Koizumi, J., Okamoto, Y., Onogi, H., Mayeda, A., Krainer, A. R., Hagiwara, M. (April 1999) The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs). Journal of Biological Chemistry, 274 (16). pp. 11125-11131. ISSN 0021-9258
Abstract
Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co-purified from HeLa cells with a 95-kDa protein, which was recognized by an anti SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs, Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs, Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)(8) repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF, Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF, Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.
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