PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest (original) (raw)

Cell-cycle deregulation is a hallmark of cancer. Loss of cytokinemediated G1 arrest may confer an advantage during malignant progression. Resistance to the antiproliferative effects of transforming growth factor-β (TGF-β) often occurs despite intact TGFβ signaling and such cells may manifest resistance to multiple inhibitory cytokines, suggesting underlying alterations in cellcycle controls 1,2. Cyclin-dependent kinases (cdks) are regulated by cyclin binding, phosphorylation and by two families of cdk inhibitors 3. G1 progression is governed by D-type and E-type cyclin-cdk complexes. The inhibitors of cdk4 (INK4) family includes p15 INK4B and the kinase inhibitor protein (KIP) family comprises p21 Cip1 , p27 Kip1 and p57 Kip2 (ref. 4). In addition to inhibition of cyclin E-cdk2, p21 and p27 also facilitate assembly and activation of cyclin D-cdks in early G1 (refs. 5,6). p27 Kip1 was first identified as a mediator of TGF-β1-induced G1 arrest 7-9. TGF-β induces expression of the gene encoding p15 INK4B. p15 INK4B binds and inhibits cdk4 facilitating dissociation of p27 and cyclin D1 from cyclin D1-cdk4-p27 complexes and p27 binds and inhibits cyclin E-cdk2, leading to G1 arrest 10-12. p27 also mediates G1 arrest induced by IL-6 (ref. 13). p27 is a nuclear protein whose frequent deregulation in human cancers may confer resistance to antiproliferative signals. In cMyc or MAPK activated cancer-derived lines, cyclin D1-cdk4/6 complexes sequester p27, and cyclin E-cdk2-inhibition is impaired 14-16. cMyc inhibits p15 induction by TGF-β (ref. 17) and may also induce a factor that inactivates p27 18. In up to 50% of human cancers, reduced p27 protein is associated with a poor prognosis 19. In some tumors, p27 is mislocalized to the cytoplasm 19,20 , however; the mechanism and significance of this has not been elucidated. In human cancers, constitutive activation of phosphoinositol 3′ kinase (PI3K) and its effector PKB/Akt arise through oncogenic receptor tyrosine kinase activation, Ras activation, mutational loss of PTEN, or through activating mutation of the PI3K effector, protein kinase B (PKB)/Akt (hereafter termed Akt) itself 21,22. Akt can increase cyclin D1 levels 23 and downregulate p27 by increasing p27 proteolysis 24 or repressing p27 expression through Akt phosphorylation of a forkhead transcription factor 25. However, in most cancers, reduced p27 does not result from transcriptional silencing 19. Here we show that Akt causes resistance to cytokine-mediated G1 arrest. p27 phosphorylation by Akt impairs its nuclear import and leads to cytoplasmic p27 accumulation. In human breast cancers, cytoplasmic mislocalization of p27 is associated with Akt activation, loss of differentiation and poor patient outcome. Activation of Akt in lines resistant to G1 arrest by TGF-β Ras has been shown to confer TGF-β resistance. While investigating mechanisms of TGF-β resistance, we found that two TGF-β PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest