The protein kinase Clk/Sty directly modulates SR protein activity: Both hyper- and hypophosphorylation inhibit splicing

The splicing of mammalian mRNA precursors requires both protein phosphoryla tion and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identifie d that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splici ng in vivo. But evidence that a specific kinase can directly affect the spl icing activity of SR proteins has been lacking. Here, by using purified rec ombinant Clk/Sty, a catalytically inactive mutant, and individual SR protei ns, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR p rotein splicing activity, repressing constitutive splicing and sl,itching a lternative splice site selection. These findings indicate that Clk/Sty dire ctly and specifically influences the activity of SR protein splicing factor s and, importantly, show that both under- and overphosphorylation of SR pro teins can modulate splicing.