A New Role for IQ Motif Proteins in Regulating Calmodulin Function

John A Putkey,Quinn Kleerekoper,Tara R Gaertner,M Neal Waxham

doi:10.1074/jbc.c300372200

Abstract

IQ motifs are found in diverse families of calmodulin (CaM)-binding proteins. Some of these, like PEP-19 and RC3, are highly abundant in neuronal tissues, but being devoid of catalytic activity, their biological roles are not understood. We hypothesized that these IQ motif proteins might have unique effects on the Ca2+ binding properties of CaM, since they bind to CaM in the presence or absence of Ca2+. Here we show that PEP-19 accelerates by 40 to 50-fold both the slow association and dissociation of Ca2+ from the C-domain of free CaM, and we identify the sites of interaction between CaM and PEP-19 using NMR. Importantly, we demonstrate that PEP-19 can also increase the rate of dissociation of Ca2+ from CaM when bound to intact CaM-dependent protein kinase II. Thus, PEP-19, and presumably similar members of the IQ family of proteins, has the potential to alter the Ca2+-binding dynamics of free CaM and CaM that is bound to other target proteins. Since Ca2+ binding to the C-domain of CaM is the rate-limiting step for activation of CaM-dependent enzymes, the data reveal a new concept of importance in understanding the temporal dynamics of Ca2+-dependent cell signaling.

Highlights

From the ‡Departments of Biochemistry and Molecular Biology, and ¶Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77030
The magnitude of this effect is dependent on a given target enzyme and can be so large as to promote constitutive association of CaM with targets even at basal Ca2ϩ levels. It would be of great functional significance if proteins were discovered that could increase, rather than decrease, the rate of dissociation of Ca2ϩ from free or target-bound CaM or potentially modulate the rate of Ca2ϩ association. This would be important for the C-domain of CaM, which exhibits Ca2ϩ binding kinetics that are too slow to respond to rapid Ca2ϩ transients such as those found in excitable cells
The effect of PEP-19 on the C-domain of CaM, and localization of PEP-19 and other SNIQs to neurons, suggest that these proteins play a role in modulating the Ca2ϩ binding properties of CaM so it can respond to challenging

Summary

Accelerated Publication

Like PEP-19 and RC3, are highly abundant in neuronal tissues, but being devoid of catalytic activity, their biological roles are not understood We hypothesized that these IQ motif proteins might have unique effects on the Ca2؉ binding properties of CaM, since they bind to CaM in the presence or absence of Ca2؉. It would be of great functional significance if proteins were discovered that could increase, rather than decrease, the rate of dissociation of Ca2ϩ from free or target-bound CaM or potentially modulate the rate of Ca2ϩ association This would be important for the C-domain of CaM, which exhibits Ca2ϩ binding kinetics that are too slow to respond to rapid Ca2ϩ transients such as those found in excitable cells. CaM, calmodulin; SNIQ, small, neuronal IQ motif protein; Nm, neuromodulin; Ng, neurogranin; MOPS, 4-morpholinepropanesulfonic acid; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid; NOESY, nuclear Overhauser effect spectroscopy; CKII␣, CaM-dependent protein kinase II ␣; IAEDANS, 5-[N-(iodoacetamidoethyl)amino]naphthalene-1-sulfonate

EXPERIMENTAL PROCEDURES

New Role for IQ Motif Proteins

Keq koff kon

DISCUSSION