title: Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors.
abstract: Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.
authors.affiliation: Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla 92093-0651.
authors.forename: J F
authors.initials: JF
authors.lastname: Gui
authors.forename: H
authors.initials: H
authors.lastname: Tronchère
authors.forename: S D
authors.initials: SD
authors.lastname: Chandler
authors.forename: X D
authors.initials: XD
authors.lastname: Fu
mesh_terms: D000595:Amino Acid Sequence; D000939:Epitopes; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000939:Epitopes; D010750:Phosphoproteins; D011994:Recombinant Proteins; C087728:SRPK1 protein, human; D017346:Protein Serine-Threonine Kinases