PHOSPHORYLATION OF THE BETA SUBUNIT OF CASEIN KINASE-II IN HUMAN-A431 CELLS - IDENTIFICATION OF THE AUTOPHOSPHORYLATION SITE AND A SITE PHOSPHORYLATED BY P34CDC2

被引:0
作者
LITCHFIELD, DW
LOZEMAN, FJ
CICIRELLI, MF
HARRYLOCK, M
ERICSSON, LH
PIENING, CJ
KREBS, EG
机构
[1] UNIV WASHINGTON, DEPT PHARMACOL, MAIL STOP SL-15, SEATTLE, WA 98195 USA
[2] UNIV WASHINGTON, DEPT BIOCHEM, SEATTLE, WA 98195 USA
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中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
To examine the phosphorylation of casein kinase II in cells, the enzyme was isolated by immunoprecipitation from metabolically labeled human epidermal carcinoma A431 cells using polyclonal antipeptide antibodies specific for either the alpha-subunit or the beta-subunit of the enzyme. When isolated from P-32-labeled cells, the beta-subunit was found to be significantly labeled on serine residues whereas only minimal labeling was associated with the a subunit. In vitro, the beta-subunit of purified bovine casein kinase II was autophosphorylated, also on serine residues. Cleavage of the beta-subunit, that had been autophosphorylated in vitro, at tryptophan 9 and tryptophan 12 using N-chlorosuccinimide demonstrated that the autophosphorylation site is located near the amino terminus of the protein, most likely at serine 2 and serine 3. Two-dimensional maps of phosphopeptides generated by digestion of the beta-subunit with endoproteinase Glu-C indicted that the majority of the phosphate that was incorporated into the protein in cells was at sites that were indistinguishable from the sites that were autophosphorylated in vitro. In addition to phosphorylation at the autophosphorylation site, the beta-subunit is also phosphorylated at an additional site, serine 209, in intact cells. This residue, which is near the carboxyl terminus of the protein, can be phosphorylated in vitro by p34cdc2.
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页码:20380 / 20389
页数:10
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