RUNX1/AML1 is a key transcriptional mediator of hematopoiesis and leukemogenesis. AML1 regulates myeloid and lymphoid differentiation via activation of lineage-specific genes such as those encoding myeloperoxidase or the T cell receptor δ and participates in apoptotic response pathways via its ability to tranactivate the p14/p19ARF gene. In addition, AML1 accelerates G1 to S cell cycle progression, via activation of the cyclin D3 and potentially the cdk4 genes. CBF oncoproteins such as AML1-ETO or CBFβ-SMMHC interfere with the activities of AML1 and block myeloid differentiation and slow cell cycle progression, and mutations such as loss of p16 which accelerate G1 prevent cell cycle inhibition and cooperate with CBF oncoproteins to induce acute leukemia in mice. In addition to regulation of the cell cycle by AML1, we have been interested in how AML1 activities vary and may be regulated during cell cycle progression. We recently reported that endogenous AML1 levels increase in hematopoietic cell lines as they progress from G1 to S and then diminish again at the end of mitosis (
Bernardin-Fried et al J. Biol. Chem. 279:15678, 2004
). RNA levels did not vary, but exogenous AML1 mimicked the behaviour of the endogenous protein, suggesting regulation at the level of protein stability. Mutation of two Ras-dependent phosphorylation sites, S276 and S293, to alanine did not prevent cell cycle variation. We have therefore set out to evaluate whether AML1 stability might be regulated by cyclin-dependent kinase (cdk) phosphorylation. AML1 contains 480 amino acids and binds DNA via its N-terminal Runt domain. Both the cdk6/cyclin D3 and the cdk1/cyclin B complex, expressed from baculovirus vectors, phosphorylated GST-AML1(1-290) and GST-AML1(290–480). The Runt domain alone, in GST-AML1(86–217), was not phosphorylated. Interestingly, exogenous DNA-binding domain alone did not vary during the cell cycle. This is the first demonstration that a specific kinase phosphorylates AML1 in vitro. There are three (S/T)PX(K/R) cdk consensus sites in AML1, with serines at residues 48, 303, and 424. Mutation of S424 to alanine did not prevent phosphorylation of GST-AML1(290–480). Additional mutations of these and other serines or threonines adjacent to proline are being generated to further map the cdk phosphorylation sites and to enable in vivo experiments designed to evaluate the effects of these mutations on cell cycle-specific AML1 expression. We propose a model in which accumulated phosphorylation of AML1 during the S and G2/M cell cycle phases leads to ubiquitin-mediated AML1 destabilization at the end of mitosis. The increased stability of AML1 in the presence of proteosome inhibitors supports this model. Phosphorylation-mediated destabilization of AML1 may complement the recent finding that direct interaction of cyclin D3 with AML1 inhibits its activity as a transcriptional activator. Each of these mechanisms may help regulate the proliferation of hematopoietic stem/progenitor cells. Finally, perhaps loss of destabilizing C-terminal phosphorylation sites in the AML1-ETO oncoprotein increases its ability to dominantly repress AML1-target genes during myeloid leukemogenesis.
