Mitotic control of RUNX2 phosphorylation by both CDK1/cyclin B kinase and PP1/PP2A phosphatase in osteoblastic cells.
Skeletal development and osteoblast maturation require the phenotype promoting activity of the transcription factor RUNX2, which controls both cell growth and differentiation in osteoblasts. We have recently shown that in actively proliferating cells RUNX2 regulates the expression of specific target genes as cells enter and exit mitosis. In this study, we addressed whether post-translational modifications of RUNX2 control its activity during mitotic exit. Western blot analysis of proteins from osteoblastic Saos-2 cells released from mitotic inhibition into early G(1) show a phosphatase-sensitive shift in the mobility of RUNX2 in SDS gels. The slowly migrating hyper-phosphorylated form of RUNX2 is immunoreactive with a CDK related phospho-antibody (MPM2) only in mitotic cells and is converted into a faster migrating hypo-phosphorylated RUNX2 when cells complete mitosis. This conversion is inhibited by okadaic acid, an inhibitor of protein phosphatases 1 and 2 (PP1 and PP2A), but not by deltamethrin which blocks PP2B phosphatase. Mitotic phosphorylation of RUNX2 is sensitive to the CDK inhibitors roscovitine and olomoucine. Furthermore, RUNX2 can directly interact with CDK1 and is phosphorylated in vitro by the CDK1/cyclin B kinase complex. Hence, RUNX2 is hyper-phosphorylated by CDK1/cyclin B during mitosis, and dynamically converted into a hypo-phosphorylated form by PP1/PP2A-dependent dephosphorylation after mitosis to support the post-mitotic regulation of RUNX2 target genes.
Rajgopal
Arun
A
Department of Cell Biology and Cancer Center, University of Massachusetts Medical School and Cancer Center, Worcester, Massachusetts 01655, USA.
Young
Daniel W
DW
Mujeeb
Khawaja A
KA
Stein
Janet L
JL
Lian
Jane B
JB
van Wijnen
Andre J
AJ
Stein
Gary S
GS
D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D045744:Cell Line, Tumor; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D008938:Mitosis / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D019319:Okadaic Acid / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D014162:Transfection
D050659:Core Binding Factor Alpha 1 Subunit; D019926:Cyclin B; D011687:Purines; C493709:RUNX2 protein, human; D000077546:Roscovitine; D019319:Okadaic Acid; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases