Abstract

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1d and CKle were previously shown to autophosphorylate carboxy-terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CKla and splice variant CKlaL has identified the autophosphorylation of the last four carboxyl-end serines and threonines and also for CKlaS, the same four residues plus threonine-327 and serine-332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy-terminal phosphorylation sites of CKla to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CKla and CKlaS with ? phosphatase causes an activation of 80-100% and 300%, respectively. Similar treatment fails to stimulate the CKla mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CKla and CKlaS. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CKla and CKlaS was also observed using the known CK1 protein substrates DARPP-32, ß-catenin, and CK2ß, which have different CK1 recognition sequences. Autophosphorylation effects on CKla activity are not due to changes in Kmapp for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CKla and its splice variants can be regulated by their autophosphorylation status. © 2008 Wiley-Liss, Inc.