Identification and Characterization of the CLK1 Gene Product, a Novel CaM Kinase-like Protein Kinase from the Yeast Saccharomyces cerevisiae

Abstract

The CLK1 gene of Saccharomyces cerevisiae encodes a 610-residue protein kinase that resembles known type II Ca 2؉ /calmodulin-dependent protein kinases (CaM kinases), including the CMK1 and CMK2 gene products from 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 sites) and is as similar to mammalian CaM kinase (38% identity to rat CaM kinase ␣) as it is to yeast CaM kinase (37% identity to Cmk2). However, Clk1 shares 52% identity with Rck1, another putative protein kinase encoded in the S. cerevisiae genome. Clk1 tagged with a c-myc epitope (expressed in yeast) and a GST-Clk1 fusion (expressed in bacteria) underwent autophosphorylation and phosphorylated an exogenous substrate (yeast protein synthesis elongation factor 2), primarily on Ser. Neither Clk1 activity was stimulated by purified yeast calmodulin (CMD1 gene product), with or without Ca 2؉ ; no association of Clk1 with Cmd1 was detectable by other methods. C-terminally truncated Clk1(⌬487-610) was growth-inhibitory when overexpressed, whereas catalytically inactive Clk1(K201R ⌬487-610) was not, suggesting that the C terminus is a negative regulatory domain. Using immunofluorescence, Clk1 was localized to the cytosol and excluded from the nucleus. A clk1⌬ mutant, a clk1⌬ rck1⌬ double mutant, a clk1⌬ cmk1⌬ cmk2⌬ triple mutant, and a clk1⌬ rck1⌬ cmk1⌬ cmk2⌬ quadruple mutant were all viable and manifested no other overt growth phenotype.

The Srk1 Protein Kinase Is a Target for the Sty1 Stress-activated MAPK in Fission Yeast

“…2, a computer-based amino acid sequence comparison revealed that the greatest degree of amino acid sequence identity was shared with fission yeast Cmk2 (39%), and budding yeast Rck2/Clk1 (32%) (26). The kinase domains in all of these proteins share significant homology with the mammalian Ca 2ϩ /calmodulin kinase superfamily, although the significance of this is unclear as extensive analysis of S. cerevisiae Rck2/Clk1 failed to uncover any regulation by calmodulin (36).…”

Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast

“…Despite their high similarity to Ca 2ϩ ͞calmodulin-dependent kinases, Rck2 does not respond to Ca 2ϩ signals, and its regulatory inputs have remained unknown (12). The kinases were also discovered serendipitously by complementation of Schizosaccharomyces pombe mutants deficient in the DNA damage pathway (13).…”

“…In the set of positive clones (Table 1), we found independent fusions containing sequences of the following three genes: RCK2, GLO3, and RNR4. Rck2 has been previously identified as a Ser͞Thr protein kinase (12,13). Glo3 is thought to act as GTPase-activating protein for ADP ribosylation factor Arf1, which is involved in vesicular transport (23).…”

“…Rck1 and Rck2 were first identified by reverse genetics to study Ca 2ϩ signaling in yeast (12). Despite their high similarity to Ca 2ϩ ͞calmodulin-dependent kinases, Rck2 does not respond to Ca 2ϩ signals, and its regulatory inputs have remained unknown (12).…”

The Serine/Threonine Kinase Cmk2 Is Required for Oxidative Stress Response in Fission Yeast

“…cmk2 was isolated by its sequence similarity to the yeast and mammalian calmodulin kinases. 2 It has a high degree of homology to budding yeast RCK2, previously isolated by virtue of its sequence similarity to mammalian calmodulin kinases (30). It has also been described as a suppressor of fission yeast checkpoint mutants (31) and a substrate of Hog1, the MAPK in budding yeast responsive only to osmolarity stress (32).…”