AML1/RUNX1 Phosphorylation by Cyclin-Dependent Kinases Regulates the Degradation of AML1/RUNX1 by the Anaphase-Promoting Complex

Abstract

AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Here, we report that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein which cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree.The AML1 proteins, including AML1a, AML1b, and AML1c (AML1c is also known as AML1B), are generated from one gene by alternative splicing (22). This gene has been given the names RUNX1, AML1, CBFA2, and PEBP2␣B (37). The AML1 protein is composed of a DNA binding runt homology domain located in the amino terminus followed by a transcriptional activation domain and a negative regulatory C-terminal domain (15,20,29). AML1 was initially identified during the study of breakpoint t(8;21), which is a common chromosomal translocation in acute myeloid leukemia (23). The association of AML1 with blood cell development is shown by the disruption of the AML1 gene through multiple chromosomal translocations, deletions, point mutations, or amplification in approximately 30% of human myeloid leukemias and myelodysplastic syndrome patients and a significant number of lymphoid leukemia patients (21,24,27,32). Furthermore, no detectable definitive hematopoiesis is observed in Aml1 knockout mice (26,40). The importance of AML1 in nonhematopoietic cells has also been recognized in angiogenesis, muscle function, and neurogenesis (6,12,14,35,41).AML1 is detected as a serine and threonine phosphorylated protein (9). Previous work has suggested that AML1 activity may be regulated by phosphorylation (34,45). Phosphorylation at specific serine-proline or threonine-proline sites in AML1 appears to be necessary for normal activity (45). It has also been suggested that phosphorylation releases AML1 from an association with the nuclear matrix mediated by sin3A, in turn leading to both increased activity and an increased rate of degradation (11).We have now shown that AML1 phosphorylation by cyclindependent kinases (CDKs) affects the overall stability of AML1 as well as the ability of certain ubiquitin ligase complexes, such as Cdc20-anaphase-promoting complex (APC), to target AML1 for degradation. MATERIALS AND METHODSCell culture and treatment. 293T and NIH 3T3 cells were grown...

Aurora kinase-induced phosphorylation excludes transcription factor RUNX from the chromatin to facilitate proper mitotic progression

“…1C, lanes 7 and 8). This result is consistent with studies that showed that the C termini of RUNX1 and RUNX2 harbor mitotic kinase CDK1 phosphorylation sites (13)(14)(15)(16).…”

“…CDK1-mediated phosphorylation of RUNX2 enhanced DNA binding activity, suggesting a role for RUNX2 in G 2 /M progression (15). In addition, CDK1/2 phosphorylates RUNX1, promoting its degradation by CDC20-associated anaphase-promoting complex during the late stages of mitosis (13,16). However, despite these findings, the significance of RUNX hyperphosphorylation in mitosis remains unclear.…”

PEBP2-β/CBF-β–dependent phosphorylation of RUNX1 and p300 by HIPK2: implications for leukemogenesis

“…In a recent study, Ser 276 was reported to be also a target of Cdk1 and Cdk2 phosphorylation, which marks RUNX1 for Cdc20-dependent anaphase-promoting complex degradation. 49 Interestingly, in their system, the authors reported residual phosphorylation of RUNX1 persisted in the presence of multiple Cdk-specific inhibitors, suggesting that Ser 276 can serve concurrently as the target for several kinases. 49 In a related study, Aikawa et al recently reported the phosphorylation of RUNX1 and p300 by HIPK2 and HIPK1.…”

“…49 Interestingly, in their system, the authors reported residual phosphorylation of RUNX1 persisted in the presence of multiple Cdk-specific inhibitors, suggesting that Ser 276 can serve concurrently as the target for several kinases. 49 In a related study, Aikawa et al recently reported the phosphorylation of RUNX1 and p300 by HIPK2 and HIPK1. 28 Using a proteomic approach, they observed the phosphorylation of Ser 249 and Ser 276 in phosphorylated RUNX1 proteins isolated from myeloid cells.…”

“…28 Although the p300 mapping data are largely complimentary, significant differences between the 2 experimental approaches should be noted. In their study, Aikawa et al performed immunoprecipitation experiments to delineated p300 regions interacting with RUNX1 and HIPK2, 49 whereas we chose a functional readout in our mapping experiments, namely, the detection of phosphorylation-induced band shift. The strength in this approach is that it takes into account the participation of other, yet undetermined upstream events and interactions.…”