Cell cycle-dependent phosphorylation of C/EBPbeta mediates oncogenic cooperativity between C/EBPbeta and H-RasV12.
CCAAT/enhancer binding protein beta (C/EBPbeta) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPbeta is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPbeta in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPbeta activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPbeta, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPbeta as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPbeta on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.
Laboratory of Protein Dynamics and Signaling, NCI-Frederick, Frederick, MD 21702-1201, USA.
Jon D
JD
Shuman
Thomas
T
Sebastian
Philipp
P
Kaldis
Terry D
TD
Copeland
Songyun
S
Zhu
Robert C
RC
Smart
Peter F
PF
Johnson
D000595:Amino Acid Sequence; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D002471:Cell Transformation, Neoplastic; D051357:Cyclin-Dependent Kinase 2; D004791:Enzyme Inhibitors / Q000378:metabolism; D011905:Genes, ras; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D022782:CCAAT-Enhancer-Binding Protein-beta; D004791:Enzyme Inhibitors; D013912:Threonine; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins