Nucleic Acids Research, 1993, Vol. 21, No. 12 2815-2822
© 1993
MOLECULAR BIOLOGY |
Identification of an snRNP-associated kinase activity that phosphorylates arginine/serine rich domains typical of splicing factors
Institut für Molekularbiologie und Tumorforschung Emil Mannkopff Straße 2, D-3550 Marburg, Germany 1Department of Biological Sciences, Columbia University New York, NY 10027, USA
*To whom correspondence should be addressed
Received April 6, 1993. Revised May 19, 1993. Accepted May 19, 1993.
The U1 snRNP-specific 70K protein is one of the few snRNP proteins from higher eukaryotic cells that is phosphorylated In vivo (1,2). Immunoaffinity purified spliceosomal snRNPs (U1, U2, U5, and U4/U6) were tested for their ability to phosphorylate In vitro the U1-specific 70K protein. An snRNP-associated kinase activity which phosphorylates all U1-70K isoelectric variants was identified. Like Its In vivo counterpart, this snRNP-associated enzyme phosphorylates solely serine residues of the 70K protein, preferentially utilizing ATP as a phosphodonor. Tryptic phosphopep-tide analysis revealed an overlapping set of at least four radiolabeled peptides in the In vivo and In vitro phosphorylated protein, suggesting that the snRNP-associated serine kinase is responsible, at least in part, for the 70K protein phosphorylation observed In vivo. Chymotryptic digestion of In vitro, 32P-labeled 70K protein and In vitro phosphorylation studies with a synthetic peptide, indicated that the multiple 70K phosphorylation sites are limited to a highly charged, C-terminal domain of the protein. In vitro phosphorylation studies with the splicing factor ASF/SF2 and several deletion mutants demonstrated that, similar to the U1-70K protein, the snRNP-associated serlne kinase phosphorylates the carboxy terminal RS-rich domain of ASF/SF2. A potential general role for this enzyme in the phosphorylation of splicing factors and its consequences for pre-mRNA splicing regulation are discussed.
+Present address: Neurcx Co., 3760 Haven Avenue, Menlo Park, CA 94025, USA
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