Identification of cellular substrate for CaMKII using unnatural amino acid incorporation

钙调素激酶II （Calmodulin-kinase II, CaMKII）在学习和记忆中起着重要的作用。虽然CaMKII调控突触的可塑性，但CaMKII的直接作用靶点尚未明确。为了获得细胞CaMKII底物，我们尝试采用非天然氨基酸掺入法。在CaMKII的催化结构域中制备了几个苯丙氨酸残基，用于随后掺入非天然氨基酸pBpa。pBpa成功地结合到HEK293细胞的CaMKII上。在表达CaMKII突变体的细胞中，特别是残基F16、F157、F171，在紫外线照射下形成共价键。这些突变体是鉴定CaMKII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.