We have previously demonstrated that epidermal growth factor induced a rapid, transient decrease in gap junctional communication and increase in serine phosphorylation on the connexin-43 gap junction protein in T51B rat liver epithelial cells. The kinase(s) responsible for phosphorylation and specific serine targets in connexin-43 have not been identified. There are three consensus mitogen-activated protein (MAP) kinase serine phosphorylation sequences in the carboxyl-terminal tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitro on tryptic peptides that comigrated with a subset of peptides from connexin-43 phosphorylated in vivo in cells treated with epidermal growth factor. These data suggested that MAP kinase may phosphorylate connexin-43 directly in vivo. We have utilized a glutathione S-transferase fusion protein containing the cytoplasmic tail of connexin-43 to characterize MAP kinase phosphorylation. Site-directed mutagenesis, phosphotryptic peptide analysis, and peptide sequencing have confirmed that MAP kinase can phosphorylate connexin-43 at Ser255, Ser279, and Ser282, which correspond to the consensus sites recognized earlier. Characterization of MAP kinase-mediated phosphorylation of connexin-43 has defined potential targets for phosphorylation in vivo following activation of the epidermal growth factor receptor and has provided the basis for studies of the effects of phosphorylation, at specific molecular sites, on the regulation of gap junctional communication.

The ®broblast growth factor receptor (FGFR) family members mediate a number of important cellular processes, and are mutated or overexpressed in several forms of human cancer. Mutation of Lys650?Glu in the activation loop of the FGFR3 kinase domain causes the lethal human skeletal disorder thanatophoric dysplasia type II (TDII) and is also found in patients with multiple myeloma, bladder and cervical carcinomas. This mutation leads to constitutive activation of FGFR3. To compare the signaling activity of FGFR family members, this activating mutation was generated in FGFR1, FGFR3, and FGFR4. We show that the kinase domains of FGFR1, FGFR3, and FGFR4 containing the activation loop mutation, when targeted to the plasma membrane by a myristylation signal, can transform NIH3T3 cells and induce neurite outgrowth in PC12 cells. Phosphorylation of Shp2, PLC-g, and MAPK was also stimulated by all three`TDII-like' FGFR derivatives. Additionally, activation of Stat1 and Stat3 was observed in cells expressing the activated FGFR derivatives. Finally, we demonstrate that FGFR1, FGFR3, and FGFR4 derivatives can stimulate PI-3 kinase activity. Our comparison of these activated receptor derivatives reveals a signi®cant overlap in the panel of eector proteins used to mediate downstream signals. This also represents the ®rst demonstration that activation of FGFR4, in addition to FGFR1 and FGFR3, can induce cellular transformation. Moreover, our results suggest that Stat activation by FGFRs is important in their ability to act as oncogenes. Oncogene (2000) 19, 3309 ± 3320.

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)...

Growth factors regulate cellular proliferation and differentiation by activating plasma membrane tyrosine kinase receptors and triggering a cascade of events mediated by intracellular signaling proteins. The mechanism underlying growth factor modification of cellular functions, such as gap-junctional communication (gjc), has not been established clearly. Addition of epidermal growth factor (EGF) to T51B rat liver epithelial cells resulted in the rapid activation of EGF receptor tyrosine kinase activity followed by a transient dose-dependent disruption of gjc. This change did not result from the gross disturbance of membrane gap junction plaques as measured by immunofluorescence microscopy, but instead correlated with markedly elevated phosphorylation of the connexin43 (cx43) gap junction protein, a profound shift to predominantly phosphorylated forms of cx43, and the appearance of a novel phosphorylated cx43 protein. These changes in cx43 phosphorylation involved only serine residues. On restoration of gjc, these alterations in cx43 phosphorylation reverted to the pre-EGF treatment state. Both events were inhibited by the serine/threonine protein phosphatase inhibitor, okadaic acid. Therefore, unlike the case for pp60v-src, EGF-induced disruption of gjc is not associated with tyrosine phosphorylation of cx43, but instead may result from phosphorylation of cx43 by activated intracellular signaling serine protein kinase(s).