TY - JOUR
T1 - Identification of casein kinase I substrates by in vitro expression cloning screening
AU - Gao, Zhong Hua
AU - Metherall, James
AU - Virshup, David M.
N1 - Funding Information:
We thank Erica Vielhaber for purification of CKIδΔ, Kathy Waugh for technical assistance with library normalization, and Andrew Thorburn for helpful discussions. This work was supported by R01CA71074 from the National Institutes of Health, and a University of Utah Pilot project grant.
PY - 2000/2/16
Y1 - 2000/2/16
N2 - Casein kinase I (CKI) is a widely expressed protein kinase family implicated in diverse processes including membrane trafficking, DNA repair, and circadian rhythm. Despite the large number of CKI genes, few biologically relevant substrates have been identified. As an approach to better defining the spectrum of CKI substrates, we extended a recently described in vitro expression cloning (IVEC) strategy. Polypeptides pools were screened for kinase-dependent electrophoretic mobility shifts. Ten putative CKI substrates were isolated from an initial sample of 3000 random cDNA clones. Candidate substrates include proteins involved in RNA metabolism (a putative RNA helicase, the nucleolar protein hNOP56, and hnRNP A1, and ribosomal proteins L4, L8, and L13), as well as keratin 17, a necdin-related protein, and the calcium-binding proteins desmoglein 2 and annexin II. The same pools were also screened with active ERK2, and four substrates identified: aldolase, NSD-like protein, uracil-DNA glycosylase, and HHR23A. IVEC is an effective method to identify novel protein kinase substrates. (C) 2000 Academic Press.
AB - Casein kinase I (CKI) is a widely expressed protein kinase family implicated in diverse processes including membrane trafficking, DNA repair, and circadian rhythm. Despite the large number of CKI genes, few biologically relevant substrates have been identified. As an approach to better defining the spectrum of CKI substrates, we extended a recently described in vitro expression cloning (IVEC) strategy. Polypeptides pools were screened for kinase-dependent electrophoretic mobility shifts. Ten putative CKI substrates were isolated from an initial sample of 3000 random cDNA clones. Candidate substrates include proteins involved in RNA metabolism (a putative RNA helicase, the nucleolar protein hNOP56, and hnRNP A1, and ribosomal proteins L4, L8, and L13), as well as keratin 17, a necdin-related protein, and the calcium-binding proteins desmoglein 2 and annexin II. The same pools were also screened with active ERK2, and four substrates identified: aldolase, NSD-like protein, uracil-DNA glycosylase, and HHR23A. IVEC is an effective method to identify novel protein kinase substrates. (C) 2000 Academic Press.
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U2 - 10.1006/bbrc.2000.2168
DO - 10.1006/bbrc.2000.2168
M3 - Article
C2 - 10679243
AN - SCOPUS:0034673236
SN - 0006-291X
VL - 268
SP - 562
EP - 566
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -