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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 pp60v-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 protein kinases, growth factors, and oncogenes frequently correlates with enhanced mitogenesis. The oncogene v-src appears to cause acute closure of gap junction channels. Tyr265 in the COOH-terminal tail of connexin 43 (Cx43) has been implicated as a potential target of v-src, although v-src action has also been associated with changes in serine phosphorylation. We have investigated the mechanism of this acute regulation through mutagenesis of Cx43 expressed in Xenopus laevis oocyte pairs. Truncations of the COOH-terminal domain led to an almost complete loss of response of Cx43 to v-src, but this was restored by coexpression of the independent COOH-terminal polypeptide. This suggests a ball and chain gating mechanism, similar to the mechanism proposed for pH gating of Cx43, and K+ channel inactivation. Surprisingly, we found that v-src mediated gating of Cx43 did not require the tyrosine site, but did seem to depend on the presence of two potential SH3 binding domains and the mitogen-activated protein (MAP) kinase phosphorylation sites within them. Further point mutagenesis and pharmacological studies in normal rat kidney (NRK) cells implicated MAP kinase in the gating response to v-src, while the stable binding of v-src to Cx43 (in part mediated by SH3 domains) did not correlate with its ability to mediate channel closure. This suggests a common link between closure of gap junctions by v-src and other mitogens, such as 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; Po, open probability; PKC, Ca2+-dependent protein kinase; pp60v-src, Rous sarcoma virus oncogene; SH, Src homology.

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our sincere appreciation to Steve Taffet and Mario Delmar for provision of many of the mutants used and sharing of unpublished data, and to Marilyn Resh for provision of the v-src construct.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


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