Zhou L , Kasperek EM , Nicholson BJ .

CA 48049 NCI NIH HHS , GM 48773 NIGMS NIH HHS , R01 CA048049 NCI NIH HHS

	Figure 8. Association of v-src with various Cx43 mutants. Oocytes were injected with [35S]-Met and cRNA for wild-type Cx43 or the various mutant Cx43 constructs indicated, with (+) or without (−) pp60v-src cRNA. Immunoprecipitation was performed with anti-Cx43 antiserum. The amount of associated v-src protein with Cx43 tyrosine mutants (A), deletion mutants (B), and serine/proline mutants (C) could be compared with the amount of v-src protein associated with wild-type Cx43 (A, first two lanes). The most dramatic reductions in pp60v-src binding to Cx43 were evident in the overlapping Δ261–280 and Δ241–280 deletion mutants, and in the Y247/265F mutant.
	Figure 1. Cx43 protein turnover in Xenopus oocytes. Oocytes were injected with Cx43 cRNA and labeled with [35S]met as described in Materials and Methods. After 6–7 h, each oocyte was injected with 40 nl of 2.5 mM L-methionine and treated with cycloheximide (15 μg/ml). At the time intervals noted, Cx43 was precipitated from the oocyte lysate, and the levels quantitated by PhosphoImage analysis on an SDS gel (normalized to protein level at 0 h). Data from three independent experiments are shown, along with a line derived from a linear fit that suggests a half-life of ∼22 h.
	Figure 2. A ball and chain model for v-src induced gating of Cx43 channels. Xenopus oocytes were first injected with cRNAs encoding the connexin construct indicated, and paired to allow gap-junctional channels to form as described in Materials and Methods. Initial conductance recording by dual cell voltage clamp was followed by a secondary injection of cRNA for pp60v-src, or pp60v-src (+) the COOH-terminal tail of Cx43 (C-TERM244: residues 244– 382). Conductance was recorded again after ∼6 h incubation, and expressed as a fraction of the conductance before v-src injection. Expression of pp60v-src caused almost complete loss of coupling in oocytes expressing wild-type Cx43 (>200-fold reduction), but only slightly decreased conductances of Cx32 channels. COOH-terminal truncation of Cx43 (Δ245) eliminated the response to v-src. This was mostly restored by coexpression of C-TERM244 with pp60v-src. Expression of the same domain with pp60v-src had no effect on Cx32 channels.
	Figure 4. Effects of pp60v-src on Cx43 truncation and deletion mutants. The ratio of conductances post- and pre-src injection on the indicated constructs of Cx43 were compared with wild-type Cx43 and Cx32, the mean conductances and standard errors are indicated on the right. Truncation of Cx43 at residue 257 (Δ257) reduced sensitivity to v-src compared with wild-type Cx43 (but to a lesser degree than in Cx43 Δ245; see Fig. 2). Coexpression of the truncated COOH- terminal domain (C-TERM258, 258–382) rescued the response of Δ257 to v-src completely, but only partially restored the response of the more severe Cx43 Δ245 truncation. Deletion of residues between 281–320 had no effect on v-src induced gating of Cx43 channels, while both Δ241–260 and Δ261–280 deletions showed significantly reduced responses to v-src. A combined deletion of these two regions (Δ241–280) showed no response to v-src beyond that of the Cx32 negative control.
	Figure 6. Effects of v-src on Cx43 serine and proline mutants. The ratio of conductances post- and pre-src injection on the indicated Cx43 mutants were compared with wild-type Cx43 and Cx32, the mean conductances and standard errors of which are indicated on the right. Proline mutants (P253/256A and P277/ 280A) that should disrupt both the MAP kinase recognition site and the consensus SH3 binding motif showed significantly reduced sensitivity to v-src. Mutations of potential MAP kinase phosphorylation sites (S255/257A and S279/282A) showed similar reduced responses to v-src. A combined mutant of all four serines showed even less inhibition by v-src (not significantly different to Cx32), indicating that these sites act synergistically.
	Figure 9. Inhibition of MAPK activation blocks v-src induced cell uncoupling. (A) LA25 cells were treated with DMSO alone (left column) or MEK inhibitor in DMSO (right column) at 40°C for 1 h before analysis of coupling by dye-transfer. LY was injected into a single LA25 cell of a monolayer at 0, 30, and 60 min (top to bottom) after transfer to the permissive temperature of 33°C for v-src activation. LY diffusion into adjacent cells was counted as digital images from a CCD camera 2 min after injection. (B) The complete time course of coupling (normalized to the value seen before v-src activation) is shown for both LA25 cells (filled symbols) and a Cx32 expressing LA25 clone (LA25-O25; open symbols) in the presence (triangles) or absence (squares) of MEK inhibitor.
	Figure 3. Potential v-src regulatory sites on Cx43-mutagenic strategy. A schematic diagram showing sequences on the COOH-terminal tail of Cx43 related to direct and possible downstream effectors of v-src. Tyr265 and Tyr247 are potential tyrosine phosphorylation sites of pp60v-src. Proline-rich sequences, 253PLSP256 and 277PLSPMSP283, represent possible SH3 binding domains, which also contain documented substrate sites of MAP kinase (S255, S279, and S282). Also indicated are structures of the truncation (Δ245 and Δ257) and systematic deletion constructs (Δ241–260, Δ261–280, Δ241–280, Δ281–300, and Δ301– 320) used in the current study.
	Figure 7. (A) Association of v-src with Cx43 and its COOH-terminal tail (258–382). [35S]-Met labeled oocytes were injected with cRNAs for Cx43 (lanes 2 and 5) or pp60v-src alone (lanes 1 and 6), or in combination (lane 3). The Cx43 COOH-terminal domain was also expressed in the presence of pp60v-src (lane 4). Lysates of the oocytes were precipitated with either anti-Cx43 antiserum (lanes 1–4) or anti-src mAb (lanes 5 and 6), separated by SDS-PAGE, and the dried gel exposed to X-ray film. The position of pp60v-src, Cx43, and its COOH-terminal tail are indicated. In the latter two cases, bands that correspond to phosphorylated (P), nonphosphorylated (NP), and partially degraded (*) products are indicated. (B) Dephosphorylation of Cx43/COOH-tail by alkaline phosphatase. [35S]-Met labeled oocytes were injected with cRNAs for either Cx43 (lanes 1–4) or Cx43 COOH-tail (residues 244–382; lanes 5–8), alone (lanes 1, 2, 5, and 6) or with pp60v-src (lanes 3, 4, 7, and 8). Lysates of the oocytes were precipitated with anti-Cx43 antiserum (all lanes) followed by treatment with alkaline phosphatase (lanes 2, 4, 6, and 8) or phosphatase reaction buffer (lanes 1, 3, 5, and 7).

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