SRPK1 and Clk/Sty Protein Kinases Show Distinct Substrate Specificities for Serine/Arginine-rich Splicing Factors

doi:10.1074/jbc.271.40.24569

SRPK1 and Clk/Sty Protein Kinases Show Distinct Substrate Specificities for Serine/Arginine-rich Splicing Factors*

From the ^‡ Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, the
^§Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the
^∥ Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093-0651, and the
^‴ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208

^∥ Terry Fox Cancer Research Scientist of the NCIC. To whom correspondence should be addressed:
Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario M5G 1X5, Canada
. Tel.: 416-586-8262; Fax: 416-586-8857; E-mail:Pawson{at}MSHRI.ON.CA

Abstract

Serine/arginine-rich (SR) proteins are essential for pre-mRNA splicing, and modify the choice of splice site during alternative splicing in a process apparently regulated by protein phosphorylation. Two protein kinases have been cloned that can phosphorylate SR proteins in vitro: SRPK1 and Clk/Sty. Here, we show that these two kinases phosphorylate the same SR proteins in vitro, but that SRPK1 has the higher specific activity toward ASF/SF2. SRPK1, like Clk/Sty, phosphorylates ASF/SF2 in vitro on sites that are also phosphorylated in vivo. Tryptic peptide mapping of ASF/SF2 revealed that three of the phosphopeptides from full-length ASF/SF2 phosphorylated in vitro contain consecutive phosphoserine-arginine residues or phosphoserine-proline residues. In vitro, the Clk/Sty kinase phosphorylated Ser-Arg, Ser-Lys, or Ser-Pro sites, whereas SRPK1 had a strong preference for Ser-Arg sites. These results suggest that SRPK1 and Clk/Sty may play different roles in regulating SR splicing factors, and suggest that Clk/Sty has a broader substrate specificity than SRPK1.

Footnotes

↵^¶ Supported by a studentship from the MRC.
↵^″ Supported by a postdoctoral fellowship from NIH.
↵^# Searle Scholar.
↵* This work was supported in part by grants from the Medical Research Council of Canada (MRC) and the National Cancer Institute of Canada (NCIC), by a Howard Hughes International Research Scholar Award to T.P., and by a National Institutes of Health (NIH) Grant (to X.-D. F.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SR

serine/arginine-rich

RS

arginine/serine-rich

PP1

protein phosphatase 1

PP2A

protein phosphatase 2A

GST

glutathione S-transferase.
↵² J. Prasad, K. Colwill, T. Pawson, and J. L. Manley, unpublished results.
- Received April 18, 1996.
- Revision received July 1, 1996.