Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.
The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.
Corti
C
C
Protein Chemistry Laboratory, Department of Biology, Swiss Federal Institute of Technology, (ETH) Zürich.
Leclerc L'Hostis
E
E
Quadroni
M
M
Schmid
H
H
Durussel
I
I
Cox
J
J
Dainese Hatt
P
P
James
P
P
Carafoli
E
E
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000302:isolation & purification / Q000378:metabolism
D002147:Calmodulin; D005456:Fluorescent Dyes; D010455:Peptides; D011506:Proteins; D014443:Tyrosine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D019061:src-Family Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000252:Calcium-Transporting ATPases