Localization of Protein Regions Involved in the Interaction between Calponin and Myosin

Abstract

Calponin is a 33-kDa smooth muscle-specific protein that has been suggested to play a role in muscle contractility. It has previously been shown to interact with actin, tropomyosin, and calmodulin. More recently we showed that calponin also interacts with myosin (Szymanski, P. T., and Tao, T. (1993) FEBS Lett. 331, 256 -259). In the present study we used a combination of co-sedimentation and fluorescence assays to localize the regions in myosin and calponin that are involved in the interaction between these two proteins. We found that recombinant chicken gizzard ␣-calponin co-sediments with myosin rod and, to a lesser extent, with light meromyosin. Fluorescently labeled recombinant calponin shows interaction with heavy meromyosin and myosin subfragment 2 but not subfragment 1. A fragment comprising residues 7-182 and a synthetic peptide spanning residues 146 -176 of calponin co-sediment with myosin, but fragments comprising residues 7-144 and 183-292 do not. Our results indicate that there are calponin binding sites in the subfragment 2 and light meromyosin regions of myosin, and that the region comprising residues 145-182 of calponin mediates its interaction with myosin. Calponin (CaP)1 is a 33-kDa smooth muscle-specific, thin filament-associated protein that has been suggested to play a role in muscle contractility (2, 3). It is capable of binding to actin (2, 4 -6), tropomyosin (7-10), and calmodulin (4, 6, 11). The binding sites for these proteins are all located in the NH 2 -terminal portion of CaP (residues 7-182) (12).CaP inhibits the actin-activated myosin ATPase in solution (3, 13), unloaded shortening velocity, and, to a lesser extent, isometric force in permeabilized smooth muscle fibers (14 -17). It is commonly assumed that these effects are exerted via CaP binding to actin and competition of CaP with myosin heads for binding to actin. On the other hand, observations that CaP inhibits actin translocation over myosin heads (18,19) and enhances the affinity between actin and myosin in in vitro motility assays (19) suggest that CaP interacts simultaneously with both actin and myosin. Previous studies showed that CaP indeed binds to isolated smooth muscle myosin (1,20). This interaction was found to be reversible upon the addition of Ca 2ϩ -calmodulin (1) and partially abolished upon phosphorylation of CaP by protein kinase C. In this study we further investigated the interaction between CaP and myosin. Using a combination of co-sedimentation and fluorescence assays, we localized the regions in these two proteins that are involved in their interaction. Fragments of chicken gizzard myosin and recombinant chicken gizzard ␣-CaP (R␣CaP) were generated by proteolytic digestion. This allowed us to isolate the major functional portions of these two proteins, viz. S1, S2, rod, HMM, and LMM of myosin and the NH 2 -terminal fragment (residues 7-182), the NH 2 -terminal fragment without the central portion (residues 7-144), and the COOH-terminal fragment (residues 183-292) of CaP.Our data show that there are CaP bi...

Potent Inhibition of Arterial Smooth Muscle Tonic Contractions by the Selective Myosin II Inhibitor, Blebbistatin

“…In this model, cross-links between actin and myosin are proposed to form with time during muscle stimulation and to be of sufficient strength to permit force maintenance while impeding cross-bridge cycling. (Ϫ)Blebbistatin binds myosin in the 50-kDa cleft of the motor domain (Allingham et al, 2005), a location far removed from the sites at which caldesmon or calponin are thought to bind (Ikebe and Reardon, 1988;Hemric and Chalovich, 1990;Szymanski and Tao, 1997;Szymanski, 2004). Thus, (Ϫ)blebbistatin is unlikely to alter putative cross-links between actin and myosin formed by proteins such as caldesmon.…”

Calponin isoforms CNN 1, CNN 2 and CNN 3: Regulators for actin cytoskeleton functions in smooth muscle and non-muscle cells

“…Calponin also binds or interacts with many other cytoskeleton and related proteins, including tropomyosin (Takahashi et al, 1988a; Childs et al, 1992), myosin (Szymanski and Tao, 1997), tubulin (Fujii et al, 1999), desmin (Wang and Gusev, 1996; Mabuchi et al, 1997), gelsolin (Ferjani et al, 2006), Ca 2+ -calmodulin (Takahashi et al, 1986), Ca 2+ -S100 (Fujii et al, 1994), and phospholipids (Bogatcheva and Gusev, 1995). Calponin was also reported to interact with caldesmon (Graceffa et al, 1996) and α-actinin (North et al, 1994), although these observations may reflect a co-localization on actin filaments (Czurylo et al, 1997; Leinweber et al, 1999a).…”

SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum

“…The mechanism underlying the latch state is unknown. Several regulatory mechanisms have been hypothesized including altered kinetics of phosphorylated vs. dephosphorylated myosin cross bridges (7,21), cytoskeletal remodeling (19,26,35,41,45), calponin-or caldesmon-dependent actin-to-myosin crosslinks (57,59), second messenger pathway regulation of MLCK/MLCP activity (24,28,47,48,53,63), and the kinetic properties of actomyosin ATPase of different myosin isoforms [nonmuscle (NM) and smooth muscle (SM) myosin II isoforms; Refs. 16, 32, 33, 37-39, 42, 51, 65].…”