Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5 but not ERK1/2 kinase activation.
The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5alpha, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.
Cameron
Scott J
SJ
Department of Pharmacology/Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Malik
Sundeep
S
Akaike
Masashi
M
Lerner-Marmarosh
Nicole
N
Yan
Chen
C
Lee
Jiing-Dwan
JD
Abe
Jun-Ichi
J
Yang
Jay
J
D015153:Blotting, Western; D002460:Cell Line; D018031:Connexin 43 / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D017629:Gap Junctions / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D015151:Immunoblotting; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D048054:Mitogen-Activated Protein Kinase 7; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D018031:Connexin 43; D004791:Enzyme Inhibitors; D005419:Flavonoids; D012694:Serine; D004815:Epidermal Growth Factor; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048054:Mitogen-Activated Protein Kinase 7; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one