Corti C, Leclerc L'Hostis E, Quadroni M, Schmid H, Durussel I, Cox J, Dainese Hatt P, James P, Carafoli E
Protein Chemistry Laboratory, Department of Biology, Swiss Federal Institute of Technology, (ETH) Zürich.
Eur J Biochem. 1999 Jun;262(3):790-802. doi: 10.1046/j.1432-1327.1999.00441.x.
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.
测试了酪氨酸99位点磷酸化的钙调蛋白（PCaM）对六种靶酶的激活作用及其各自钙调蛋白结合域的结合亲和力。这六种酶分别是：肌球蛋白轻链激酶（MLCK）、3'-5'-环核苷酸磷酸二酯酶（PDE）、质膜（PM）Ca2+ -ATP酶、Ca2+ -钙调蛋白依赖性蛋白磷酸酶2B（钙调神经磷酸酶）、神经元型一氧化氮合酶（NOS）和II型Ca2+ -钙调蛋白依赖性蛋白激酶（CaM激酶II）。总体而言，酪氨酸磷酸化导致钙调蛋白（CaM）的激活特性增强。对于质膜（PM）Ca2+ -ATP酶、PDE和CaM激酶II，主要影响是达到最大速度一半时的浓度（Kact）降低。相反，对于钙调神经磷酸酶和NOS，CaM的磷酸化显著增加了Vmax。然而，对于MLCK，酪氨酸磷酸化既不影响Vmax也不影响Kact。通过荧光技术直接测定与六种分析酶的CaM结合域对应的合成肽的解离常数，结果表明CaM上酪氨酸99位点的磷酸化通常会增加其对肽的亲和力。
