Growth factor dependent regulation of p70S6 kinase Evaluation of domain specific signals sensitive to rapamycin

Abstract

newline Phosphorylation dependent regulation of S6K1 has been proposed in accordance with newlinethe strategy of activation adapted by AGC family kinases. Accordingly three newlinephosphorylations at the turn motif, HM and AL are considered critical for S6K1 newlineregulation , while the turn motif phosphorylation is thought to be constitutive , the newlineburden of the regulation is more or less entirely shared by the phosphorylations turn newlineover at HM and AL of the enzyme. Although both phosphorylations are equally newlineimportant without a question their individual roles in contributing to activation and newlinemediating rapamycin sensitivity remains controversial. Phosphorylations of the newlineenzyme at its HM brought about by rapamycin sensitive mTOR kinase is thought to newlineprime the enzyme for PDK1 dependent phosphorylations at AL, in accordance with newlinethe prevalent dogma of sequential phosphorylations. Accordingly inactivation of newlinemTOR by rapamycin resulting in loss of HM phosphorylation and resultant loss of newlinephosphorylation at the AL is the model put forward to explain inhibition by newlinerapamycin. The attribution of mTOR as the kinase responsible to engender HM newlinephosphorylation, is however, based on scanty evidence carried forward simply on the newlinebasis of the ability of mTOR to bind rapamycin. In fact S6K1 mutants supposedly newlineincapacitated for TOR kinase recruitment continue to be phosphorylated at the HM to newlinesuggest that effects attributed to TOS motif deletion may simply be conformational newlineand not attributable to the loss of TOR kinase input at the HM. Further the newlineimplication of kinases other than TOR in mediating HM phosphorylation compound newlinethe ambiguity associated with TOR kinase involvement in mediating rapamycin newlineinhibition. This study therefore, attempts to understand as to how the dynamics of the newlinetwo phosphorylations in general and TOR mediated phosphorylation in particular newlinerelates to rapamycin mediated inhibition of S6K1. newlineWe present evidence that baculovirus mediated expression of S6K1 in insect cells newlineresults in the failure of the enzyme to engender both HM and AL phosphorylations. newlineThe enzyme surprisingly continues to be inhibited by rapamycin. Evidence indicating newlineredundant role of phosphorylation in general and TOR mediated phosphorylation in newlineparticular for S6K1 inhibition by rapamycin challenge the prevailing dogma. We also newlineAbstract newlineDepartment of Biotechnology University of Kashmir newlineprovide evidence about the interdependent turnover of the two phosphorylations such newlineas to exhibit all or none characteristics. The evidence that both AL and HM kinases newlineare completely insensitive to rapamycin, suggests that their co-ordinate loss to newlinerapamycin cannot be mechanistic and therefore has to be consequential. We finally newlineshow that the loss of the two phosphorylations is also associated with inhibition of newlineS6K1 by agents like bupivacaine and lidocaine that target signalling pathways other newlinethan TOR, to strongly suggest that the loss of HM and AL phosphorylations is a newlinegeneral feature of S6K1 inhibition not necessarily governed by TOR inactivation.