Genetically engineered calmodulins differentially activate target enzymes.
Three mutant calmodulin (CaM) genes together with the normal chicken CaM cDNA have been expressed in bacteria for the purpose of determining structure/function relationships in CaM. The mutant CaM genes were generated by in vitro recombination between a chicken CaM cDNA and a processed pseudogene that encodes a full-length CaM but with 19 amino acid substitutions as compared to authentic vertebrate CaM. The calmodulin-like (CaML) proteins derived from the pseudogene are called CaML19, CaML16, and CaML3 and contain 19, 16, and 3 amino acid substitutions, respectively. CaML3 is functionally identical to CaM by all criteria tested. The functional characteristics of CaML16 and CaML19 are also indistinguishable yet quite different from normal CaM. CaML19 and CaML16 will maximally activate myosin light chain kinase but will only half-maximally activate calcineurin and CaM-dependent multiprotein kinase. In addition, CaML16 and CaML19 do not activate phosphorylase kinase. The differential activation of these enzymes does not result from the loss of Ca2+-binding sites, since CaML16 binds four Ca2+ with affinity similar to CaM or CaM23. It is more likely that the functional characteristics of the mutant proteins result from an altered tertiary structure, since the Ca2+-dependent enhancement of tyrosine fluorescence and limited proteolysis pattern of CaML16 are different from that of CaM. The data demonstrate that the nature of the interaction of CaM with myosin light chain kinase is different from its interaction with calcineurin, CaM-dependent multiprotein kinase, and phosphorylase kinase and may involve different functional domains in CaM.
Putkey
J A
JA
Draetta
G F
GF
Slaughter
G R
GR
Klee
C B
CB
Cohen
P
P
Stull
J T
JT
Means
A R
AR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002645:Chickens; D004247:DNA / Q000378:metabolism; D005818:Genetic Engineering; D008958:Models, Molecular; D009154:Mutation; D009219:Myosin-Light-Chain Kinase; D010764:Phosphorylase Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004247:DNA; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin; D002118:Calcium