Analysis of calmodulin-kinase cascade by using functional proteomics.

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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