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Direct comparison of Ca2+ requirements for calmodulin interaction with and activation of protein phosphatase.

Abstract：

:The mechanism of Ca2+-dependent protein-protein interaction and enzyme activation by calmodulin was investigated with the phosphoprotein phosphatase, calcineurin. Dimethylaminonaphthalene (dansyl)-calmodulin, a fluorescent derivative used to monitor complex formation, produced similar maximal activation (10- to 12-fold) with a Ca2+ dependence (Ka = 17 microM) identical to that of native calmodulin. The Ca2+-dependent increase in fluorescence intensity of dansyl-calmodulin was enhanced 100-150% by calcineurin, indicating complex formation; the concentration of Ca2+ required for a half-maximal increase in fluorescence was the same (K1/2 approximately equal to 7 microM) with and without calcineurin. Since the Ca2+ concentration required for activation appeared to differ from that necessary for protein-protein interaction, a method was devised to measure both the formation of complexes between dansyl-calmodulin and calcineurin and enzyme activity in the same samples. Direct comparison of interaction (measured by polarization of fluorescence) and enzyme activity demonstrated different Ca2+ requirements for the two events. Whereas dansyl-calmodulin-calcineurin interaction, measured in the presence of phosphoprotein substrate, exhibited very little cooperativity (Hill coefficient = 1.2, Ca2+ concentration required for the half-maximal increase in fluorescence, K1/2, approximately equal to 6 microM), phosphatase activation was highly cooperative (Hill coefficient = 3.5) and required 3 times higher Ca2+ concentration for half-maximal stimulation. Equivalent results were obtained with p-nitrophenyl phosphate as substrate. These data are consistent with a sequential mechanism for interaction and activation wherein filling of perhaps two Ca2+ sites permits calmodulin interaction with the phosphatase; this complex is inactive, requiring further binding of Ca2+ for activation. Such a scheme would provide a sensitive switch for control of enzyme activity within a narrow range of free Ca2+ concentration.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Kincaid RL,Vaughan M

doi

10.1073/pnas.83.5.1193

subject

Has Abstract

pub_date

1986-03-01 00:00:00

pages

1193-7

issue

eissn

0027-8424

issn

1091-6490

journal_volume

pub_type

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Direct comparison of Ca2+ requirements for calmodulin interaction with and activation of protein phosphatase.