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

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Abstract
The role played by the phosphorylation sites of calmodulin on its ability to activate the human erythrocyte Ca2+‐transporting ATPase (Ca2+‐ATPase) was evaluated. Phosphorylation of mammalian calmodulin on serine/threonine residues by casein kinase II decreased its affinity for Ca2+‐ATPase by twofold. In contrast, tyrosine phosphorylation of mammalian calmodulin by the insulin‐receptor kinase did not significantly alter calmodulin‐stimulated Ca2+‐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 calmodulin which has tyrosine at position 139. The concentrations of [F138]calmodulin and wheat germ calmodulin required for half‐maximal activation of Ca2+‐ATPase were tenfold and fourfold higher, respectively, than mammalian calmodulin.Phosphorylation at Tyr99 of [F138]calmodulin shifted its affinity for Ca2+‐ATPase towards that of mammalian calmodulin. However, phosphorylation at Tyr139 of wheat germ calmodulin had essentially no effect on its interaction with Ca2+‐ATPase. Thus, all of the observed effects of both phosphorylation and substitution of residues of calmodulin are on its affinity for Ca2+‐ATPase, not on Vmax. 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 secondary conformational change in the C‐terminal half of calmodulin, the region that is important in its activation of Ca2+‐ATPase.

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