Analysis of calmodulin-kinase cascade by using functional proteomics.

为了寻找Ca^<2+>/钙调素依赖性蛋白激酶I （CaM-KI）的底物，我们使用未磷酸化或磷酸化（在Thr^<177>） gst融合的CaM-KI催化结构域（残基1-293，Lys^<49>Glu）作为亲和配体进行亲和层析纯化。然后进行蛋白质组学分析以确定相互作用的蛋白质。除了检测两种已知的CaM-KI底物（CREB和synapsin I）外，我们还从大鼠组织中鉴定了两种Numb家族蛋白（Numb和Numbl）。这些蛋白未被磷酸化，仅与Thr^<177>-磷酸化的CaM-KI催化结构域结合。这一发现与在体外被活化的CaM-KI和另外两种CaM-Ks （CaM-KII和CaM-KIV）在等效Ser残基（Numb中的Ser^<264>和Numbl中的Ser^<304>）有效和化学计量磷酸化的结果一致。利用抗phosphonumb /Numbl抗体，我们在不同的大鼠组织提取物中观察到…More Numb家族蛋白的磷酸化，并在COS-7细胞中检测到离子霉素诱导的内源性Numb Ser^<264>位点的磷酸化。目前的研究结果表明，Numb家族蛋白在体内和体外都被磷酸化。此外，我们发现14-3-3蛋白的募集是Numb家族蛋白磷酸化的功能结果。14-3-3蛋白与磷酸化的Numbl相互作用阻断Ser^<304>的去磷酸化。Numb被认为通过直接与网格蛋白相关的接头复合物AP-2相互作用参与网格蛋白依赖的内吞作用，尽管潜在的机制尚不清楚。下拉实验表明，Numb的磷酸化破坏了其与AP-2复合物的结合，同时在体外募集14-3-3蛋白。根据对Numb突变体的实验，初始磷酸化Ser^<264>和随后磷酸化Ser^<283>都足以消除Numb与AP-2的结合，并促进与14-3-3蛋白的相互作用。这些发现提示了一种新的机制来调节麻木介导的内吞作用，即通过直接磷酸化。少

To search for the substrates of Ca^<2+>/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr^<177>) GST-fused CaM-KI catalytic domain (residues 1-293, Lys^<49>Glu) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr^<177>-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser^<264> in Numb and Ser^<304> in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phosphoNumb/Numbl antibody, we observed the phosphorylation of … More Numb family proteins in various rat tissue extracts and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser^<264> in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl blocked dephosphorylation of Ser^<304>. Numb is thought to participate in clathrin-dependent endocytosis by directly interacting with the clathrin-associated adaptor complex AP-2, although the underlying mechanisms are unknown. Pull-down experiments showed that the phosphorylation of Numb impaired its binding to the AP-2 complex and simultaneously recruited 14-3-3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser^<264> and the subsequent phosphorylation of Ser^<283> are sufficient to abolish the binding of Numb to AP-2 and to promote the interaction with 14-3-3 protein. These findings suggest a novel mechanism for the regulation of Numb-mediated endocytosis, namely through direct phosphorylation. Less