Mammalian target of rapamycin complex 1 signaling opposes the effects of anchorage loss, leading to activation of Cdk4 and Cdc6 stabilization

Abstract

MINT‐7890626: cdc27 (uniprotkb:Q4V8A2) physically interacts (MI:0915) with Cyclin‐A (uniprotkb:Q6AY13) by anti bait coimmunoprecipitation (MI:0006)

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). Consistently, Cdk2 activity markedly diminished at 36 -48 h of the MC.…”

“…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.…”

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.…”

“…In Vitro Cdk2 Reactivation Assay-Proliferating REF cells were cultured in methylcellulose medium (MC) for 12 h, lysed with lysis buffer (14), and immunoprecipitated for Cdk2 with the agarose beads-conjugated anti-Cdk2 antibody. The Cdk2-bound agarose beads were then incubated at 30 ºC in 20 l of 50 mM Tris-HCl (pH 7.5) buffer containing 30 mM MgCl 2 and 10 mM ATP for 30 min with the addition of 1 l of either Escherichia coli-expressed active ROCK1 (17) or its control preparation and then for another 30 min with the addition of 1 l of either recombinant Cdc6/Cdc6 WB /Cdc6 Cy or its control preparation.…”

“…Inactivation of Cdk4/Cdk6 results in activation of retinoblastoma protein (Rb) and its cognates, which in turn inactivates the E2F transcriptional factors to shut down a subset of genes essential or important for S phase onset, such as Cdc6, cyclin A, and E2F1 (13). Furthermore, Cdc6 protein is rapidly eliminated by proteasomal degradation executed mainly by the APC/ C CDH1 ubiquitin ligase (14). The evolutionarily conserved Tsc1/Tsc2-Rheb-mTORC1 pathway mediates growth and metabolic signals to control cell proliferation (15,16).…”