Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation
Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation
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DOI：
10.1074/jbc.m610541200
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发表时间：
2007-01-19
影响因子：
4.8
通讯作者：
Hisanaga, Shin-ichi
中科院分区：
文献类型：
--
作者：
Kamei, Hirotsugu;Saito, Taro;Hisanaga, Shin-ichi
Cdk5 是一种脯氨酸导向的 Ser/Thr 蛋白激酶，与其激活剂 p35 一起主要在有丝分裂后神经元中表达。钙蛋白酶将 p35 N 端截短为 p25，导致 Cdk5 失调，并导致与多种神经退行性疾病相关的神经元细胞死亡。之前我们报道过p35作为磷蛋白出现，磷酸化p35水平随着神经元成熟而变化，并且p35的磷酸化影响其对钙蛋白酶裂解的脆弱性。在这里，我们确定 p35 残基 Ser(8) 和 Thr(138) 是 Cdk5 磷酸化的主要位点。将这些位点诱变为非磷酸化的丙氨酸，增加了培养细胞和神经元对钙蛋白酶的敏感性，同时将它们转变为拟磷酸化谷氨酸减弱的裂解。此外，针对这些位点的磷酸化状态特异性抗体显示，尽管 Ser(8) 和 Thr(138) 在产前大脑中均被磷酸化，但成年大鼠中的 Thr(138) 被去磷酸化。在培养的神经元中，抑制蛋白磷酸酶可将磷酸-Ser(8) p35 转化为双磷酸-Ser(8)/Thr(138) p35，并赋予对钙蛋白酶裂解的抗性。这些结果表明 Thr138 的磷酸化主要决定了 p35 对钙蛋白酶依赖性裂解的敏感性，并且该位点的去磷酸化是 Cdk5-p25 诱导的与神经变性相关的细胞死亡的关键决定因素。
Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser(8) and Thr(138) as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr(138) was dephosphorylated in adult rat, although both Ser(8) and Thr(138) were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser(8) p35 to dual phospho-Ser(8)/Thr(138) p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr138 predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration.