δ Ca2+/calmodulin-dependent protein kinase II isozyme-specific induction of neurite outgrowth in P19 embryonal carcinoma cells
δ Ca2+/calmodulin-dependent protein kinase II isozyme-specific induction of neurite outgrowth in P19 embryonal carcinoma cells
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DOI：
10.1046/j.1471-4159.2000.0752380.x
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发表时间：
2000-12-01
影响因子：
4.7
通讯作者：
Tombes, RM
中科院分区：
文献类型：
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作者：
Johnson, LD;Willoughby, CA;Tombes, RM
Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMK-II）与前神经元细胞的分化诱导有关。在这些细胞中，δ同工酶代表大多数表达的CaMK-Ⅱ，并被分化刺激激活。为了确定δ CaMK-II是否与体外分化有关或一致，我们使用高效转染在小鼠P19和NIH/3 T3细胞中过表达δ和γ CaMK-II同工酶的野生型、组成型活性和C-末端结构域。在转染后1-2天，只有组成型活性δ CaMK-II同工酶在两种细胞类型中诱导分支细胞延伸。在P19细胞中，视黄酸诱导神经突起延伸3-4天后，这些扩展与内源性CaMK-II活性增加四倍。由视黄酸和δ CaMK-II诱导的延伸包含不连续或珠状模式的III类β-微管蛋白。C-末端CaMK-II结构破坏了内源性CaMK-II自磷酸化的能力，并阻断了视黄酸诱导的分化。发现δ CaMK-II沿着延伸，而γ CaMK-II表现出更弥散的胞质定位。这些数据不仅支持CaMK-II在促进神经突生长中的核内作用，而且还证明了CaMK-II同工酶在神经元分化的这些早期步骤中的特异性。
Ca2+/calmodulin-dependent protein kinase II (CaMK-II) has been linked to the induction of differentiation in preneuronal cells. In these cells, delta isozymes represent the majority of CaMK-IIs expressed and are activated by differentiation stimuli. To determine whether delta CaMK-IIs are causative or coincident with in vitro differentiation, we overexpressed wild-type, constitutively active, and C-terminal domains of delta and gamma CaMK-II isozymes in mouse P19 and NIH/3T3 cells using high-efficiency transfections. At 1-2 days after transfection, only constitutively active delta 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 delta CaMK-IIs contained class III beta -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. delta CaMK-II was found along extensions, whereas gamma 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.