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Volume 271, Number 47,
Issue of November 22, 1996
pp. 29958-29968
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of the CLK1 Gene
Product, a Novel CaM Kinase-like Protein Kinase from the Yeast
Saccharomyces cerevisiae
(Received for publication, September 10, 1996)
Marc L.
Melcher
and
Jeremy
Thorner
From the Department of Molecular and Cell Biology, Division of
Biochemistry and Molecular Biology, University of California,
Berkeley, California 94720-3202
The CLK1 gene of Saccharomyces
cerevisiae encodes a 610-residue protein kinase that resembles
known type II Ca2+/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 Ca2+; 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.

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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.
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