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Reports
Cell cycle-dependent phosphorylation of human CDC5 regulates RNA processing
Remo Gräub, Hope Lancero, Anissa Pedersen, Meihua Chu, Krishnan Padmanabhan, Xiao-Qin Xu, Paul Spitz, Robert Chalkley, Alma L. Burlingame, David Stokoe and Harold S. Bernstein
volume 7 | issue 12
15 June 2008Pages: 1795 - 1803
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CDC5 proteins are components of the pre-mRNA splicing complex and essential for cell cycle progression in yeast, plants and mammals. Human CDC5 is phosphorylated in a mitogen-dependent manner, and its association with the spliceosome is ATP-dependent. Examination of the amino acid sequence suggests that CDC5L may be phosphorylated at up to 28 potential consensus recognition sequences for known kinases, however, the identity of actual phosphorylation sites, their role in regulating CDC5L activity, and the kinases responsible for their phosphorylation have not previously been determined. Using two-dimensional phosphopeptide mapping and nanoelectrospray mass spectrometry, we now show that CDC5L is phosphorylated on at least nine sites in vivo. We demonstrate that while CDC5L is capable of forming homodimers in vitro and in vivo, neither homodimerization nor nuclear localization is dependent on phosphorylation at these sites. Using an in vitro splicing assay, we show that phosphorylation of CDC5L at threonines 411 and 438 within recognition sequences for CDKs are required for CDC5L-mediated pre-mRNA splicing. We also demonstrate that a specific inhibitor of CDK2, CVT-313, inhibits CDC5L phosphorylation in both in vitro kinase assays and in vivo radiolabeling experiments in cycling cells. These studies represent the first demonstration of a regulatory role for phosphorylation of CDC5L, and suggest that targeting these sites or the implicated kinases may provide novel strategies for treating disorders of unguarded cellular proliferation, such as cancer.
Authors
Remo Gräub
University of California at San Francisco; San Francisco, CA
Hope Lancero
University of California at San Francisco; San Francisco, CA
Anissa Pedersen
University of California at San Francisco; San Francisco, CA
Meihua Chu
University of California at San Francisco; San Francisco, CA
Krishnan Padmanabhan
University of California at San Francisco; San Francisco, CA
Xiao-Qin Xu
University of California at San Francisco; San Francisco, CA
Paul Spitz
University of California at San Francisco; San Francisco, CA
Robert Chalkley
University of California at San Francisco; San Francisco, CA
Alma L. Burlingame
University of California at San Francisco; San Francisco, CA
David Stokoe
University of California at San Francisco; San Francisco, CA
Harold S. Bernstein
University of California at San Francisco; San Francisco, CA