Identification of cellular substrate for CaMKII using unnatural amino acid incorporation

钙调素激酶II（CaMKII）在学习记忆中起重要作用。虽然突触的可塑性受CaMK Ⅱ的调节，但CaMK Ⅱ的直接靶点还不清楚。为了获得细胞CaMKII底物，我们尝试应用非天然氨基酸掺入法。在CaMKII的催化结构域中的几个苯丙氨酸残基用于随后掺入非天然氨基酸pBpa。在HEK293细胞中，pBpa成功地掺入到CaMK II上。在表达CaMKII突变体，特别是残基F16、F157、F171的细胞中，在UV照射后形成共价键。这些突变体是鉴定CaMK II的细胞底物的有用分子。

It is widely accepted that Calmodulin-kinase II (CaMKII) plays important roles in learning and memory. Although plasticity of synapse regulated by CaMKII, direct targets of CaMKII has poorly identified. To obtain cellular CaMKII substrates, we tried to apply unnatural amino acid incorporation method. Several phenylalanine residues in catalytic domain of CaMKII were made for subsequent incorporation of an unnatural amino acid pBpa. The pBpa were successfully incorporated on CaMKII in HEK293 cells. The covalent bonds were formed upon UV irradiation in cells expressing CaMKII mutants, especially residues, F16, F157, F171. This mutants are useful molecules to identify cellular substrate for CaMKII.