Signaling Effects of Menadione: From Tyrosine Phosphatase Inactivation to Connexin Phosphorylation

The chapter explains the methods used to delineate the preceding pathway leading from protein tyrosine phosphatase (PTPase) inhibition to connexin43 (Cx43) phosphorylation to deal with (1) receptor tyrosine kinase phosphorylation and PTPase inhibition, (2) extracellular signal-regulated kinases (ERK) activation and the use of pharmacological inhibitors, and (3) Cx43 phosphorylation and gap junctional communication (GJC). These methods are helpful in analyzing menadione-induced signaling pathways in rat liver epithelial cells. The chapter shows that the exposure of cells to menadione leads to the activation of a signaling pathway that results in the activation of ERK 1/2, entailing the phosphorylation of Cx43 and a decrease in gap junctional intercellular communication in rat liver epithelial cells. These effects are brought about by the activation of the EGFR, probably because of the inactivation of a not–yet-identified PTPase regulating the receptor. Menadione is a naphthoquinone derivative (2-methyl-1, 4-naphthoquinone) that is used clinically because of its vitamin K–like properties (it is also termed vitamin K3). It is enzymatically converted to menaquinone-4(2-methyl-3-geranyl-geranyl-1, 4-naphthoquinone, a form of vitamin K2) by mammals. Menadione is capable of inducing phosphorylation of ERK 1/2 and Cx43, resulting in attenuated GJC, which is reversed in the presence of inhibitors of MAPK/ERK kinase (MEK) 1 and MEK 2 (the kinases directly upstream of ERK 1/2) and the epidermal growth factor receptor (EGFR) tyrosine kinase.