A dynamic equilibrium between CDKs and PP2A modulates phosphorylation of pRB, p107 and p130.
It is thought that G(1) cyclin/CDK mediated phosphorylation of pocket proteins from mid G(1) to mitosis is reversed via dephosphorylation in mitosis. We examined the mechanisms involved in the unexpectedly rapid dephosphorylation of the pocket proteins induced via inhibition of cellular protein synthesis by cycloheximide (CHX) as well as direct inhibition of CDKs by flavopiridol. CHX and flavopiridol-induced dephosphorylation of pocket proteins is attributable to inactivation of D-type cyclin/CDKs and G(1)/S CDKs, respectively, which unmasks a phosphatase activity that targets the three pocket proteins apparently throughout the cell cycle. Treatment of cells with phosphatase inhibitors at concentrations selective for PP2A inhibition prevents CHX and flavopiridol-mediated dephosphorylation of pocket proteins in vivo. Also, ectopic expression of SV40 small t antigen, which inhibits PP2A via disruption of trimeric PP2A holoenzymes, delays CHX-induced pocket protein dephosphorylation. Moreover, dephosphorylation of p130 and p107 in cell extracts is inhibited by concentrations of okadaic acid known to inhibit PP2A, but not PP1. Finally, the PP2A catalytic subunit (PP2A/C) specifically interacts with both p130 and p107 in quiescent cells as well as cells progressing throughout the cell cycle. Together, these results demonstrate that the overall phosphorylation state of pocket proteins is determined, at least in part, by a dynamic equilibrium between CDKs and PP2A, or a closely related PP2A-like enzyme. These findings have important implications, as cell cycle or checkpoint-dependent inhibition of CDK activities counteracted by an active PP2A should have imminent effects on the phosphorylation state and activities of pocket proteins.
Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Judit
J
Garriga
Arun L
AL
Jayaraman
Ana
A
Limón
Girish
G
Jayadeva
Elena
E
Sotillo
May
M
Truongcao
Antonia
A
Patsialou
Brian E
BE
Wadzinski
Xavier
X
Graña
D000957:Antigens, Viral, Tumor / Q000378:metabolism; D001665:Binding Sites; D002453:Cell Cycle; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D016160:Retinoblastoma Protein / Q000378:metabolism*; D050720:Retinoblastoma-Like Protein p107 / Q000378:metabolism*; D050717:Retinoblastoma-Like Protein p130 / Q000378:metabolism*
D000957:Antigens, Viral, Tumor; D016160:Retinoblastoma Protein; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D019938:Cyclin D1; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases