Studies on the substrate specificity of Ca2+/calmodulin-dependent protein kinase kinase alpha.
Ca2+/calmodulin-dependent protein kinase (CaM-kinase) kinase a, which is known to activate CaM-kinases IV and I by phosphorylation of Thr196 and Thr177, respectively, can only phosphorylate Thr196 among many phosphorylation sites of CaM-kinase IV [Kitani, T., Okuno, S., and Fujisawa, H. (1997) J. Biochem. 121, 804-810], indicating its high degree of substrate specificity. In the present study, the substrate specificity of CaM-kinase kinase a was examined using various proteins and synthetic peptides as substrates as a means to address its physiological function. Among a number of proteins and synthetic peptides, including several known as good substrates for various protein kinases, only CaM-kinases IV and I and peptides containing the sequence surrounding Thr196 of CaM-kinase IV or Thr177 of CaM-kinase I were significantly phosphorylated by CaM-kinase kinase alpha, while the heat-denatured (at 60 degrees C for 5 min) CaM-kinases IV and I were not phosphorylated. Peptides containing the phosphorylation site of CaM-kinase IV or I were far less active as substrates for CaM-kinase kinase a than were native CaM-kinase IV or I. Thus, CaM-kinase kinase a showed a high degree of substrate specificity, recognizing not only specific amino acid sequences but also the native conformation of CaM-kinases IV and I.
Okuno
S
S
Department of Biochemistry, Asahikawa Medical College.
Kitani
T
T
Fujisawa
H
H
D000255:Adenosine Triphosphate; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002457:Cell Extracts; D002540:Cerebral Cortex / Q000201:enzymology; D004789:Enzyme Activation; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
D002457:Cell Extracts; D009419:Nerve Tissue Proteins; D010455:Peptides; D013912:Threonine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517599:Camk1 protein, rat; C517686:Camk4 protein, rat; C517701:Camkk1 protein, rat; C517682:Pnck protein, rat; D002118:Calcium