QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 19, Issue 7, 2907-2915, July 2008

This Article Full Text Full Text (PDF) Supplemental Materials All Versions of this Article: E07-07-0679v1 19/7/2907    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Borysov, S. I. Articles by Guadagno, T. M. PubMed PubMed Citation Articles by Borysov, S. I. Articles by Guadagno, T. M.

A Novel Role for Cdk1/Cyclin B in Regulating B-Raf Activation at Mitosis

Sergiy I. Borysov^*,, and Thomas M. Guadagno^*

*Molecular Oncology Program, H. Lee Moffitt Cancer and Research Institute, Tampa, FL 33612; and Johnnie B. Byrd, Sr. Alzheimer's Center and Research Institute, Tampa, FL 33613

Submitted July 18, 2007; Revised March 10, 2008; Accepted April 16, 2008
Monitoring Editor: Yixian Zheng

MAPK activity is important during mitosis for spindle assembly and maintenance of the spindle checkpoint arrest. We previously identified B-Raf as a critical activator of the MAPK cascade during mitosis in Xenopus egg extracts and showed that B-Raf activation is regulated in an M-phase–dependent manner. The mechanism that mediates B-Raf activation at mitosis has not been elucidated. Interestingly, activation of 95-kDa B-Raf at mitosis does not require phosphorylation of Thr-599 and Ser-602 residues (Thr-633 and Ser-636 in Xenopus B-Raf), previously shown to be essential for B-Raf activation by Ras. Instead, we provide evidence for Cdk1/cyclin B in mediating mitotic activation of B-Raf. In particular, Cdk1/cyclin B complexes associate with B-Raf at mitosis in Xenopus egg extracts and contribute to its phosphorylation. Mutagenesis and in vitro kinase assays demonstrated that Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144, which is part of a conserved Cdk1 preferential consensus site (S¹⁴⁴PQK). Importantly, phosphorylation of Ser-144 is absolutely required for mitotic activation of B-Raf and subsequent activation of the MAPK cascade. However, substitution of a phospho-mimicking amino acid at Ser-144 failed to produce a constitutive active B-Raf indicating that, in addition of Ser-144 phosphorylation, other regulatory events may be needed to activate B-Raf at mitosis. Taken together, our data reveal a novel cell cycle mechanism for activating the B-Raf/MEK/MAPK cascade.

Address correspondence to: Thomas Guadagno (Thomas.Guadagno{at}moffit.org)

Abbreviations used: CR, conserved regions; CSF, cytostatic factor; GST, glutathione S-transferase; IP, immunoprecipitation; IPed, immunoprecipitated; WT, wild type.