Does Calponin Interact with Caldesmon?

Abstract

The roles of calponin and caldesmon and their interaction in regulation of smooth muscle contraction are controversial. Recently, strong binding between these two proteins has been reported (Graceffa, P., Adam, L. P., and Morgan, K. G. (1996) J. Biol. Chem. 271, 30336 -30339). Results in this paper fail to confirm their data and are consistent with the concept of independent functions for calponin and caldesmon.To examine the ability of duck gizzard caldesmon to interact with calponin, three caldesmon derivatives, each containing a different sulfhydryl-specific reporter probe (6-acryloyl-2-dimethylaminonaphtalene, N-(1-pyrenyl)iodoacetamide, and N-iodoacetyl-N-(5-sulfo-1-naphtylo)ethylenediamine) attached to a single cysteine located in the C-terminal domain, were synthesized. Addition of calponin to labeled caldesmon at both low and physiological salt concentrations did not induce any changes in fluorescence intensity or maximum shift. Under the same conditions, calmodulin and tropomyosin (known to bind to the C terminus of caldesmon) produced substantial changes in these spectral parameters. Gel filtration of an equimolar caldesmon-calponin mixture on a fast protein liquid chromatography Superose-12 column revealed two base-line-separated peaks, the first containing only caldesmon and the second only calponin, thus confirming the lack of any interaction between these two proteins. Also, the addition of calponin did not change the fluorescence parameters of labeled caldesmon in complexes with F-actin and F-actin-tropomyosin.The primary mechanism for smooth muscle regulation, necessary to initiate contraction, involves phosphorylation of the 20-kDa myosin light chains by a specific Ca 2ϩ /calmodulin-dependent myosin light chain kinase (for reviews see Refs. 1-3). It is postulated that the specific thin filament-associated proteins caldesmon and calponin take part in the secondary mechanism of regulation, probably during the relaxation phase. This view is supported by in vitro experiments showing that both of these proteins inhibit actin-activated ATPase activity of myosin, mobility of actin filaments over immobilized myosin in motility assay, and skinned muscle contraction. This inhibition can be reversed by Ca 2ϩ /calmodulin, or other Ca 2ϩ -binding proteins (like S100 or caltropin), and by phosphorylation with protein kinase C or casein kinase II (Refs. 3-6 and references therein).Studies by Makuch et al. (7) revealed that calponin and caldesmon compete for the binding sites on actin filament, calponin being more effective at displacing caldesmon than vice versa. Similar results were obtained in the Chalovich laboratory (8). This suggested that calponin and caldesmon do not form a mutual complex on actin and reside on different populations of thin filaments in vivo. Immunofluorescence and immunogold electron microscopy confirmed this suggestion showing that, whereas caldesmon is present exclusively in the contractile domain, calponin is primarily, although not exclusively, located in the cytoskeletal domain of...

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

“…An N-terminal 22-kDa fragment of calponin was reported to interact with phosphatidylserine and phosphatidylinositol. *Caldesmon was reported to possibly interact with calponin but no definitive site was identified (Graceffa et al, 1996; Czurylo et al, 1997). …”

“…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). Functional significance of these biochemically detected bindings of calponin to cytoskeleton proteins or regulatory molecules is largely unknown and merits further investigation.…”

Smoothelin contains a novel actin cytoskeleton localization sequence with similarity to troponin T

“…Two independently working proteins, caldesmon and calponin, are involved in regulation of contraction [Czurylo et al, 1997]. Both caldesmon and calponin inhibit the actin regulated ATPase activity of myosin in a Ca 2 -calmodulin dependent manner [Marston et al, 1994;Winder and Walsh, 1996].…”

Motifs of the caldesmon family.

“…All CaDs are rich in ionisable amino-acid residues (202 acidic and 188 basic for chicken, 191 acidic and 206 basic for human H-CaDs). Thus, on average, every other residue carries a charge causing CaD to interact with other biomolecules, including many proteins, so readily that it is hard to distinguish between specific and nonspecific interactions (Czury³o et al, 1991;Czury³o et al, 1997b;Marston et al, 1998;Marston & Huber, 1996). The large number of charged residues prevents the CaD molecule from folding into a globule and forces its extended conformation (Czury³o et al, 1997a;Czury³o et al, 1993;Mabuchi & Wang, 1991).…”