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Regulation of protein kinases during postnatal development of rat heart

University of British Columbia

The loss of ability to proliferate, the shift in the energy dependency from carbohydrate to fatty acids and the reduction in capability to resist ischemia are some of the key phenomena observed during postnatal development of the heart. These distinct phenotypic changes can be attributed to the regulation of intracellular signal transduction proteins. To investigate how these protein kinases are expressed during postnatal development, 44 different protein kinases were studied by Western blotting analysis. Among them protein kinases, implicated in cell proliferation and cell cycle including protein kinase C, cyclin-dependent protein kinases and mitogenic MAP kinases, declined during the postnatal development. The specific activities of Erkl and Erk2 declined with age, which was concurrent to the decrease of CDK1 activity. The protein kinases involved in stress-responses such as Mlk3, Mekkl, Sekl, Mkk3 and Mapkapk2, increased in expression during postnatal development. The proto-oncogene-encoded kinases Mos and Tpl2 were up-regulated. The expression of the insulin-activated kinases (PI3K, PKB, S6K and CK2) was downregulated by 40-60% with age. The activities of PKB, S6K and CK2 were also decreased with age. Tissue distribution studies of adult rats revealed that most of the protein kinases that were up-regulated during heart development tended to be preferentially expressed in the heart, whereas the down-regulated protein kinases were generally expressed in the heart at relatively lower amounts than in most other tissues. To investigate the signalling pathways of these kinases, isolated cardiomyocytes or isolated whole hearts were treated with various agonists. These results indicate that Erkl can be regulated via PKC but not by cAPK nor cGPK, whereas Mapkapk2 can be controlled by cGPK but not by the other two kinases. Erkl and Erk2 were also activated by endothelin-1 and adenosine in isolated cardiomyocytes. Insulin led to activation of PKB, S6K and CK2, whereas Erk1 and Erk2 were not stimulated. To investigate novel protein kinases which undergo developmental changes, the extracts of rat heart ventricles were incubated in the presence of [y-³²P]ATP and then the phosphoproteins were resolved by using SDS-PAGE. This assay revealed at least 8 phosphoproteins that underwent change during development. Among them, a 40-kDa phosphoprotein appeared to be an autophosphorylating protein-histidine kinase, which may represent the first of its kind detected in a mammalian system.

Thesis/Dissertation

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2009-06-20

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http://hdl.handle.net/2429/9503

Pharmaceutical Sciences

University of British Columbia

UBCV

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