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

Abstract

The mechanism of Ca2+-dependent proteinprotein 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 Ca2l dependence (Ka = 17 ,uM) 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 7 ,.M) 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. Calmodulin, a highly conserved Ca2"-binding protein (1), has four Ca2"-binding sites that apparently differ in affinity (2, 3).At physiologic ionic strength and Mg2+ concentration, the two higher affinity sites are reported to have dissociation constants of 3-8 ,uM while the other sites have estimated binding constants of 20-150 ,uM (2-5). In the Ca2+-bound state, calmodulin can interact with and activate a number of enzymes (for reviews see refs. 6 and 7). A question of primary importance in understanding the function of this signalling system relates to the Ca2+ requirement for the effect of calmodulin; that is, must all Ca2+ sites be filled to produce activation or must only specific sites be filled? In this regard, the activation by calmodulin of several of its target enzymes, cyclic nucleotide phosphodiesterase (4,8), phosphorylase b kinase (9), Ca2+-dependent, Mg2+-activated ATPase (10), and myosin light chain kinase (11), has been examined using kinetic analysis of enzyme stimulation. These studies focused on the Ca2+ binding to calmodulin that is required to produce activation and, in each case, it was concluded that occupancy of three or four sites was necessary for activation. In several of the studies, enzyme activation and interaction with calmodulin were assumed to be equivalent; certainly, it was not possible to discriminate between the two events since, in most of these, only enzyme activity was monitored. It is, of course, feasible that interaction of calmodulin with a target enzyme is not sufficient for activation; i.e., an enzymatically inactive complex might be formed at subsaturating amounts of Ca2", requiring further Ca2" for activation.To investigate this possibility, we employed a fluorescent derivative of calmodulin, dimethylaminonaphthalene (dansyl)-calmodulin, that has been used to monitor the Ca2+-dependent interaction with several calmodulin-binding proteins (12, 13) or model peptides (14) and the calmodulinactivated protein phosphatase calcineurin (15). By measuring the amount of calmodulin-calcineurin complex in the presence of enzyme substrate, ...

An allosteric model of calmodulin explains differential activation of PP2B and CaMKII

“…A sequential model fails to explain why calmodulin can activate PP2B or CaMKII when bound to less than four calcium ions (17,18). Some models assume that calmodulin can activate PP2B with two, three, or four calcium ions bound but requires four calcium ions bound to activate CaMKII (49).…”

“…Each of these models reflects some properties of calmodulin and is reasonably applicable in contexts in which only these properties are relevant. However, none of these models can satisfactorily account for all of the observed properties of calmodulin such as cooperativity of calcium binding and different affinities for different calcium-binding sites (16), activation of targets by unsaturated calmodulin (17,18), and increased affinity for calcium upon binding to targets (18)(19)(20). We propose an alternative model, based on a biophysical description of the conformational transitions.…”

Na+/H+ Exchanger 1 Directly Binds to Calcineurin A and Activates Downstream NFAT Signaling, Leading to Cardiomyocyte Hypertrophy

“…Therefore, the highly cooperative activation of CaN by Ca 2ϩ (Fig. 6B) is partly due to the cooperative binding of Ca 2ϩ to the 4 EF-hand domains of CaM (8,13,25). CaN is also activated in response to increasing pH, which increases the affinity of CaN for Ca 2ϩ (Fig.…”

De Novo Design of Peptides Targeted to the EF Hands of Calmodulin

“…Another intriguing question is how an interaction with only one of the two domains leads to a CaM conformation that is capable of activating PDE. This may be related to intermediary conformations of CaM having some enzyme-activating ability (65).…”