activates both M K K 7 and M K K 4 (Fig. 5.17), an observation recently reported by Wu et al. (Wu et al., 1997). M K K 4 has also been recently 142 reported to be activated in normal bone marrow-derived macrophages treated with TNFa , supporting the notion that these kinases will be regulated differently depending on their cellular context (Winston et al., 1997). Furthermore, Wu et al. have reported that ASK1 and G C K activate M K K 7 in preference to M K K 4 , whereas M E K K 1 and M E K K 2 activate both M K K 4 and M K K 7 to comparable levels (Wu et al., 1997). The functional significance of the activation of M K K 7 is unclear but its activation by physiological stimuli such as IL-3 (Fig. 5.14 and 5.16), by ligation of immunoregulators such as FcR (Fig. 5.18), CD40, BCR, CD3 (Foltz et al., 1998, M . Luckach and R. Salmon, unpublished observations), and by the GTPases Ras, Rac and Cdc42 (Fig. 5.19 and 5.20) suggests that the role of M K K 7 will not be confined to stress responses. The existence of an ortholog of M K K 7 in C. elegans and our failure to identify an ortholog in S. cerevisiae suggest that M K K 7 arose in evolution during the transition from single cellular to multicellular organisms. The notion that M K K 7 is involved in processes important for multicellular organisms, such as embryonic development, chemotaxis or apoptosis, is in keeping with evidence that mutations in dHep and Bsk, the Drosophila homologue of J N K l , result in a similar failure of epithelial cell movement and dorsal closure (Glise et al., 1995; Riesgo-Escovar et al., 1996; Sluss et al., 1996). Holland et al. have demonstrated that M K K 7 is able to partially complement a deficiency of dHep in Drosophila (Holland et al., 1997). This demonstrates that M K K 7 is highly conserved functionally and suggests that it may play a role in embryological development in mammals. The embryonic lethality resulting from disruption of the M K K 4 gene in mice also points to the importance of normal JNK signalling during embryonic development (Ganiatsas et al., 1998; Nishina et al., 1997; Yang et al., 1997), and indicates that M K K 4 and M K K 7 have discrete physiological functions. Future work detailing the function of individual activators of JNK is likely to reveal roles in multiple aspects of development and other physiological processes, including hematopoiesis and immune responses. 143 C H A P T E R 6 - Conclusion The stress-activated protein kinases, p38 M A P K and JNK, are activated by diverse stimuli acting on a cell. When I began working in this field, these kinases were known to be activated by cellular insults including hyperosmotic shock, U V irradiation, heat shock and the pro-inllammatory cytokines TNFa and IL-1. In contrast, growth factors such as EGF and PDGF failed to activate these enzymes to comparable levels. We had some biochemical evidence that a protein with similar isoelectric point and electrophoretic mobility as p38 M A P K was tyrosine phosphorylated by the hematopoietic growth factors, IL-3 and SLF (Welham and Schrader, 1992), and we hypothesized this unknown phosphoprotein was p38 M A P K . I demonstrated that p38 M A P K was indeed tyrosine phosphorylated and activated by these growth factors. Furthermore, we demonstrated that p38 M A P K activity was required for the activation of M A P K A P kinase-2 by these cytokines. Previous work in our laboratory indicated that IL-4 was unable to activate E R K M A P K or Ras (Duronio et al., 1992; Welham et al., 1992; Welham et al., 1994), and we were interested to examine the ability of IL-4 to activate p38 M A P K . I found that IL-4 was unable to activate p38 M A P K , or M A P K A P kinase-2, consistent with the notion that M A P K A P kinase-2 was a substrate of p38 M A P K in vivo. Growth factors are important not only for the proliferation, but also for the survival of hematopoietic cells. We hypothesized that p38 M A P K would regulate some aspect of cytokine action. Indeed, we demonstrated using the inhibitor, SB 203580, that the activity of p38 M A P K was required for D N A synthesis. However, an SB 203580-resistant mutant of p38a M A P K failed to restore DNA synthesis, suggesting that p38a M A P K was not sufficient for DNA synthesis. The activation of M A P K A P kinase-2 was completely restored by the SB 203580-resistant mutant of p38a M A P K after hyperosmotic shock, suggesting that M A P K A P kinase-2 activity was not required for DNA synthesis. However it is not clear if p38a M A P K activity was completely restored under normal cell culture conditions. Since SB 203580 also inhibits p38p M A P K , these data suggest a role for p38p M A P K in D N A synthesis, or perhaps another target of this inhibitor. 