IDENTIFICATION AND CHARACTERIZATION OF THE CLK1 GENE-PRODUCT, A NOVELCAM KINASE-LIKE PROTEIN-KINASE FROM THE YEAST SACCHAROMYCES-CEREVISIAE

The CLK1 gene of Saccharomyces cerevisiae encodes a 610-residue protei n kinase that resembles known type II Ca2+/calmodulin-dependent protei n kinases (CaM kinases), including the CMK1 and CMK2 gene products fro m the same yeast, The Clk1 kinase domain is preceded by a 162-residue N-terminal extension, followed by a 132-residue C-terminal extension ( which contains a basic segment resembling known calmodulin-binding sit es) and is as similar to mammalian CaM kinase (38% identity to rat CaM kinase cu) as it is to yeast CaM kinase (37% identity to Cmk2). Howev er, Clk1 shares 52% identity with Rck1, another putative protein kinas e encoded in the S. cerevisiae genome, Clk1 tagged with a c-myc epitop e (expressed in yeast) and a GST-Clk1 fusion (expressed in bacteria) u nderwent autophosphorylation and phosphorylated an exogenous substrate (yeast protein synthesis elongation factor 2), primarily on Ser. Neit her Clk1 activity was stimulated by purified yeast calmodulin (CMD1 ge ne product), with or without Ca2+; no association of Clk1 with Cmd1 wa s detectable by other methods, C-terminally truncated Clk1(Delta 487-6 10) was growth-inhibitory when overexpressed, whereas catalytically in active Clk1(K201R Delta 487-610) was not, suggesting that the C termin us is a negative regulatory domain. Using immunofluorescence, Clk1 was localized to the cytosol and excluded from the nucleus, A clk1 Delta. mutant, a clk1 Delta rck1 Delta double mutant, a clk1 Delta cmk1 Delt a cmk2 Delta triple mutant, and a clk1 Delta rck1 Delta cmk1 Delta cmk 2 Delta quadruple mutant were all viable and manifested no other overt growth phenotype.