Identification of casein kinase I substrates by in vitro expression cloning screening

Casein kinase I (CKI) is a widely expressed protein kinase family implicate d in diverse processes including membrane trafficking, DNA repair, and circ adian rhythm. Despite the large number of CRI genes, few biologically relev ant substrates have been identified. As an approach to better defining the spectrum of CKI substrates, we extended a recently described in vitro expre ssion cloning (IVEC) strategy. Polypeptides pools were screened for kinase- dependent electrophoretic mobility shifts. Ten putative CKI substrates were isolated from an initial sample of 3000 random cDNA clones. Candidate subs trates include proteins involved in RNA metabolism (a putative RNA helicase , the nucleolar protein hNOP56, and hnRNP Al, and ribosomal proteins L4, L8 , and L13), as well as keratin 17, a necdin-related protein, and the calciu m-binding proteins desmoglein 2 and annexin II. The same pools were also sc reened with active ERK2, and four substrates identified: aldolase, NSD-like protein, uracil-DNA glycosylase, and HHR23A. IVEC is an effective method t o identify novel protein kinase substrates, (C) 2000 Academic Press.