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

Abstract

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...

PCP4 (PEP19) overexpression induces premature neuronal differentiation associated with Ca2+/Calmodulin‐Dependent kinase II‐δ activation in mouse models of down syndrome

“…The CaMKIIc antibody was raised against a peptide that is totally conserved across species. This antibody recognized a single band at 61 kDa on Western blot of E11.5 proteins as expected from a previous report on this CaMKII isoform in mouse embryonic cells (Johnson et al, 2000).…”

“…Interestingly, in a model of P19 differentiation based on transfection with different CaMKII isoforms, CaMKIIc isoforms were found to have a diffuse cytosolic distribution and to be absent from cellular extensions, whereas CaMKIId isoforms had a more filamentous cytoskeletal distribution and promoted neurite outgrowth when overproduced (Johnson et al, 2000;Caran et al, 2001). In the P19 model, CaMKIId has a higher capacity for autophosphorylation during retinoic acid-induced differentiation and is associated with an increase in bIII-tubulin levels that may result from direct association between CaMKIId and bIIItubulin (Johnson et al, 2000). Thus, in vivo, the higher levels of bIII-tubulin at E11.5 in TgPCP4 and Ts1Cje mice may result partly from activation of CaMKIId, via pcp4-induced Ca 2þ -CaM activation.…”

The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry

“…3A). K 43 A CaMK-II is not gene specific, as it can hetero-oligomerize with any endogenous CaMK-II and decrease inter-subunit autophosphorylation (Johnson et al, 2000). Targeted K 43 A CaMK-II induced fewer defects than did CaMK-II MOs, which is consistent with an endodermal/KV-specific role for CaMK-II in asymmetry, in addition to its many other developmental roles.…”

CaMK‐II promotes focal adhesion turnover and cell motility by inducing tyrosine dephosphorylation of FAK and paxillin

“…CaMK-II can be made constitutively active (CON) through a phosphomimetic point mutation (T 287 D) in the autoinhibitory domain [Johnson et al, 2000]. Cells which were transfected with GFP-tagged CON δ C CaMK-II, sub-cultured on FN-coated coverslips and imaged using Fm and TIRFm exhibited a rounded morphology (Fig.…”