Tyrosine Phosphorylation of Connexin 43 by v-Src Is Mediated by SH2 and SH3 Domain Interactions

Abstract

Reduction of gap junctional communication in v-The Rous sarcoma virus transforming protein, v-Src, and its cellular homologue, c-Src, are protein tyrosine kinases associated with the plasma membrane (1). Expression of v-Src leads to neoplastic cell transformation, accompanied by increased tyrosine phosphorylation of cellular proteins (2-4). Signaling through Src is due, in part, to protein-protein interactions mediated by SH2 1 and SH3 domains (5-7). Mutations in either the SH2 or SH3 domain of c-Src can lead to increased tyrosine kinase activity and oncogenic potential (5,8,9). SH2 domains bind Tyr(P) residues in target proteins. The specificity of binding is determined by residues immediately C-terminal to the Tyr(P) (6, 10, 11). SH3 domains bind short proline-rich peptide motifs (6,(12)(13)(14). Studies using phage display libraries to identify peptides that bind SH3 domains showed that a minimal PXXP consensus sequence is required for binding (15,16).In addition to playing a role in cell growth and transformation, the Src protein tyrosine kinase has been implicated in regulating GJC (17)(18)(19)(20). Gap junctions are membrane channels which mediate the intercellular passage of ions, second messengers, and small molecules (21). It has been proposed that growth regulatory molecules pass between cells through gap junctions (22)(23)(24)(25).Gap junctions are formed by specialized proteins termed connexins, arranged in the cell membrane so that each connexin has four membrane spanning regions, a cytoplasmic loop, two extracellular loops, and cytoplasmic N-and C-terminal ends. Among the 13 connexin family members identified to date, the C-terminal tail is the most divergent region. In some cases this region contains consensus protein kinase phosphorylation sites (26 -28). Connexins 32 and 43 are phosphoproteins (29 -32), however, Cx43 is phosphorylated on tyrosine by Src, but Cx32 is not (32). Comparison of the amino acid sequences of connexins 32 and 43 revealed that putative SH3-binding regions and tyrosine phosphorylation sites in Cx43 were absent in connexin 32 (26).Reduced GJC is a characteristic of cells transformed by several oncogenes (24), including SV-40 (33), polyomavirus middle T antigen (34), v-ras (35, 36), v-mos (37), and v-fps (38), as well as v-src. The effects of Src on GJC have been studied extensively (17-20, 30, 32, 39 -41). Several lines of evidence suggest that tyrosine phosphorylation of Cx43 is important in the regulation of GJC by v-Src. First, cells infected with temperaturesensitive Rous sarcoma virus show reduced levels of GJC (17,19), correlated with a rapid increase in tyrosine phosphorylation of Cx43 (40), at the permissive temperature. Second, expression of v-Src in communication-competent paired Xenopus oocytes expressing Cx43, but not in oocytes expressing Cx32, leads to reduced GJC, which depends on phosphorylation of Cx43 on tyrosine 265 (32). Finally, purified Src phosphorylates Cx43 in vitro at sites that are phosphorylated in vivo in v-src transformed Rat-1 fibroblasts (41)...

Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1

“…Previous studies have indicated that the SH3 domain of activated Src binds the CT domain of Cx43 near Pro-280 (44,45). Our data indicate that Src SH3 binding induces major structural changes in Cx43CT region Lys-264 -Lys-287, which extends upstream to the SH2 binding domain; presumably, these are the structural changes necessary for Tyr-265 phosphorylation.…”

“…3). Only the region Lys-264 -Lys-287 contains the amino acids previously reported to act as an SH3 binding domain (43)(44)(45). Although all changes were consistent with SH3 binding and altering the structure of Cx43CT, the resonance peaks were affected in three different ways.…”

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells

“…By use of mutant src genes, several cellular proteins have been described as transformation-relevant substrates of v-Src kinase. These include; b-catenin (Hamaguchi et al, 1993a); glycoproteins of 95 kDa and 130 kDa (Hamaguchi et al, 1990;Kozuma et al, 1990); connexin-43 (Crow et al, 1992;Kanemitsu et al, 1997); 120-kDa protein (Linder and Burr, 1988). Thus, mutant src genes have been utilized as an e cient tool to characterize substrate proteins.…”

v-Src SH3-enhanced Interaction with Focal Adhesion Kinase at β1 Integrin-containing Invadopodia Promotes Cell Invasion

“…Substitution of murine c-Src RT loop residues (Trp at Arg-97 and Ile at Thr-98) did not disrupt the binding of normal c-Src SH3 targets such as p130Cas (6). Instead, RT loop residue changes at Trp-97 promoted the binding of proteins such as connexin 43 and FAK to the c-Src SH3 domain (6,7). In FAK, the v-Src SH3 domain binding sites were mapped to three proline-rich motifs conforming to a PXXPXX consensus where is a hydrophobic residue (6).…”

“…In FAK, the v-Src SH3 domain binding sites were mapped to three proline-rich motifs conforming to a PXXPXX consensus where is a hydrophobic residue (6). This extended PXXPXX motif differs from c-Src class I or class II SH3 binding motifs (5) and is conserved in other v-Src SH3 domain-binding proteins (7,8). Since the v-Src SH3 domain binds to additional targets compared with the c-Src SH3 domain, the RT loop substitutions can be considered gain-of-function mutations.…”