Colwill K, Feng L L, Yeakley J M, Gish G D, Cáceres J F, Pawson T, Fu X D
Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
J Biol Chem. 1996 Oct 4;271(40):24569-75. doi: 10.1074/jbc.271.40.24569.
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 specific activity toward ASF/SF2. SRPK1, like Clk/Sty, phosphorylates ASF/SF2 in vitro on sites that are also phosphorylated in vivo. Tryptic peptide mapping of ASF/SF2 revealed that three of the phosphopeptides from full-length ASF/SF2 phosphorylated in vitro contain consecutive phosphoserine-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 results suggest that SRPK1 and Clk/Sty may play different roles in regulating SR splicing factors, and suggest that Clk/Sty has a broader substrate specificity than SRPK1.
富含丝氨酸/精氨酸（SR）的蛋白质对于前体mRNA剪接至关重要，并在一个明显受蛋白质磷酸化调控的过程中，在可变剪接期间改变剪接位点的选择。已经克隆出两种能在体外使SR蛋白磷酸化的蛋白激酶：SRPK1和Clk/Sty。在此，我们表明这两种激酶在体外能使相同的SR蛋白磷酸化，但SRPK1对ASF/SF2具有更高的比活性。与Clk/Sty一样，SRPK1在体外使ASF/SF2磷酸化的位点也是其在体内被磷酸化的位点。对ASF/SF2进行胰蛋白酶肽图谱分析显示，体外磷酸化的全长ASF/SF2的三个磷酸肽含有连续的磷酸丝氨酸-精氨酸残基或磷酸丝氨酸-脯氨酸残基。在体外，Clk/Sty激酶使丝氨酸-精氨酸、丝氨酸-赖氨酸或丝氨酸-脯氨酸位点磷酸化，而SRPK1强烈偏好丝氨酸-精氨酸位点。这些结果表明，SRPK1和Clk/Sty在调节SR剪接因子方面可能发挥不同作用，并且表明Clk/Sty比SRPK1具有更广泛的底物特异性。
