Submitted August 28, 2007
Accepted November 9, 2007
Cyclin-dependent kinase phosphorylation of RUNX1/AML1 on three sites increases trans-activation potency and stimulates cell proliferation
Linsheng Zhang, Florence Bernardin Fried, Hong Guo, and Alan D. Friedman*
Division of Pediatric Oncology, Johns Hopkins University, Baltimore, MD, United States
* Corresponding author; email: afriedm2{at}jhmi.edu.
RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and stimulates G1 cell cycle progression. Within RUNX1, S48, S303, and S424 fit the cyclin-dependent kinase (cdk) phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cdks on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now utilize an in vitro kinase assay, phosphopeptide-specific antiserum, and the cdk inhibitor roscovitine to demonstrate that S48 and S424 are also phosphorylated by cdk1 or cdk6 in hematopoietic cells. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation of the cdk sites to the partially phosphomimetic aspartic acid mildly reduced DNA-affinity while progressively increasing trans-activation of a model reporter. Mutation to alanine increased DNA-affinity, suggesting that in other gene or cellular contexts phosphorylation of RUNX1 by cdks may reduce trans-activation. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBF
-SMMHC more effectively than the tripleA mutant. Together these findings indicate that cdk phosphorylation of RUNX1 potentially couples stem/progenitor proliferation and lineage progression.
