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J. Biol. Chem., Vol. 281, Issue 32, 22586-22596, August 11, 2006

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B-Raf Is Critical For MAPK Activation during Mitosis and Is Regulated in an M Phase-dependent Manner in Xenopus Egg Extracts^*

Sergiy I. Borysov, Anthony W. M. Cheng, and Thomas M. Guadagno¹

From the Molecular Oncology Program, Department of Interdisciplinary Oncology, H. Lee Moffitt Comprehensive Cancer Center and Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612

Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. Here we purified and characterized the MEK kinase activity present in Xenopus M phase-arrested egg extracts. Our results show that B-Raf was the critical MEK kinase required for M phase activation of the MAPK pathway. Consistent with this, B-Raf was activated and underwent hyperphosphorylation in an M phase-dependent manner. Interestingly B-Raf hyperphosphorylation at mitosis occurred, at least in part, as a consequence of a feedback loop involving MAPK-mediated phosphorylation within a conserved C-terminal SPKTP motif. The kinase activity of a B-Raf mutant defective at both phosphorylation sites was substantially greater than its wild type counterpart when incubated in Xenopus M phase egg extracts. Furthermore suppression of MAPK feedback at mitosis enhanced B-Raf activity, whereas constitutive activation of MAPK at mitosis strongly suppressed B-Raf activity. These results suggest that feedback phosphorylation by MAPK negatively regulates B-Raf activity at mitosis. Collectively our data demonstrate for the first time a role for B-Raf at mitosis and provide new insight into understanding the regulation and function of B-Raf during cell proliferation.

Received for publication, February 14, 2006 , and in revised form, May 24, 2006.

^* This work was supported in part by National Institutes of Health Grant GM62542 and by funding from the American Cancer Society-Florida (to T. M. G.) and a predoctoral fellowship from the American Heart Association-Florida (to S. I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ097958 [GenBank] .

¹ Supported as a special fellow of the Leukemia and Lymphoma Society. To whom correspondence should be addressed: H. Lee Moffitt Comprehensive Cancer Center, Bldg. MRC 3 annex, 12902 Magnolia Dr., Tampa, FL 33612. Tel.: 813-745-6818; Fax: 813-745-6817; E-mail: guadagnt{at}moffitt.usf.edu.

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				S. I. Borysov and T. M. Guadagno A Novel Role for Cdk1/Cyclin B in Regulating B-Raf Activation at Mitosis Mol. Biol. Cell, July 1, 2008; 19(7): 2907 - 2915. [Abstract] [Full Text] [PDF]