The subcellular localization of SF2/ASF is regulated by direct interactionwith SR protein kinases (SRPKs)

Serine/arginine-rich (SR) proteins play an important role in constitutive a nd 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-af finity chromatography, the SF2/ASF kinase activity was co-purified from HeL a cells with a 95-kDa protein, which was recognized by an anti SR protein k inase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to an d phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that i dentical sites were phosphorylated in the pull-down kinase reaction with He La extracts and by recombinant SRPKs, Epitope-tagged SF2/ASF transiently ex pressed in COS7 cells co-immunoprecipitated with SRPKs, Deletion analysis m apped the phosphorylation sites to a region containing an (Arg-Ser)(8) repe at beginning at residue 204, and far-Western analysis showed that the regio n is required for binding of SRPKs to SF2/ASF, Further binding studies show ed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylate d SF2/ASF, Expression of an SRPK2 kinase-inactive mutant caused accumulatio n of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphoryla tion state of SF2/ASF, may have regulatory roles in the assembly and locali zation of this splicing factor.