Selective blockade of axonogenesis in cultured hippocampal neurons by the tyrosine phosphatase inhibitor orthovanadate.

Protein tyrosine phosphorylation has been implicated in several aspects of neurite outgrowth regulation. To address specific roles in early neuronal morphogenesis, hippocampal neurons in culture were treated with the tyrosine phosphatase inhibitor orthovanadate. This treatment completely suppressed axon formation, yet enhanced formation of minor neurites. The inhibition of axonogenesis was dose dependent and occurred in parallel with a marked increase in cellular phosphotyrosine immunoreactivity, which was especially concentrated within neuritic growth cones and showed partial colocalization with f-actin. Both the blockade of axonogenesis and the elevation of phosphotyrosine were completely reversible. An additional and unexpected effect of orthovanadate was the appearance of many binucleate neurons. Immunoblotting experiments using a phosphotyrosine-specific antibody revealed an orthovanadate-induced reversible hyperphosphorylation of several protein bands, especially of two at 115 and 125 kD. These data suggest a potentially important role for tyrosine phosphatases and their phosphoprotein substrates in axonogenesis.