Fig. 1.

a. ERK1/2 is necessary, but not sufficient, for v-src gating of Cx43: Cx43 paired oocytes were injected with water, v-src or CA-MEK1, and conductance 6 hours post-injection measured as a ratio of the pre-injection conductance. v-src induced almost complete inhibition of coupling, which could be partially prevented by inhibition of MEK1/2 with U0126. However, CA-MEK1 injection alone had no significant effect on Cx43 coupling. U0126 alone had no effect on coupling. The data represent means of six separate experiments each experiment having a minimum of 8 coupled oocyte pairs. Statistical analysis was performed by use of the Student’s t-test comparing each group to the Cx43 expressing oocytes (* *= P<0.001). In this and all subsequent graphs, bars represent mean values with standard errors indicated.

b. CA-MEK induces phosphorylation of Cx43: Cx43 coupled oocytes, subjected to various treatments as indicated, were lysed and then immunoprecipitated with anti-Cx43 polyclonal antibody, followed by analysis via Western blot probed with a Cx43 monoclonal antibody. No bands were evident in the absence of Cx43 injection (lanes 2 and 3). A doublet corresponding to P₀ and P₁, a phosphorylated form, is found in cells injected with only Cx43 (lane 1). CA-MEK induces retardation in electrophoretic mobility of Cx43, producing a more highly phosphorylated P2 form (lane 4). In contrast, v-src (lane 5) failed to induce a significant shift in mobility.

c. Src phosphorylation of Cx43 occurs partially through ERK, but with low efficiency: Cx43 coupled oocytes were co-injected with ATP- ³² and either v-src or CA-MEK1 cRNA. After 6 hrs of incubation oocytes pairs were lysed and immunoprecipitated with anti- Cx43 antibody and separated by SDS-PAGE. Incorporated ³²P was quantitated by phosphoimage analysis. Src induces phosphorylation, which is about 50% inhibited by the MEK inhibitor U0126. CA-MEK induces significantly higher phosphorylation levels than does src. Data shown represent the mean ± S.E. from three separate experiments with equal numbers of oocytes pairs in each set. Data are normalized, and compared statistically to, oocytes expressing only Cx43; statistical significance was determined by Student t-test (*P<0.05).