EPIDERMAL GROWTH-FACTOR STIMULATES THE DISRUPTION OF GAP JUNCTIONAL COMMUNICATION AND CONNEXIN43 PHOSPHORYLATION-INDEPENDENT OF 12-0-TETRADECANOYLPHORBOL 13-ACETATE-SENSITIVE PROTEIN-KINASE-C - THE POSSIBLE INVOLVEMENT OF MITOGEN-ACTIVATED PROTEIN-KINASE

被引:158
作者
KANEMITSU, MY [1 ]
LAU, AF [1 ]
机构
[1] UNIV HAWAII MANOA, SCH MED, DEPT PHYSIOL, HONOLULU, HI 96813 USA
关键词
D O I
10.1091/mbc.4.8.837
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
We previously reported that epidermal growth factor (EGF) induced the disruption of gap junctional communication (gjc) and serine phosphorylation of connexin43 (Cx43) in T51B rat fiver epithelial cells. However, the cascade of events linking EGF receptor activation to these particular responses have not been fully characterized. Furthermore, the serine kinase(s) acting directly on Cx43 remain unidentified. In the current study, we demonstrate that downmodulation of 12-0-tetradecanoylphorbol 13-acetate (TPA)-sensitive protein kinase C (PKC) activity does not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43 in T51B cells. EGF in the presence or absence of chronic TPA treatment stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. EGF stimulated activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 on serine residues in vitro. Two-dimensional tryptic and chymotryptic phosphopeptide mapping demonstrated that the in vitro phosphopeptides represented a specific subset of the in vivo phosphopeptides produced in response to EGF after chronic TPA treatment. Therefore, EGF-induced disruption of gjc and phosphorylation of Cx43 may be mediated in part by MAP kinase in vivo.
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页码:837 / 848
页数:12
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