Phosphorylation of RUNX1 by Cyclin-dependent Kinase Reduces Direct Interaction with HDAC1 and HDAC3

Abstract

RUNX1 regulates formation of the definitive hematopoietic stem cell and its subsequent lineage maturation, and mutations of RUNX1 contribute to leukemic transformation. Phosphorylation of Ser-48, Ser-303, and Ser-424 by cyclin-dependent kinases (cdks) increases RUNX1 trans-activation activity without perturbing p300 interaction. We now find that endogenous RUNX1 interacts with endogenous HDAC1 or HDAC3. Mutation of the three RUNX1 serines to aspartic acid reduces co-immunoprecipitation with HDAC1 or HDAC3 when expressed in 293T cells; mutation of these three serines to alanine increases HDAC interaction, and mutation of each serine individually to aspartic acid also reduces these interactions. GST-RUNX1 isolated from bacterial extracts bound in vitro translated HDAC1 or HDAC3, and these interactions were weakened by mutation of Ser-48, Ser-303, and Ser-424 to aspartic acid. The ability of RUNX1 phosphorylation and not only serine to aspartic acid conversion to reduce HDAC1 binding was demonstrated using wild-type GST-RUNX1 phosphorylated in vitro using cdk1/cyclinB and by exposure of 293T cells transduced with RUNX1 and HDAC1 to roscovitine, a cdk inhibitor. Finally, RUNX1 or RUNX1(tripleD), in which Ser-48, Ser-303, and Ser-424 are mutated to aspartic acid, stimulated proliferation of transduced, lineage-negative murine marrow progenitors more potently than did RUNX1(tripleA), in which these serines are mutated to alanine, suggesting that stimulation of RUNX1 trans-activation by cdk-mediated reduction in HDAC interaction increases marrow progenitor cell proliferation. RUNX1 and its heterodimeric partner CBF␤ direct emergence of adult hematopoietic stem cells (HSC)2 from hemogenic endothelium during embryogenesis and participate in subsequent lymphoid, myeloid, or megakaryocyte lineage maturation in adult marrow (1-3). RUNX1 is commonly mutated or inhibited in acute myeloid or lymphoid leukemias, and RUNX1 is overexpressed as well in a subset of acute lymphoblastic leukemias (4, 5).RUNX1 also directly regulates G 1 to S cell cycle progression. The myeloid oncoproteins CBF␤-SMMHC or RUNX1-ETO dominantly inhibit RUNX1 activities and slow G 1 progression in hematopoietic cell lines or in murine or human marrow progenitors (6 -9). cdk4, cyclin D2, or c-Myc overcome inhibition of proliferation by these CBF oncoproteins (8, 10, 11); exogenous RUNX1 stimulates G 1 progression in 32Dcl3 or Ba/F3 cells (9, 10, 12), and stimulation of G 1 via deletion of the p16INK4a/p19ARF locus or expression of the viral E7 protein (which inactivates retinoblastoma protein) cooperates with CBF␤-SMMHC or TEL-RUNX1 to induce acute leukemia (13,14). Induction of cdk4 or cyclin D3 transcription may underlie stimulation of G 1 progression by RUNX1 (10,15).Regulation of cell proliferation by RUNX proteins represents an evolutionarily conserved activity. In the sea urchin Strongylocentrotus purpuratus, depletion of the RUNX ortholog SpRunt-1 reduces blastocyst cell proliferation and inhibits expression of cyclinD, whose promoter binds SpRunt-1...

PMA-induced GCMa phosphorylation stimulates its transcriptional activity and degradation

“…The expression level of AML1 is regulated by acetylation controlled by p300/CBP and HDAC, as well as GCMa (3,22). Although the function of phosphorylation is dependent on the site(s), the phosphorylation of AML1 on serines 249 and 266 changes the interaction between AML1 and the corepressor mSin3A, which causes activation of transcriptional activity and degradation of AML1 (17,20). We suspect the phosphorylation of GCMa may be related to an interaction between GCMa and the other protein(s), leading to enhancement of its degradation and transcriptional activity.…”

Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis

“…Plasmids-(Runx1) 4 TKLUC, CMV-␤Gal, pCEFL-Src(E381G), pBabePuro-Runx1-ER(T), CMV-CBF␤, CMV-FLAG-HDAC1, CMV-FLAG-HDAC3, CMV-HA-Cdc20, CMV-HA-Cdh1, pMIG, and pMIGC were previously described (4,16,23,26,27,30,31). CMV-HA-ubiquitin was obtained commercially (Addgene).…”

“…Runx1 and its variants were inserted into pMIGC as XhoI/EcoRI fragments. Protein and RNA Expression-Total cellular proteins were generated in Laemmli sample buffer and subjected to Western blotting as described (23). Antibodies used were as follows: C/EBP␣ (14AA), PU.1 (D-19), HA (Y-11), ER␣ (MC-20), or lamin B (M-20; Santa Cruz Biotechnology); Runx1 (Active Motif); Tyr(P) (4G10; Millipore); HDAC1 (Ab7028) or HDAC3 (3G6; Abcam); HA (16B12; Biolegend); FLAG (M2) or ␤-actin (AC-15; Sigma); GAPDH (14C10; Cell Signaling); or CBF␤ (32).…”

“…CBF␤ also stabilizes Runx1 by inhibiting its ubiquitin-mediated proteolysis (20). Once bound to DNA, Runx1 activates or represses transcription dependent on gene and cellular context, in part mediated by interaction of a central trans-activation domain with the p300/CBF co-activators and of a C-terminal repression domain with HDAC1 or HDAC3 (21)(22)(23).…”

“…Several post-translational modifications have been found to regulateRunx1activity (15).Forexample,Ser-249/Ser-266phos-phorylation by ERK or Lys-206/Lys-210 methylation by PRMT1 reduces interaction with mSin3a to increase trans-activation (24,25), and Ser-21/Ser-249/Ser-266/Ser-397 phos-phorylation by cyclin-dependent kinase increases Runx1 transactivation potency by reducing HDAC 2 interaction and prevents ubiquitin-mediated Runx1 degradation (23,26,27).…”