SRPK1 AND CLK STY PROTEIN-KINASES SHOW DISTINCT SUBSTRATE SPECIFICITIES FOR SERINE/ARGININE-RICH SPLICING FACTORS/

Serine/arginine-rich (SR) proteins are essential for pre-mRNA splicing , and modify the choice of splice site during alternative splicing in a process apparently regulated by protein phosphorylation, Two protein kinases have been cloned that can phosphorylate SR proteins in vitro: SRPK1 and Clk/Sty. Here, we show that these two kinases phosphorylate the same SR proteins in. vitro, but that SRPK1 has the higher specifi c activity toward ASF/SF2. SRPK1, like Clk/Sty, phosphorylates ASF/SF2 in vitro on sites that are also phosphorylated in vivo. Tryptic pepti de mapping of ASF/SF2 revealed that three of the phosphopeptides from full-length ASF/SF2 phosphorylated in vitro contain consecutive phosph oserine-arginine residues or phosphoserine-proline residues. In vitro, the Clk/Sty kinase phosphorylated Ser-Arg, Ser-Lys, or Ser-Pro sites, whereas SRPK1 had a strong preference for Ser-Arg sites. These result s suggest that SRPK1 and Clk/Sty may play different roles in regulatin g SR splicing factors, and suggest that Clk/Sty has a broader substrat e specificity than SRPK1.