SITE AND CONSEQUENCES OF THE AUTOPHOSPHORYLATION OF CA(2-DEPENDENT PROTEIN-KINASE TYPE-GR() CALMODULIN)

CaM kinase-Gr is a Ca2+/calmodulin-dependent protein kinase that is en riched in brain and thymus. The enzyme was isolated from rat cerebellu m, which contained alpha (M(r) 65,000) and beta (M(r) 67,000) polypept ides, and rat forebrain, which contained only the alpha polypeptide. B oth enzyme preparations readily underwent autophosphorylation with dra matic up-regulation of their Ca2+/calmodulin-dependent, as well as -in dependent, activity. Autophosphorylation also caused a characteristic retardation in the electrophoretic gel mobility of the alpha and beta polypeptides. Treatment of autophosphorylated CaM kinase-Gr with acid phosphatase fully dephosphorylated the enzyme and reversed the changes in electrophoretic migration of both polypeptides. Phosphopeptide map ping indicated that the alpha and beta polypeptides were phosphorylate d on identical or homologous sites, which probably induces similar str uctural and catalytic modifications in the two polypeptides. The actua l site(s) of autophosphorylation was determined by the purification an d amino acid sequencing of tryptic peptides from P-32-labeled CaM kina se-Gr. The major site of autophosphorylation was localized to a novel N-terminal domain, which is rich in Ser/Thr/Pro residues. The function al and structural studies on CaM kinase-Gr autophosphorylation imply t hat the enzyme is comprised of two regulatory domains, one on either s ide of a catalytic domain, followed by a C-terminal, putative associat ion domain. The properties of such a structural model are discussed.