Mirk is a constitutively active protein kinase which can mediate cell survival in the absence of mitogens. Transiently transfected mirk activates the erk subfamily of MAP kinases in the absence of growth factors or serum. Stable mirk transfectants, under serum-free conditions in which they grow and survive, and vector control cells do not, exhibit a low constitutive activation of erks, 5-fold over that of vector control cells, and more response to mitogens. These observations are likely to be physiologically relevant, not simply an artifact of overexpression or an adaptation of transfectants to culture, because mirk is required for one of the known survival factors, insulin-like growth factor 1 (IGF-1) to function as a mitogen. When mirk protein levels in NIH3T3 cells were decreased by phosphorothiolated antisense oligonucleotides, IGF-1 no longer was a mitogen for these cells. Varying the dosage of the antisense oligos caused a dose-dependent decrease in IGF-1 response, so the more mirk was reduced, the less mitogenic IGF-1 was. Therefore, endogenous levels of mirk are necessary for mitogenic response in vivo. There is also reciprocal regulation between erks and mirk: overexpressed mirk activates erks, whereas sustained activation of erks downregulate mirk. Mirk is relevant to cancer. Stable overexpression of mirk occurs in vivo in a large subset of colon cancers which exhibit mirk levels 5-40-fold those in paired normal colon. If wild-type mirk is stably overexpressed in 2 colon cancer cell lines, cells grow and survive in serum-free conditions in a mirk-dependent manner, with no effect of kinase-inactive mirk.
Aim 1 :analysis of the role of mirk in erk activation. The regions of mirk necessary to activate erks in transient transfection experiments will be determined by mutation/deletion analysis. Mirk may activate erks by phosphorylating some component of the erk signaling cascade. This in vivo substrate will be found by using mirk as """"""""bait"""""""" in a yeast two-hybrid complementation assay to screen a human skeletal muscle cDNA library. Mirk may modulate erk signaling so the capacity of mirk to activate promoter elements linked to reporter genes will be assayed by transient co-transfections.
Aim 2 : analysis of mirk regulation by erks. Whether erk regulation is transcriptional or post-transcriptional will be determined using conditions in which (a) erk activation is blocked and mirk protein levels rise several fold, (b) erks undergo a sustained activation and mirk levels decrease several fold. Mirk is a MAP kinase substrate with erk phosphorylation sites in its C'terminus. The role of erks, if any, in generation of a C'terminal deleted 57 kDa nuclear mirk species will be determined, as will the biological properties of this C'terminal deleted mirk.
Aim 3 : measurement of mirk protein expression by immunohistochemistry in human cancer tissues.
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