Ca2؉ /calmodulin-dependent protein kinase II (CaMK-II) isozyme variability is the result of alternative usage of variable domain sequences. Isozyme expression is cell type-specific to transduce the appropriate Ca 2؉ signals. We have determined the subcellular targeting domain of ␦ E CaMK-II, an isozyme that induces neurite outgrowth, and of a structurally similar isozyme, ␥ C CaMK-II, which does not induce neurite outgrowth. ␦ E CaMK-II co-localizes with filamentous actin in the perinuclear region and in cellular extensions. In contrast, ␥ C CaMK-II is uniformly cytosolic. Constitutively active ␦ E CaMK-II induces F-actin-rich extensions, thereby supporting a functional role for its localization. C-terminal constructs, which lack central variable domain sequences, can oligomerize and localize like full-length ␦ E and ␥ C CaMK-II. Central variable domains themselves are monomeric and have no targeting capability. The C-terminal 95 residues of ␦ CaMK-II also has no targeting capability but can efficiently oligomerize. These findings define a targeting domain for ␥ and ␦ CaMK-IIs that is in between the central variable and association domains. This domain is responsible for the subcellular targeting differences between ␥ and ␦ CaMK-IIs.

Ca 2ϩ /calmodulin-dependent protein kinase II (CaMK-II) has been linked to the induction of differentiation in preneuronal cells. In these cells, ␦ isozymes represent the majority of CaMK-IIs expressed and are activated by differentiation stimuli. To determine whether ␦ CaMK-IIs are causative or coincident with in vitro differentiation, we overexpressed wild-type, constitutively active, and C-terminal domains of ␦ and ␥ CaMK-II isozymes in mouse P19 and NIH/3T3 cells using highefficiency transfections. At 1-2 days after transfection, only constitutively active ␦ 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 ␦ CaMK-IIs contained class III ␤-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. ␦ CaMK-II was found along extensions, whereas ␥ 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. Key Words: Ca 2ϩ /calmodulin-dependent protein kinase II-Isozyme -Neurites-P19 embryonal carcinoma cells-Retinoic acid-Green fluorescent protein. J. Neurochem. 75, 2380 -2391 (2000).Ca 2ϩ /calmodulin (CaM)-dependent protein kinase II (CaMK-II) is activated in cells induced to undergo differentiation (MacNicol et al., 1990;Tombes et al., 1999). Overexpression and inhibition studies have also linked CaMK-II with axon extension, guidance, and arborization (MacNicol et al., 1990;Kelly, 1991;Solem et al., 1995;VanBerkum and Goodman, 1995;Williams et al., 1995;Tashima et al., 1996;Massé and Kelly, 1997;Wang et al., 1997). It is also evident that the spectrum of CaMK-II isozymes is influenced by the state of cell growth and differentiation (Scholz et al., 1988;Brocke et al., 1995;Bayer et al., 1999;Tombes et al., 1999). Taken together, these findings suggest that neuronal differentiation is influenced by the enzymatic activity of specific CaMK-II isozymes.We have directly tested this hypothesis by overexpressing only those CaMK-II isozymes that had previously been identified in preneuronal cells (Tombes et al., 1999). Almost 30 different isozymes of mammalian CaMK-II are produced from the expression of only four genes (␣, ␤, ␥, and ␦). ␣ CaMK-II is expressed exclusively in fully differentiated brain tissue (Tobimatsu and Fujisawa, 1989;Bayer et al., 1999), whereas ␤, ␥, and ␦ CaMK-IIs are expressed in both neuronal and nonneuronal cell types (Nghiem et al., 1993;Edman and Schulman, 1994;Urquidi and Ashcroft, 1995;Singer et al., 1997). CaMK-II isozyme variability (50 -65 kDa) is the result of alternatively spliced domains found in the central variable region (Karls...