PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoprote ins 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 proper ties of SR protein kinase 1 (SRPK1), a kinase specific for SR family m embers. 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) produ ced in bacteria, suggesting that SRPK1 is responsible for the generati on of the mAb 104-specific phosphoepitope in vivo. Finally, we have co rrelated the effects of mutagenesis in the SR domain of SF2/ASF on spl icing with those on phosphorylation of the protein by SRPK1, suggestin g that phosphorylation of SR proteins is required for splicing.