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 CK1 delta and CK1 epsilon were previously shown to autophosphorylate carboxy-terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CK1 alpha and splice variant CK1 alpha L has identified the autophosphorylation of the last four carboxyl-end serines and threonines and also for CK1 alpha S, 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 CK1 alpha to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CK1 alpha and CK1 alpha S with X phosphatase causes an activation of 80-100% and 300%, respectively. Similar treatment fails to stimulate the CK1 alpha mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CK1 alpha and CK1 alpha S. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CK1 alpha and CK1 alpha S was also observed using the known CK1 protein substrates DARPP-32, beta-catenin, and CK2 beta, which have different CK1 recognition sequences. Autophosphorylation effects on CK1 alpha activity are not due to changes in Km(app) for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CK1 alpha and its splice variants can be regulated by their autophosphorylation status. J. Cell. Biochem. 106: 399-408, 2009. (C) 2008 Wiley-Liss, Inc.