Increased MAP1B expression without increased phosphorylation in manganese-treated PC12(Mn) cells

Regulation of MAP1B expression and phosphorylation is thought to play an im portant role in neuronal development, particularly with regard to axon grow th. The present work utilizes a novel PC12 cell variant [26] which exhibits many of the early morphological features of neurite outgrowth when stimula ted with manganese chloride. Expression of MAP1B was determined by immunobl ots and phosphorylation was assessed by metabolic radiolabeling with [P-32] orthophosphate or with a phospho-specific antibody. The results indicate th at MAP1B protein levels rise within 12 to 24 h, but there is no significant change in the phosphorylation of MAP1B. The latter conclusion is based on (i) experiments utilizing SMI 31, a monoclonal antibody that specifically r eacts with phospho-MAP1B and (ii) assessments of both MAP1B phosphorylation and MAP1B protein within that same isloated protein band on Western blots. Thus, manganese increases MAP1B expression without affecting its relative phosphorylation. Although manganese does not cause neurite formation in the parental PC12 cell line, manganese is capable of inducing transient neurit e regeneration from NG;F-primed cells. These studies provide further eviden ce that the onset of neurite outgrowth may proceed without increased phosph orylation of MAP1B. During sustained neurite regeneration, however, NGF inc reases phosphate incorporation into MAP1B. Based on all of these findings, we conclude that early phases of neurite outgrowth (cell spreading and form ation of short tapered extensions) do not necessarily require elevated phos phorylation of MAP1B. (C) 1998 Academic Press.