Calponin-calmodulin interaction: Properties and effects on smooth and skeletal muscle actin binding and actomyosin ATPase

Abstract

Smooth muscle calponin bound to the biologically active fluorescent calmodulin [2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin] (MIANS.CaM) with a Kd of 80 nM and produced a 3.4-fold fluorescence enhancement. PKC-phosphorylated calponin (1.3 mol of Pi/mol) bound to CaM with approximately 15-fold lower affinity. Calponin inhibited CaM (10 nM) activation of the Ca(2+)-/CaM-activated cyclic nucleotide phosphodiesterase (PDE) with an IC50 of 138 nM. The calponin-CaM interaction was Ca(2+)-dependent: half-maximal binding of calponin to MIANS.CaM occurred at pCa 6.6 with a Hill coefficient of 2.4. Stopped-flow fluorescence kinetic analysis demonstrated that EGTA chelation of Ca2+ from CaM disrupted the MIANS.CaM-calponin complex at a rate of 1 s-1. Calponin bound MIANS.CaM at a rate of (6.0 +/- 1.8) x 10(6) M-1s-1, and melittin and unlabeled brain CaM both disrupted the MIANS.CaM-calponin complex at a rate of 0.3 +/- 0.1 s-1. These studies suggest that calponin binds CaM with 80-fold lower affinity than myosin light-chain kinase and that calponin associates with CaM much slower than it associates with caldesmon or myosin light-chain kinase. The physiological relevance of the CaM-calponin interaction was evaluated by analysis of the effects of Ca(2+)-CaM on (i) the interaction of calponin with actin and (ii) calponin-mediated inhibition of actin-activated myosin MgATPase activity. Ca(2+)-CaM half-maximally inhibited calponin (2 microM) binding to smooth and skeletal muscle actins (9 microM) at 5.4 and 11 microM CaM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic Control of the Dissociation Pathway of Calmodulin-Peptide Complexes

“…Calmodulin labeled at Cys-27 with the fluorescent probe MI-ANS has been used to study the EGTA-induced dissociation of the CaM complexes with the proteins caldesmon, calponin, and sm-MLCK. The rate determined for the calponin complex (K d Ϸ nM) was 1 s Ϫ1 (21). The rates for the complexes with sm-MLCK (K d ϭ 1.1 nM) and caldesmon (K d ϭ 108 nM) were 3.5 and 13.5 s Ϫ1 (20).…”

“…Although slow processes attributed to conformational changes have been observed in studies of the association of fluorescently labeled CaMs with peptides and proteins (22,33), no slow processes were observed in the association reactions studied here. Values of k on for the interaction of CaM with proteins tend to be lower (20,21), but a value of 5.3 ϫ 10 8 M Ϫ1 ⅐s Ϫ1 was measured for caldesmon (20).…”

Calcium/calmodulin‐dependent regulation of the NH2‐terminal F‐actin binding domain of utrophin

“…Several actin binding proteins have been shown to be regulated by Ca2+-calmodulin; caldesmon [18], calponin [19], MAP1 and tau [20,21] and the heat shock proteins HSP90 and HSPI00 [22,23] for example. It is therefore not inconceivable that the utrophin-actin interaction could also be regulated by calmodulin.…”

Functional Analysis of Rat Acidic Calponin.

“…33) Interaction of Recombinant Acidic Calponin with Basic Calponin-Related Components Basic calponin has been reported to interact with F-actin, microtubules, desmin filaments, tropomyosin, calmodulin, S100 and PS vesicles. [1][2][3][4][5][6][15][16][17][18][19][20][21][22][23][24]27,28) We examined whether the recombinant acidic calponin interacts with these proteins and PS vesicles using sedimentation and bead assays (Fig. 1).…”

“…S, supernatant; P, pellet; aCaP, acidic calponin; Tub, tubulin. F-actin up a molar ratio of acidic calponin to actin monomers of 1 : 2 and with an apparent Kd value of 4ϫ10 5 …”