Identification of Clk/Sty as a serine/arginine-rich protein kinase

grantor: University of TorontoI have investigated the function of the protein kinase Clk/Sty. To discover proteins that may interact with Clk/Sty, I performed a two-hybrid screen in yeast using Clk/Sty as bait. Twenty-six mouse cDNA clones were identified in the screen. Four of these clones encode known transcription factors c-Myc, ZFP38, N-CoR, as well as a novel zinc finger protein. Other clones identified in the screen suggest a role for Clk/Sty in RNA processing. For instance, one of the clones, CARS-Cyp, encodes a novel arginine/serine-rich (RS) cyclophilin which co-localizes with splicing factors to nuclear speckles. Furthermore, six of the two-hybrid clones encode RNA-binding proteins. One of these clones is a mammalian homologue of the Drosophila splicing regulator tra-2. Three of the remaining five RNA-binding proteins are members of the SR family of essential splicing factors. I focused my studies on one member of the SR family, ASF/SF2, and found that Clk/Sty phosphorylated ASF/SF2 in vitro on sites that are also phosphorylated in vivo. As well, a catalytically inactive Clk/Sty co-localized to nuclear speckles with SR family members. Interestingly, overexpression of wild-type Clk/Sty led to disassembly of nuclear speckles resulting in redistribution of SR proteins. Therefore, Clk/Sty regulates the intranuclear distribution of SR proteins, presumably by phosphorylation. SRPK1 was the first kinase to be cloned and identified as an SR protein kinase. I found that SRPK1 phosphorylated RSRS repeats in ASF/SF2. Clk/Sty, on the other hand, phosphorylated SPR and KSKS repeats as well as RSRS repeats. Therefore, Clk/Sty displays a broader substrate specificity than SRPK1 in vitro, suggesting that Clk/Sty may phosphorylate more substrates than SRPK1 does in vivo. Pre-mRNA splicing is a complex process that is executed with high fidelity. In order to choose splice sites appropriately, the splicing machinery must be responsive to its external environment. I propose that Clk/Sty functions to transduce signals to this machinery enabling proper splice site selection.Ph.D