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Molecular and Cellular Biology, September 2004, p. 7380-7391, Vol. 24, No. 17
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.17.7380-7391.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cell Cycle-Dependent Phosphorylation of C/EBPß Mediates Oncogenic Cooperativity between C/EBPß and H-RasV12

Jon D. Shuman,1,{dagger} Thomas Sebastian,1 Philipp Kaldis,2 Terry D. Copeland,1 Songyun Zhu,3 Robert C. Smart,3 and Peter F. Johnson1*

Laboratory of Protein Dynamics and Signaling,1 Mouse Cancer Genetics Program, National Cancer Institute— Frederick, Frederick, Maryland,2 Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina3

Received 9 April 2004/ Returned for modification 3 May 2004/ Accepted 26 May 2004

CCAAT/enhancer binding protein ß (C/EBPß) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPß 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/EBPß 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/EBPß 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/EBPß, 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/EBPß as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPß on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.

* Corresponding author. Mailing address: Laboratory of Protein Dynamics and Signaling, NCI—Frederick, Frederick, MD 21702-1201. Phone: (301) 846-1627. Fax: (301) 846-5991. E-mail: johnsopf{at}ncifcrf.gov.

{dagger} Present address: Brewton-Parker College, Mount Vernon, Ga.

Molecular and Cellular Biology, September 2004, p. 7380-7391, Vol. 24, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.17.7380-7391.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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