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J. Biol. Chem., Vol. 280, Issue 50, 41761-41768, December 16, 2005

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Mass Spectrometric and Kinetic Analysis of ASF/SF2 Phosphorylation by SRPK1 and Clk/Sty^*

Adolfo Velazquez-Dones12, Jonathan C. Hagopian¶13, Chen-Ting Ma¶3, Xiang-Yang Zhong, Huilin Zhou, Gourisankar Ghosh, Xiang-Dong Fu4, and Joseph A. Adams¶5

From the Department of Cellular and Molecular Medicine, the Department of Chemistry and Biochemistry, and the ^¶Department of Pharmacology, University of California, San Diego, La Jolla, California 92093

Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.

Received for publication, April 18, 2005 , and in revised form, October 12, 2005.

^* This work was supported in part by National Institutes of Health (NIH) Grant GM67969 and National Science Foundation Grant MCB-111068 (to J. A. A.) and by NIH Grant GM52872 (to X.-D. F.). 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.

¹ Both authors contributed equally to this work.

² Supported by a minority supplement to NIH Grant GM52872.

³ Supported by NIH Training Grant GM07752.

⁴ To whom correspondence may be addressed. Tel.: 858-534-4937; Fax: 858-534-8549; E-mail: xdfu{at}ucsd.edu.

⁵ To whom correspondence may be addressed. Tel.: 858-822-3360; Fax: 858-822-3361; E-mail:joeadams{at}chem.ucsd.edu.

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