Connexin 43 in LA-25 cells with active v-src is phosphorylated on Y247, Y265, S262, S279/282, and S368 via multiple signaling pathways

Abstract

Modulation of gap junction structures and gap junctional communication is important in maintaining tissue homeostasis and can be controlled via phosphorylation of connexin 43 (Cx43) through several different signaling pathways. Transformation of cells by v-src has been shown to down-regulate gap junction communication coincident with an increase in tyrosine phosphorylation on Cx43. Activation of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) also lead to down-regulation via phosphorylation on specific serine residues. Using phosphospecific anti-Cx43 antibodies generated by the authors' laboratory to specific tyrosines (src substrates) and serine residues (MAPK and PKC substrates) to probe LA-25 cells (which express temperature-sensitive v-src), the authors show that distinct tyrosine and serines residues are phosphorylated in response to v-src activity. They show that tyrosine phosphorylation appears to occur predominantly in gap junction plaques when src is active. In addition, src activation led to increased phosphorylation of apparent MAPK and PKC sites in Cx43. These results indicate all three signaling pathways could contribute to gap junction down-regulation during src transformation in LA-25 cells.

Figure 1. Phosphotyrosine antibodies are specific for Cx43 phosphorylated on Y247 or Y265

Immunoblot analysis of LA-25 cell lysates grown at the non-permissive (40) or permissive (35) temperature. Replicate immunoblots were probed simultaneously with a monoclonal antibody (NT1) to Total Cx43 (upper panel) and antibody against Cx43 phosphorylated on Y247 (Ab pY247) or Y265 (Ab pY265) (lower panel) as indicated. Antibodies were co-incubated with immunizing peptides (Pt Y247 and Pt Y265) as indicated to demonstrate the specificity of each phosphospecific antibody. Note that antibody binding in the western blots was detected via fluorescence with the Odyssey imaging system allowing simultaneous detection of the phosphospecific and total Cx43 antibodies resulting in a white on black image.

Figure 2. pY247 and pY265 antibodies recognize Cx43 in a subset of gap junction plaques

Immunofluorescence was performed on LA-25 cells at the permissive (35°C) or non-permissive temperature (40°C). Large panels show a field of cells labeled with antibody to total Cx43 in green and antibodies to phosphotyrosine 247 (pY247) or 265 (pY265) in red, overlay (Ovl) is seen as yellow. Small panels to the right show a magnified view of boxed areas with individual channels separated to highlight the co-localization of the Cx43 in plaques (magnification bar=25μm).

Figure 3. pS279/282 specifically recognizes Cx43 phosphorylated on S279/282 in response to PDGF, EGF or TPA stimulation

Upper panel- Immunoblot analysis of CHO cell lysates after 10 min treatment with PDGF, EGF or TPA (all 10 ng/ml). Blots were probed with antibody to total Cx43 (NT1 signal) and pS279/282 (pS279/282 signal). Lower panel- MDCK cells were transfected with wild type Cx43 (WT43) or Cx43 containing serine to alanine mutations at S279, S282 or both (S279A, S282A and S279/282A, respectively). Blots were probed with antibody to total Cx43 (NT1 signal) or pS279/282 (pS279/282 signal). Signals were quantified and ratios of pS279/282 to total Cx43 were calculated (graph, lower panel). Error bars depict standard error and differences between WT43 and all 3 mutants were statistically significant (Student’s t-test p-value<0.0001, n=3)

Figure 4. Active v-src leads to increased phosphorylation on specific tyrosine and serine residues in Cx43

Immunoblot analysis of LA-25 cells grown at the permissive (35) and non-permissive (40) temperature (Temp). blots were probed with antibodies to total Cx43 (NT1) and a series of phospho-specific antibodies. Upper panels - NT1 and phosphotyrosine antibodies pY247 (A) and pY265 (B), CT1, recognizing unmodified S364/365 (C) and phosphorylated S262 (pS262) (D). Lower panels-NT1 and antibodies recognizing phosphorylated S279/282 (pS279) (E) and S368 (pS368) (F). Signals were quantified and the ratio of phosphospecific antibodies to total Cx43 were calculated (G). Differences between permissive and non-permissive temperature was significant for both the pY247 and pY265 antibodies (Student’s t-test p-value<0.001, n=3).

Figure 5. Model of Cx43 with phosphorylated residues and SH3 binding domains indicated

(modified from (Lampe and Lau 2000). Residues to which phospho-specific antibodies have been made are shown in large bold print. Other serine residues are circled. A SH3 binding domain involved in v-src interaction is noted (Kanemitsu et al. 1997).