144 When I began working in the stress-activated protein kinase field, all stimuli that activated p38 M A P K also activated JNK. I hypothesized that the hematopoietic growth factors that activated p38 M A P K would also activate JNK. Indeed, I demonstrated that IL-3, GM-CSF or SLF activated both 45 and 55 kDa isoforms of JNK1 and JNK2. Consistent with the failure of IL-4 to activate p38 M A P K , I also hypothesized that IL-4 would fail to activate JNK. The inability of IL-4 to activate Ras also supported the notion that IL-4 would fail to activate JNK as growth factors such as EGF activated JNK in a Ras-dependent fashion. Indeed, I found that IL-4 failed to activate JNK in hematopoietic cells. Together, these results indicated that IL-4 was unique among cytokines that we examined in its failure to activate any M A P K family member, or the small GTPase Ras. These findings lead to many important biochemical questions, including what is the role of Ras for the activation of p38 M A P K , do these hematopoietic growth factors activate Rac or Cdc42, and what are the biological implications of IL-4 failing to activate Ras or any M A P kinases? The JNK family of protein kinases was known to be regulated by a single M A P K kinase, M K K 4 , at the time I was studying the activation of JNK by hematopoietic growth factors. Therefore, we presumed the activation of JNK by hematopoietic growth factors was through M K K 4 . Consistent with this hypothesis, we detected an increase in the threonine phosphorylation of M K K 4 after stimulation with GM-CSF or SLF. However, the phosphorylation was greater with SLF than with GM-CSF or NaCl despite the fact that these stimuli activate JNK to a similar extent. We hypothesized this finding reflected the existence of another unidentified JNK kinase in MC/9 mast cells that was activated in cells after treatment with GM-CSF or NaCl. Several independent reports supported the existence of another JNK kinase (Meier et al., 1996; Moriguchi et al., 1995; Nishina et al., 1997a; Yang et al., 1997b). We screened the expressed sequence tags database and identified a novel JNK kinase, M K K 7 , with a great degree of homology to M K K 4 . We demonstrated this enzyme was activated by NaCl and IL-3, a cytokine that signals through a receptor that is shared with GM-CSF. Furthermore, M K K 7 was expressed in MC/9 mast cells, 145 implicating M K K 7 as the molecule we hypothesized to be activated after cells were treated with hyperosmolarity or GM-CSF. My demonstration that JNK and p38 M A P K were activated by hematopoietic growth factors provided the first evidence that these enzymes were strongly activated by growth factors, in contrast to previous findings with EGF or PDGF. The activation of both of these enzymes by GM-CSF and SLF were on the same order of magnitude as that seen with hyperosmotic shock. Since then several physiologically relevant stimuli have been identified as activators of JNK and p38 M A P K including the hematopoietic growth factors Erythropoietin and Thrombopoietin, and receptors involved in the regulation of the immune system including the Fc receptors for IgG or IgE, the B cell antigen receptor and CD3, a component of the T cell antigen receptor. These findings are consistent with the notion that these enzymes have important physiological functions beyond simply a response to stress, and are supported by proposed roles for these kinases in cytokine production, apoptosis, proliferation, tumorigenesis, embryogenesis and organogenesis. However, the specific functions of these enzymes in mammalian cells, and indeed in the whole organism is poorly understood. Most of our understanding of the function of these enzymes are inferred through the use of over-expressed dominant negative mutants that act to sequester either upstream activators or downstream effectors of these kinases. This approach is useful as it can potentially tell us something about enzyme functions, but it has inherent problems as one never really knows if the dominant negative specifically inhibits the intended pathway. The ability to use both dominant negative proteins and a specific inhibitor has allowed a rapid determination of the functions of p38a M A P K and p38(3 M A P K . However, the inhibitor also has inherent problems as unknown targets for the compound might confound the interpretation of data. The production of new p38 M A P K mutants that are resistant to SB 203580, but retain their substrate specificity, will hopefully provide more plausible data on the biological function of these proteins and maybe differentiate between functions of p38a M A P K and p38|3 M A P K isoforms in vivo. 146 Genetic analysis of the function of these enzymes is certainly a major focus of the future research in this field. Mice lacking M K K 4 provided the first evidence for the non-redundant functions for M K K 4 and M K K 7 , as cells lacking M K K 4 were still able to activate JNBC (Nishina et al., 1997). However, these mice die in utero, and as such did not provide that much information on the role of JNK kinase activity in vivo. A better understanding of the role of M K K 4 in adult mice will likely require tissue specific or conditional disruption of this gene using the Cre recombinase. Mice lacking JNK and p38 M A P K are also being generated. As there are three J N K genes and four p38 M A P K genes, the determination of in vivo function may require the disruption of multiple genes. However, mice with the JNK1, the JNK2 or the JNK3 gene disrupted had phenotypes, indicating that these enzymes are not entirely redundant at least in some tissues (Yang et al., 1997a; Yang et al., 1998a, Dong et al., 1998). The field is moving rapidly, and as our understanding grows, better experiments are being devised to address these fundamental questions. 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