Site and consequences of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase type "Gr".
CaM kinase-Gr is a Ca2+/calmodulin-dependent protein kinase that is enriched in brain and thymus. The enzyme was isolated from rat cerebellum, which contained alpha (M(r) 65,000) and beta (M(r) 67,000) polypeptides, and rat forebrain, which contained only the alpha polypeptide. Both enzyme preparations readily underwent autophosphorylation with dramatic up-regulation of their Ca2+/calmodulin-dependent, as well as-independent, 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 mapping indicated that the alpha and beta polypeptides were phosphorylated on identical or homologous sites, which probably induces similar structural and catalytic modifications in the two polypeptides. The actual site(s) of autophosphorylation was determined by the purification and amino acid sequencing of tryptic peptides from 32P-labeled CaM kinase-Gr. The major site of autophosphorylation was localized to a novel N-terminal domain, which is rich in Ser/Thr/Pro residues. The functional and structural studies on CaM kinase-Gr autophosphorylation imply that the enzyme is comprised of two regulatory domains, one on either side of a catalytic domain, followed by a C-terminal, putative association domain. The properties of such a structural model are discussed.
McDonald
O B
OB
Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Merrill
B M
BM
Bland
M M
MM
Taylor
L C
LC
Sahyoun
N
N
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002531:Cerebellum / Q000201:enzymology*; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D016548:Prosencephalon / Q000201:enzymology; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014357:Trypsin
D007527:Isoenzymes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin; D003488:Cyanogen Bromide