Ph.D. - Biomedical Sciences (Physiology - Cell, Molecular and Neuro Sciences)

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    Characterization of epidermal growth factor-induced disruption of gap junctional intercellular communication and connexin43 phosphorylation
    ( 1993) Kanemitsu, Martha Yasuko
    Binding of a growth factor such as epidermal growth factor (EGF), to its specific cell surface receptor results in numerous intracellular responses, including increased DNA synthesis, regulation of epithelial proliferation and differentiation. The events linking EGF-induced receptor activation to these responses are still largely unknown. My work shows that EGF treatment of T51 B rat liver epithelial cells resulted in a rapid and transient disruption of gap junctional communication (gjc). EGF also stimulated the activation of its receptor tyrosine kinase in a time-dependent manner which occurred prior to the disruption of gjc. These results suggested that EGF receptor activation may be the first of a cascade of events mediating the disruption of gjc. EGF's ability to disrupt gjc was not due to any gross alterations in gap junctional plaques or cell-cell contacts between adjacent cells. Coincident with the disruption of gjc was the serine phosphorylation of connexin43 (Cx43). In addition to this kinetic similarity, EGF's effects were dose-dependent which also suggested that a functional relationship may exist. This hypothesis was further substantiated when okadaic acid, a serine/threonine phosphatase inhibitor prevented the dephosphorylation and concomitant restoration of gjc. Therefore, it appeared that EGF-induced disruption of gjc functionally depends on the post-translational serine phosphorylation of Cx43. While the signaling cascade(s) mediating EGF's effects has not yet been defined, down-modulation of TPA-sensitive PKC activity did not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43. This suggested that TPA-sensitive PKC may not be required. EGF, in the presence or absence of chronic TPA treatment, stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. It is possible that Cx43 may be phosphorylated and/or regulated by multiple serine kinases. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. Thus, MAP kinase may mediate, in part, EGF's ability to disrupt gjc and phosphorylate Cx43. EGF stimulated the tyrosine phosphorylation and activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 in vitro. Two-dimensional tryptic phosphopeptide mapping demonstrated that the in vitro tryptic phosphopeptides represented a specific subset of the in vivo tryptic phosphopeptides produced in response to EGF following chronic TPA treatment. Therefore, EGF-induced disruption of gjc may be a functional consequence of Cx43 phosphorylation.