δ Ca2+/Calmodulin-Dependent Protein Kinase IIIsozyme-Specific Induction of Neurite Outgrowth in P19 Embryonal Carcinoma Cells

Authors


  • Lippincott Williams & Wilkins, Inc., Philadelphia

  • Abbreviations used: ATRA, all-trans-retinoic acid; CaM,calmodulin; CaMK-II, Ca2+/calmodulin-dependent protein kinase II;EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein;TBST, Tris-buffered saline (pH 7.4) and 0.1% Tween 20; βIII tubulin,class III β-tubulin.

Address correspondence and reprint requests to Dr. R. M. Tombes at Departmentof Biology, Virginia Commonwealth University, Richmond, VA 23284-2012, U.S.A.E-mail:rtombes@hsc.vcu.edu

Abstract

Abstract: Ca2+/calmodulin-dependent protein kinase II(CaMK-II) has been linked to the induction of differentiation in preneuronalcells. In these cells, δ isozymes represent the majority of CaMK-IIsexpressed and are activated by differentiation stimuli. To determine whetherδ CaMK-IIs are causative or coincident with in vitro differentiation, weoverexpressed wild-type, constitutively active, and C-terminal domains ofδ and γ CaMK-II isozymes in mouse P19 and NIH/3T3 cells usinghigh-efficiency transfections. At 1-2 days after transfection, onlyconstitutively active δ CaMK-II isozymes induced branched cellularextensions in both cell types. In P19 cells, retinoic acid induced neuriteextensions after 3-4 days; these extensions were coincident with a fourfoldincrease in endogenous CaMK-II activity. Extensions induced by both retinoicacid and δ CaMK-IIs contained class III β-tubulin in adiscontinuous or beaded pattern. C-terminal CaMK-II constructs disrupted theability of endogenous CaMK-II to autophosphorylate and blocked retinoicacid-induced differentiation. δ 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 promotingneurite outgrowth, but also demonstrate CaMK-II isozyme specificity in theseearly steps of neuronal differentiation.

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