Analysis of phosphorylation and mutation of tyrosine residues of calmodulin on its activation of the erythrocyte Ca(2+)-transporting ATPase.
The role played by the phosphorylation sites of calmodulin on its ability to activate the human erythrocyte Ca(2+)-transporting ATPase (Ca(2+)-ATPase) was evaluated. Phosphorylation of mammalian calmodulin on serine/threonine residues by casein kinase II decreased its affinity for Ca(2+)-ATPase by twofold. In contrast, tyrosine phosphorylation of mammalian calmodulin by the insulin-receptor kinase did not significantly alter calmodulin-stimulated Ca(2+)-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 Ca(2+)-ATPase were tenfold and fourfold higher, respectively, than mammalian calmodulin. Phosphorylation at Tyr99 of [F138]calmodulin shifted its affinity for Ca(2+)-ATPase towards that of mammalian calmodulin. However, phosphorylation at Tyr139 of wheat germ calmodulin had essentially no effect on its interaction with Ca(2+)-ATPase. Thus, all of the observed effects of both phosphorylation and substitution of residues of calmodulin are on its affinity for Ca(2+)-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 Ca(2+)-ATPase.
Sacks
D B
DB
Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA.
Lopez
M M
MM
Li
Z
Z
Kosk-Kosicka
D
D
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013552:Swine; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D000252:Calcium-Transporting ATPases