Features of calmodulin that are important in the activation of the catalytic subunit of phosphorylase kinase.
Calmodulin (CaM) is an integral subunit, called delta, of the phosphorylase kinase hexadecamer, and the activity of the isolated catalytic gamma-subunit of the kinase is stimulated by CaM. We report here the first analysis of functionally important features of CaM for activation of the gamma-subunit. A set of genetically engineered CaMs, in which acidic residues in each of the four E-helices of the "EF-hands" were changed to basic lysine residues, was used to probe the relative importance of charge features in each domain of CaM. The maximal activation of the isolated gamma-subunit was diminished by all of the charge reversal mutations. The gamma-subunit was especially sensitive to reversals in the second and third E-helix of CaM (residues 45-47 and 82-84), the latter being present in the central helix. The results suggest the functional importance of electrostatics in the interactions between the delta-subunit (CaM) and the catalytic gamma-subunit of phosphorylase kinase, which is similar to results obtained with CaM-dependent myosin light chain kinase (MLCK) from chicken gizzard and CaM-dependent protein kinase II (CaMPK-II). However, novel features of the interaction between CaM and the gamma-subunit of phosphorylase kinase are the significant contribution of electrostatics throughout the CaM molecule, including residues in both halves and on more than one face of CaM, and the lack of a major effect of the CaM mutations on substrate kinetic parameters, unlike the effects observed with MLCK and CaMPK-II. These results are consistent with a model in which the delta-subunit (CaM) of phosphorylase kinase interacts with an extended region or multiple regions of the gamma-subunit and suggest that the mechanism of CaM activation of the gamma-subunit may have features that are distinct from those of MLCK and CaMPK-II.
Farrar
Y J
YJ
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163.
Lukas
T J
TJ
Craig
T A
TA
Watterson
D M
DM
Carlson
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
D002147:Calmodulin; D010455:Peptides; D010764:Phosphorylase Kinase