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

J Biol Chem. 1999 Apr 16;274(16):11125-31. doi: 10.1074/jbc.274.16.11125.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Glutathione Transferase / metabolism
  • HeLa Cells
  • Humans
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Protein Serine-Threonine Kinases / metabolism*
  • RNA Splicing
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins / metabolism
  • Serine-Arginine Splicing Factors
  • Subcellular Fractions / metabolism*

Substances

  • Nuclear Proteins
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Serine-Arginine Splicing Factors
  • Glutathione Transferase
  • SRPK1 protein, human
  • Protein Serine-Threonine Kinases