δ Ca2+/calmodulin-dependent protein kinase II isozyme-specific induction of neurite outgrowth in P19 embryonal carcinoma cells

Abstract

Ca2+/calmodulin-dependent protein kinase II (CaMK-II) has been linked to the induction of differentiation in preneuronal cells. In these cells, δ isozymes represent the majority of CaMK-IIs expressed and are activated by differentiation stimuli. To determine whether and CaMK-IIs are causative or coincident with in vitro differentiation, we overexpressed wild-type, constitutively active, and C-terminal domains of δ and γ CaMK-II isozymes in mouse P19 and NIH/3T3 cells using highefficiency transfections. At 1-2 days after transfection, only constitutively active and CaMK-II isozymes induced branched cellular extensions in both cell types. In P19 cells, retinoic acid induced neurite extensions after 3-4 days; these extensions were coincident with a fourfold increase in endogenous CaMK-II activity. Extensions induced by both retinoic acid and and CaMK-IIs contained class III β-tubulin in a discontinuous or beaded pattern. C-terminal CaMK-II constructs disrupted the ability of endogenous CaMK-II to autophosphorylate and blocked retinoic acid-induced differentiation. and CaMK-II was found along extensions, whereas γ CaMK-II exhibited a more diffuse, cytosolic localization. These data not only support an extranuclear role for CaMK-II in promoting neurite outgrowth, but also demonstrate CaMK-II isozyme specificity in these early steps of neuronal differentiation.