Analysis of Phosphorylation and Mutation of Tyrosine Residues of Calmodulin on Its Activation of the Erythrocyte Ca²⁺‐transporting ATPase

Abstract

The role played by the phosphorylation sites of calmodulin on its ability to activate the human erythrocyte Ca' +-transporting ATPase (Ca" -ATPase) was evaluated. Phosphorylation of mammalian calmodulin on serine/threonine residues by casein kinase TI decreased its affinity for Ca'+-ATPase by twofold. In contrast, tyrosine phosphorylation of mammalian calmodulin by the insulin-receptor kinase did not significantly alter calmodulin-stimulated Ca'? -ATPase activity. Two variant calmodulins, each containing only one tyrosine residue (the second Tyr is replaced by Phe) were also examined: [F138]calmodulin, a mutant containing tyrosine at position 99, and wheat germ calinodulin which has tyrosine at position 139. The concentrations of [FI 38 Jcalmodulin and wheat germ calmodulin required for half-maximal activation of Ca' ' -,4TPase were tenfold and fourfold higher, respectively, than mammalian calmodulin.Phosphorylation at Tyr99 of [F138]calmodulin shifted its affinity for Ca'+-ATPase towards that of mammalian calmodulin. However, phosphorylation at Tyrl39 of wheat germ calmodulin had essentially no effect on its interaction with Ca'+-ATPase. Thus, all of the observed effects of both phosphorylation and substitution of residues of calmodulin are on its affinity for Ca*+-ATPase, not on V,,,.,,. The effects are dependent on the site of phosphate incorporation. Replacement of tyrosine with phenylalanine has a larger effect than phosphorylation of tyrosine, suggesting that the observed functional alterations reflect a seco'ndary conformational change in the C-terminal half of calmodulin, the region that is itnportant in its activation of Ca2+-ATPase.

A Method for the Purification of Phospho(Tyr)calmodulin Free of Nonphosphorylated Calmodulin

“…However, no differential effect of both calmodulin species on calcineurin or the plasma membrane Ca 2ϩ -ATPase was noticed (2,3). In contrast, when a calmodulin mutant lacking Tyr138 and phosphorylated at Tyr99 was used, a decrease in the activation of the Ca 2ϩ -ATPase was observed compared to nonphosphorylated calmodulin (3).…”

Ca2+-Calmodulin and Janus Kinase 2 Are Required for Activation of Sodium-Proton Exchange by the Gi-Coupled 5-Hydroxytryptamine1a Receptor

“…Phosphorylation of CaM on Tyr-99 was shown to selectively attenuate the action of CaM antagonists on type 1 cyclic nucleotide phosphodiesterase activity (Saville and Houslay, 1994). In contrast, phosphorylation of Tyr-99 increases the affinity of CaM for Ca 2ϩ -ATPase (Sacks et al, 1996). To address that discrepancy, Corti et al (1999) studied the effects of CaM phosphorylation on Tyr-99 on the binding affinities and activation of six different CaM target enzymes [myosin light chain kinase, 3Ј-5Ј-cyclic nucleotide phosphodiesterase, plasma membrane Ca 2ϩ -ATPase, Ca 2ϩ -CaM-dependent protein phosphatase 2B (calcineurin), neuronal nitric-oxide synthase, and type 2 Ca 2ϩ -CaM-dependent protein kinase].…”

Bradykinin B2 Receptors Activate Na+/H+ Exchange in mIMCD-3 Cells via Janus Kinase 2 and Ca2+/Calmodulin

“…Phosphorylation of CaM on Tyr-99 was shown to selectively attenuate the action of CaM antagonists on type I cyclic nucleotide phosphodiesterase activity (51). In contrast, phosphorylation of Tyr-99 increases the affinity of CaM for Ca-ATPase (52). In order to address that discrepancy, Corti et al (44) studied the effects of CaM phosphorylated on Tyr-99 on the binding affinities and activation of six different CaM target enzymes (myosin light chain kinase MLCK, 3Ј-5Ј-cyclic nucleotide phosphodiesterase, plasma membrane Ca 2ϩ -ATPase, Ca 2ϩ -CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric-oxide synthase, and type II Ca 2ϩ / CaM-dependent protein kinase).…”