THE CLK STY PROTEIN-KINASE PHOSPHORYLATES SR SPLICING FACTORS AND REGULATES THEIR INTRANUCLEAR DISTRIBUTION/

Mammalian Clk/Sty is the prototype for a family of dual specificity ki nases (termed LAMMER kinases) that have been conserved in evolution, b ut whose physiological substrates are unknown. In a yeast two-hybrid s creen, the Clk/Sty kinase specifically interacted with RNA binding pro teins, particularly members of the serine/arginine-rich (SR) family of splicing factors, Clk/Sty itself has an serine/arginine-rich non-cata lytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/ar ginine-rich region (the RS domain). Tryptic phosphopeptide mapping dem onstrated that the sites on ASF/SF2 phosphorylated in vitro overlap wi th those phosphorylated in vivo, Immunofluorescence studies showed tha t a catalytically inactive form of Clk/Sty co-localized with SR protei ns in nuclear speckles, Overexpression of the active Clk/Sty kinase ca used a redistribution of SR proteins within the nucleus. These results suggest that the Clk/Sty kinase directly regulates the activity and c ompartmentalization of SR splicing factors.