Cell Cycle-dependent Phosphorylation of the RUNX2 Transcription Factor by cdc2 Regulates Endothelial Cell Proliferation

Abstract

RUNX2 is a member of the runt family of DNA-binding transcription factors. RUNX2 mediates endothelial cell migration and invasion during tumor angiogenesis and is expressed in metastatic breast and prostate tumors. Our published studies showed that RUNX2 DNA-binding activity is low during growth arrest, but elevated in proliferating endothelial cells. To investigate its role in cell proliferation and cell cycle regulation, RUNX2 was depleted in human bone marrow endothelial cells using RNA interference. Specific RUNX2 depletion inhibited DNA-binding activity as measured by electrophoretic mobility shift assay resulting in inhibition of cell proliferation. Cells were synchronized at the G 1 /S boundary with excess thymidine or in mitosis (M phase) with nocodazole. Endogenous or ectopic RUNX2 activity was maximal at late G 2 and during M phase. Inhibition of RUNX2 expression by RNA interference delayed entry into and exit out of the G 2 /M phases of the cell cycle. RUNX2 was coimmunoprecipitated with cyclin B1 in mitotic cells, which further supported a role for RUNX2 in cell cycle progression. Moreover, in vitro kinase assays using recombinant cdc2 kinase showed that RUNX2 was phosphorylated at Ser 451 . The cdc2 inhibitor roscovitine dose dependently inhibited in vivo RUNX2 DNAbinding activity during mitosis and the RUNX2 mutant S451A exhibited lower DNA-binding activity and reduced stimulation of anchorage-independent growth relative to wild type RUNX2. These results suggest for the first time that RUNX2 phosphorylation by cdc2 may facilitate cell cycle progression possibly through regulation of G 2 and M phases, thus promoting endothelial cell proliferation required for tumor angiogenesis.The RUNX gene family of transcriptional regulators is the mammalian homologue of the Drosophila transcription factor runt. The three RUNX factors contain an evolutionarily conserved 128-amino acid runt domain, which is responsible for DNA binding and heterodimerization with the cofactor CBF␤ (1-3). RUNX factors are essential regulators of hematopoietic (4 -6), osteogenic (7-10), and gastric development (11-13). RUNX2 promotes preosteoblast growth and osteoblast lineage commitment (14 -16). Mice nullizygous for RUNX2 lack bone matrix gene expression and exhibit no bone formation, whereas heterozygous mice display skeletal abnormalities similar to cleidocranial dysplasia in humans (17)(18)(19)(20). Increasing evidence suggests that RUNX2 exhibits oncogenic potential in a wide variety of cells (21). Expression of RUNX2 is higher in H-Ras-transformed NIH3T3 fibroblasts compared with normal cells (22) and, after retroviral integration in T-cells, RUNX2 expression cooperates with c-Myc to stimulate cell growth (23). Forced expression of Runx2 in transgenic mice was found to interfere with T-cell development and to predispose mice to lymphoma (23). Endogenous RUNX2 is expressed in malignant breast cancer cells and prostate tumors, regulates bone sialoprotein expression, and promotes osteolytic lesions in breast cancer metastases (24...

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).…”

“…RUNX2 is phosphorylated by mitotic kinase CDK1-cyclin B1 (14,15) and dephosphorylated at mitotic exit by the PP1/PP2A phosphatase (14). 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).…”

“…An intriguing observation is the hyperphosphorylation of RUNX proteins during mitosis (13,14). RUNX2 is phosphorylated by mitotic kinase CDK1-cyclin B1 (14,15) and dephosphorylated at mitotic exit by the PP1/PP2A phosphatase (14). CDK1-mediated phosphorylation of RUNX2 enhanced DNA binding activity, suggesting a role for RUNX2 in G 2 /M progression (15).…”

Cell-cycle–dependent oscillation of GATA2 expression in hematopoietic cells

“…Transfer into medium lacking excess thymidine but containing nocodazole permitted progression into S phase followed by the accumulation of cells containing 4N DNA. 40,47 DNA content analysis demonstrated a single peak in M phase, 7 hours after thymidine release. Immunoblot analysis demonstrated that after a 2-hour lag, GATA2 expression increased with S phase progression (3-5 hours) followed by a decrease of GATA2 expression (6-7 hours).…”

Stabilization of RNT-1 Protein, Runt-related Transcription Factor (RUNX) Protein Homolog of Caenorhabditis elegans, by Oxidative Stress through Mitogen-activated Protein Kinase Pathway

“…In addition, diverse cellular signaling events that modify both the transcriptional and posttranscriptional status of the RUNX genes affect the amount and the activity of RUNX proteins in response to developmental or environmental cues. Transcriptional regulation, such as chromatin remodeling (6) and DNA methylation (7)(8)(9)(10), and posttranscriptional regulation, such as acetylation (11,12), ubiquitination (13)(14)(15), and phosphorylation (16,17), have been reported as the regulation mechanisms of RUNX activity.…”