CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction
CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction
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DOI：
10.1074/jbc.m303576200
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发表时间：
2003-11-07
影响因子：
4.8
通讯作者：
Di Luca, M
中科院分区：
文献类型：
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作者：
Gardoni, F;Mauceri, D;Di Luca, M
突触相关蛋白97（Synapse-associated protein 97，SAP 97）是膜相关鸟苷酸激酶蛋白家族的成员，参与靶向突触后离子型谷氨酸受体的过程。在这里，我们表明，SAP 97是丰富的突触后密度，它与离子型谷氨酸受体和下游信号蛋白，如Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）共定位。SAP 97和alphaCaMKII在海马神经元以及转染的COS-7细胞中显示出高度共定位模式。海马培养物的代谢标记显示，N-甲基-D-天冬氨酸（NMDA）受体活化诱导SAP 97的CaMKII依赖性磷酸化;与CaMKII特异性抑制剂KN-93共孵育可将SAP 97磷酸化降低至基础水平。我们的研究结果表明，SAP 97直接相互作用与NMDA受体的NR 2A亚基在体外“拔出”试验和免疫共沉淀实验从匀浆和突触体纯化海马大鼠组织。有趣的是，在突触后密度级分中，SAP 97不能与NR 2A共沉淀。我们发现SAP 97通过其PDZ 1结构域与NR 2A直接相关，并且SAP 97-Ser-232的CaMK II依赖性磷酸化在体外拔出试验和转染的COS-7细胞中破坏NR 2A相互作用。此外，SAP 97（S232 D）突变体的表达具有与组成型激活CaMK II时观察到的那些类似的作用。我们的研究结果表明，SAP 97/NR 2A相互作用的调节CaMKII依赖的磷酸化，并提供了一种新的机制，调节突触靶向的NMDA受体亚基。
Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in coimmunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.