Dissection of the Molecular Basis of pp60v-src Induced Gating of Connexin 43 Gap Junction Channels

doi:10.1083/jcb.144.5.1033

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© The Rockefeller University Press, 0021-9525/1999//1033 $5.00
The Journal of Cell Biology, Volume 144, Number 5, , 1999 1033-1045

Regular Articles

Dissection of the Molecular Basis of pp60^v-src Induced Gating of Connexin 43 Gap Junction Channels

Lan Zhou, Eileen M. Kasperek, and Bruce J. Nicholson

Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260

Suppression of gap-junctional communication by various proteinkinases, growth factors, and oncogenes frequently correlateswith enhanced mitogenesis. The oncogene v-src appears to causeacute closure of gap junction channels. Tyr265 in the COOH-terminal tail of connexin 43 (Cx43) has been implicated as a potentialtarget of v-src, although v-src action has also been associatedwith changes in serine phosphorylation. We have investigatedthe mechanism of this acute regulation through mutagenesisof Cx43 expressed in Xenopus laevis oocyte pairs. Truncationsof the COOH-terminal domain led to an almost complete loss ofresponse of Cx43 to v-src, but this was restored by coexpressionof the independent COOH-terminal polypeptide. This suggestsa ball and chain gating mechanism, similar to the mechanismproposed for pH gating of Cx43, and K⁺ channel inactivation.Surprisingly, we found that v-src mediated gating of Cx43 didnot require the tyrosine site, but did seem to depend on the presence of two potential SH3 binding domains and the mitogen-activatedprotein (MAP) kinase phosphorylation sites within them. Furtherpoint mutagenesis and pharmacological studies in normal ratkidney (NRK) cells implicated MAP kinase in the gating responseto v-src, while the stable binding of v-src to Cx43 (in part mediated by SH3 domains) did not correlate with its abilityto mediate channel closure. This suggests a common link betweenclosure of gap junctions by v-src and other mitogens, suchas EGF and lysophosphatidic acid (LPA).

Key Words: intercellular coupling • MAP kinase • phosphorylation • v-src • Xenopus oocytes

Abbreviations used in this paper: Cx43, connexin 43; IGF, insulin-like growth factor; LPA, lysophosphatidic acid; LY, lucifer yellow dye; MAPK, mitogen-activated protein kinase; MEK, MAP kinase kinase; P_o, open probability; PKC, Ca²⁺-dependent protein kinase; pp60^v-src, Rous sarcoma virus oncogene; SH, Src homology.

We would like to extend our sincere appreciation to Steve Taffetand Mario Delmar for provision of many of the mutants usedand sharing of unpublished data, and to Marilyn Resh for provisionof the v-src construct. Steve Maricich prepared the Cx32 LA25cells transfectants that were characterized by Gary Goldbergand Mary Merritt with valuable input from Linda Musil (VollumInstitute, Portland, Oregon). Maria Garcia provided extensiveoriginal characterization of the LA25 cells and immunofluorescentlocalization of cells. We would also like to thank Gary Goldberg(State University of New York at Buffalo) and David Paul (HarvardUniversity, Cambridge, Massachusetts) for fruitful discussions of our results. Mary Merritt provided invaluable assistancein oocyte preparation and Jim Stamos aided in figure preparation.

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