Calponin 1

Calponin 1 is a basic smooth muscle protein that in humans is encoded by the CNN1 gene.^[3]

The CNN1 gene is located at 19p13.2-p13.1 in the human chromosomal genome and contains 7 exons, encoding the protein calponin 1, an actin filament-associated regulatory protein.^[4] Human calponin 1 is a 33.2-KDa protein consists of 297 amino acids with an isoelectric point of 9.1,^[5] thus calponin 1 is also known as basic calponin.

Evolution

Figure 1: Evolutionary lineage of vertebrate CNN1.

Three homologous genes, Cnn1, Cnn2 and Cnn3, have evolved in vertebrates, encoding three isoforms of calponin: calponin 1,^[5][6] calponin 2,^[7] calponin 3,^[8] respectively. Protein sequence alignment shows that calponin 1 is highly conserved in mammals but more diverged among lower vertebrates.

Smooth muscle-specific expression

The expression of CNN1 is specific to differentiated mature smooth muscle cells, suggesting a role in contractile functions. Calponin 1 is up-regulated in smooth muscle tissues during postnatal development^[9] with a higher content in phasic smooth muscle of the digestive tract.^[10]

Structure-function relationship

Figure 2. Structural and functional domains of calponin. The linear structural map summarized primarily from studies of chicken calponin 1 illustrates the structural and functional domains of calponin. The CH domain, two actin-binding sites, three repeating sequence motifs, and the C-terminal variable region are outlined. The CH domain overlaps with the ERK signaling binding region. Amino acid sequences of the two actin-binding sites in the three isoforms and the three repeating motifs of calponin 1 are shown in the insets. The regulatory phosphorylation sites Ser₁₇₅ and Thr₁₈₄ are located in the second actin-binding site that overlaps with the first repeating motif. Potentially phosphorylatable serine residues corresponding to Ser₁₇₅ are conserved in repeats 2 and 3, while a Thr₁₈₄ equivalent is conserved in repeat 2. Different from calponin 1 and calponin 3, calponin 2 has a potentially phosphorylatable additional serine at position 177.

The majority of structure-function relationship studies of calponin were with experiments using chicken calponin 1. Primary structure of calponin consists of a conserved N-terminal calponin homology (CH) domain, a conserved middle region containing two actin-binding sites, and a C-terminal variable region that contributes to the differences among there isoforms.

The CH domain

The CH domain was found in a number of actin-binding proteins (such as α-actinin, spectrin, and filamin) to form the actin-binding region or serve as a regulatory structure.^[11] However, the CH domain in calponin is not the binding site for actin nor does it regulate the modes of calponin-F-actin binding.^[12] Nonetheless, CH domain in calponin was found to bind to extra-cellular regulated kinase (ERK) for calponin to play a possible role as an adaptor protein in the ERK signaling cascades.^[13]

Actin-binding sites

Calponin binds actin to promote and sustain polymerization. The binding of calponin to F-actin inhibits the MgATPase activity of smooth muscle myosin.^{[14][15][14][16]} Calponin binds F-actin through two sites at residues 144-162 and 171-188 in chicken calponin 1. The two actin-binding sites are conserved in the three calponin isoforms.

There are three repeating sequence motifs in calponin next to the C-terminal region. This repeating structure is conserved in all three isoforms and across species. Outlined in Fig. 2, the first repeating motif overlaps with the second actin-binding site and contains protein kinase C (PKC) phosphorylation sites Ser175 and Thr184 that are not present in the first actin-binding site. This feature is consistent with the hypothesis that the second actin-binding site plays a regulatory role in the binding of calponin to the actin filament. Similar sequences as well as potential phosphorylation sites are present in repeats 2 and 3 whereas their function is unknown.

C-terminal variable region

The C-terminal segment of calponin has diverged significantly among the three isoforms. The variable lengths and amino acid sequences of the C-terminal segment produce the size and charge differences among the calponin isoforms. The corresponding charge features rendered calponin 1, 2 and 3 the names of basic, neutral and acidic calponins.^[17][18][19]

The C-terminal segment of calponin has an effect on weakening the binding of calponin to F-actin. Deletion of the C-terminal tail strongly enhanced the actin-binding and bundling activities of all three isoforms of calponin.^[20][21] The C-terminal tail regulates the interaction with F-actin by altering the function of the second actin-bing site of calponin.^[22]

Regulation of smooth muscle contractility

Numerous in vitro experimental data indicate that calponin 1 functions as an inhibitory regulator of smooth muscle contractility through inhibiting actomyosin interactions.^[4][23][24] In this regulation, binding of Ca²⁺-calmodulin and PKC phosphorylation dissociate calponin 1 from the actin filament and facilitate smooth muscle contraction.^[25]

In vivo data also support the role of calponin 1 as regulator of smooth muscle contractility. While aortic smooth muscle of adult Wistar Kyoto rats, which naturally lacks calponin 1, is fully contractile, it has a decreased sensitivity to norepinephrine activation.^[26][27] Matrix metalloproteinase-2 proteolysis of calponin 1 resulted in vascular hypocontractility to phenylephrine.^[28] Vas deferens smooth muscle from calponin 1 knockout mice showed faster maximum shortening velocity.^[29] Calponin 1 knockout mice exhibited blunted MAP response to phenylephrine administration.^[30]

Phosphorylation regulation

There is a large collection of in vitro evidences demonstrating the phosphorylation regulation of calponin. The primary phosphorylation sites are Ser175 and Thr184 in the second actin-binding site (Fig. 2). Experimental data showed that Ser175 and Thr184 in calponin 1 are phosphorylated by PKC in vitro.^[25] Direct association was found between calponin 1 and PKCα ^[31] and PKCε.^[13] Calmodulin-dependent kinase II and Rho-kinase are also found to phosphorylate calponin at Ser175 and Thr184 in vitro.^[32][33] Of these two residues, the main site of regulatory phosphorylation by calmodulin-dependent kinase II and Rho-kinase is Ser175. Dephosphorylation of calponin is catalyzed by type 2B protein phosphatase ^[34][35]

Unphosphorylated calponin binds to actin and inhibits actomyosin MgATPase. Ser175 phosphorylation alters the molecular conformation of calponin and dissociates calponin from F-actin.^[36] The consequence is to release the inhibition of actomyosin MgATPase and increase the production of force.^[16][37][38]

Despite the overwhelming evidence for the phosphorylation regulation of calponin obtained from in vitro studies, phosphorylated calponin is not readily detectable in vivo or in living cells under physiological conditions.^[39][40] Based on the observation that PKC phosphorylation of calponin 1 weakens the binding affinity for the actin filaments,^[36] the phosphorylated calponin may not be stable in the actin cytoskeleton thus be degraded in the cell.

References