Brain-derived neurotrophic factor and epidermal growth factor activate neuronal m-calpain via mitogen-activated protein kinase-dependent phosphorylation.
Brain-derived neurotrophic factor and epidermal growth factor activate neuronal m-calpain via mitogen-activated protein kinase-dependent phosphorylation.
复制标题
DOI：
10.1523/jneurosci.5120-09.2010
复制
发表时间：
2010-01-20
期刊：
影响因子：
--
通讯作者：
Baudry M
中科院分区：
文献类型：
--
作者：
Zadran S;Jourdi H;Rostamiani K;Qin Q;Bi X;Baudry M
钙蛋白酶是一种钙依赖性蛋白酶，在突触可塑性、细胞运动和神经变性中起重要作用。脑内存在两种主要的钙蛋白酶同工异构体，μ-钙蛋白酶（calpain1）需要微摩尔钙浓度激活，而m-钙蛋白酶（calpain2）需要毫摩尔钙浓度激活。最近对成纤维细胞的研究表明，表皮生长因子（EGF）可以通过丝裂原活化蛋白激酶（MAPK）介导的磷酸化，独立于钙激活m-calpain。在神经元中，MAPK被脑源性神经营养因子（BDNF）和EGF激活。因此，我们使用培养的原代神经元和HEK-TrkB细胞，研究了这些生长因子是否可以通过mapk依赖性磷酸化激活m-calpain，这两种细胞都表达BDNF和EGF受体。通过光谱降解的定量分析和基于FRET的检测来监测Calpain的激活，该检测评估了由FRET荧光团对、DABCYL和EDANS组成的Calpain特异性肽的截断。在这两种细胞类型中，BDNF和EGF都能迅速引发calpain激活，而MAPK和calpain抑制剂能完全阻断这一激活。BDNF刺激m-calpain丝氨酸磷酸化，但不刺激μ-calpain丝氨酸磷酸化，这一作用也被MAPK抑制剂阻断。值得注意的是，BDNF-和egf诱导的calpain激活优先定位于海马神经元的树突和树突棘，并与肌动蛋白聚合相关，而肌动蛋白聚合被calpain抑制所阻止。我们的研究结果表明，在培养的神经元中，BDNF和EGF都通过mapk介导的磷酸化激活m-calpain。这些结果有力地支持了钙蛋白酶在突触可塑性中的作用，并解释了为什么m-钙蛋白酶在中枢神经系统中广泛表达，却需要非生理性钙水平才能激活。
Calpain is a calcium-dependent protease that plays a significant role in synaptic plasticity, cell motility and neurodegeneration. Two major calpain isoforms are present in brain, with μ-calpain (calpain1) requiring micromolar calcium concentrations for activation while m-calpain (calpain2) needs millimolar concentrations. Recent studies in fibroblasts indicate that epidermal growth factor (EGF) can activate m-calpain independently of calcium via mitogen-activated protein kinase (MAPK)-mediated phosphorylation. In neurons, MAPK is activated by both brain-derived neurotrophic factor (BDNF) and EGF. We therefore examined whether these growth factors could activate m-calpain by MAPK-dependent phosphorylation using cultured primary neurons and HEK-TrkB cells, both of which express BDNF and EGF receptors. Calpain activation was monitored by quantitative analysis of spectrin degradation and by a FRET-based assay, which assessed the truncation of a calpain-specific peptide flanked by the FRET fluorophore pair, DABCYL and EDANS. In both cell types, BDNF and EGF rapidly elicited calpain activation, which was completely blocked by MAPK and calpain inhibitors. BDNF stimulated m-calpain but not μ-calpain serine phosphorylation, an effect also blocked by MAPK inhibitors. Remarkably, BDNF- and EGF-induced calpain activation was preferentially localized in dendrites and dendritic spines of hippocampal neurons, and was associated with actin polymerization, which was prevented by calpain inhibition. Our results indicate that in cultured neurons, both BDNF and EGF activate m-calpain by MAPK-mediated phosphorylation. These results strongly support a role for calpain in synaptic plasticity, and may explain why m-calpain, while widely expressed in CNS, requires non-physiological calcium levels for activation.