Rho-associated Kinase Connects a Cell Cycle-controlling Anchorage Signal to the Mammalian Target of Rapamycin Pathway

Abstract

When deprived of anchorage to the extracellular matrix, fibroblasts arrest in G 1 phase at least in part due to inactivation of G 1 cyclin-dependent kinases. Despite great effort, how anchorage signals control the G 1 -S transition of fibroblasts remains highly elusive. We recently found that the mammalian target of rapamycin (mTOR) cascade might convey an anchorage signal that regulates S phase entry. Here, we show that Rhoassociated kinase connects this signal to the TSC1/TSC2-RHEB-mTOR pathway. Expression of a constitutively active form of ROCK1 suppressed all of the anchorage deprivation effects suppressible by tsc2 mutation in rat embryonic fibroblasts. TSC2 contains one evolutionarily conserved ROCK target-like sequence, and an alanine substitution for Thr 1203 in this sequence severely impaired the ability of ROCK1 to counteract the anchorage loss-imposed down-regulation of both G 1 cell cycle factors and mTORC1 activity. Moreover, TSC2 Thr 1203 underwent ROCK-dependent phosphorylation in vivo and could be phosphorylated by bacterially expressed active ROCK1 in vitro, providing biochemical evidence for a direct physical interaction between ROCK and TSC2.

Cdc6 Protein Activates p27KIP1-bound Cdk2 Protein Only after the Bound p27 Protein Undergoes C-terminal Phosphorylation

“…Cell Construction-Rat embryonic fibroblasts (REFs) constitutively expressing human CDK6, mouse cyclin D3, rat Cdc6, C-terminal-truncated constitutively active human ROCK1 and/or constitutively active human Rheb from the cytomegalovirus promoter were constructed as described (14,17). The drugs used for selection were G418, hygromycin, puromycin, blasticidin, and zeocin.…”

“…Forced activation of mTORC1, however, restores only Cdk4/Cdk6 activity despite marked up-regulation of both cyclin A and D-type cyclins and additional enforced Cdc6 expression. By contrast, expression of both a constitutively active ROCK and Cdc6 stimulates not only Cdk4/Cdk6 but also Cdk2 (17). Although ROCK may participate therein, how anchorage signals regulate Cdk2 activity is unknown.…”

“…In addition, this cascade conveys a cellular anchorage signal to control the G 1 -S transition. ROCK (Rho-associated kinase) activated by an anchorage signal originated from RhoA and integrins up-regulates the mTORC1 pathway by directly phosphorylating Tsc2 at Thr 1203 (17). Consequently, when cells are deprived of anchorage, not only Cdk4/Cdk6 and Cdk2 but also mTORC1 undergo inactivation.…”

Successive Phosphorylation of p27KIP1 Protein at Serine-10 and C Terminus Crucially Controls Its Potency to Inactivate Cdk2

“…To confirm PIM1-mediated C-terminal phosphorylation of p27 under anchorage deprivation in vivo and the functional distinction between PIM1 and ROCK1, we constructed by retrovirus-mediated gene transfer and analyzed REF- by withdrawal of doxycycline were constructed as before (15,16), induced or uninduced for enforced Cdc6 expression, and analyzed for Cdk2 activity and the levels of Rb Ser-780 phosphorylation and S6K1 Thr-389 phosphorylation, hallmarks of Cdk4/Cdk6 activity and mTORC1 activity, respectively. In this cell, induced Cdc6 protein was far more unstable than in mTORC1-activated cells perhaps because of inactivation of Cdk4/Cdk6 and consequent failed induction of Emi1 the G 1 phase inhibitor of the APC/C CDH1 ubiquitin ligase that degrades Cdc6 (17).…”

“…In addition to these regulations, it was recently discovered that C-terminal Thr-197 phosphorylation of p27 is required for the activation of the p27-bound Cdk2 by Cdc6 the AAAϩ ATPase in an entirely new mechanism (15). Furthermore, this C-terminal phosphorylation was found to be exerted not only by PIM kinases but also by ROCK1 kinase that mediates an anchorage signal to promote actin fiber assembly as well as activate mTORC1 signaling (16,17). Thus, an additional biological role of C-terminal Thr-197 phosphorylation was discovered.…”