Calponin isoforms CNN1, CNN2 and CNN3: Regulators for actin cytoskeleton functions in smooth muscle and non-muscle cells

Abstract

Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and many types of non-muscle cells. Three homologous genes, CNN1, CNN2 and CNN3, encoding calponin isoforms 1, 2, and 3, respectively, are present in vertebrate species. All three calponin isoforms are actin-binding proteins with functions in inhibiting actin-activated myosin ATPase and stabilizing the actin cytoskeleton, while each isoform executes different physiological roles based on their cell type-specific expressions. Calponin 1 is specifically expressed in smooth muscle cells and plays a role in fine-tuning smooth muscle contractility. Calponin 2 is expressed in both smooth muscle and non-muscle cells and regulates multiple actin cytoskeleton-based functions. Calponin 3 participates in actin cytoskeleton-based activities in embryonic development and myogenesis. Phosphorylation has been extensively studied for the regulation of calponin functions. Cytoskeleton tension regulates the transcription of CNN2 gene and the degradation of calponin 2 protein. This review summarizes our knowledge learned from studies over the past three decades, focusing on the evolutionary lineage of calponin isoform genes, their tissue- and cell type-specific expressions, structure-function relationships, and mechanoregulation.

Keywords: Actin cytoskeleton; Calponin isoform genes; Mechanoregulation; Non-muscle cell motility; Smooth muscle.

Figure 1. Evolutionary lineage of vertebrate calponin isoforms

The phylogenetic tree of vertebrate calponin isoform genes is derived from protein sequence alignment performed with the Clustal V method using the MegAlign computer program (Lasergene; DNASTAR, lnc, Madison, WI). The degrees of evolutionary divergence are indicated by the lengths of lineage lines. Calponin isoforms 1, 2 and 3 are marked in green, blue and red, respectively. The NCBI database accession numbers of the sequences analyzed are: African clawed frog calponin 1, NP_001080753.1; African clawed frog calponin 2, NP_001085014.1; African clawed frog calponin 3, NP_001080482.1; Black flying fox calponin 1, ELK17809. 1; Black flying fox calponin 2, ELK19295.1; Black flying fox calponin 3, ELK05808.1; Brandt’s bat calponin 1, EPQ04681.1; Brandt’s bat calponin 2, EPQ08061.1; Brown tree snake calponin 2, JAG68493.1; Cattle calponin 1, NP_001039844.1; Cattle calponin 2, AAI03381.1; Cattle calponin 3, NP_001033268.1; Channel catfish, AHH43034.1; Chicken calponin 1, NP_990847.1; Chicken calponin 2, NP_001135728.1; Chimpanzee calponin 1, NP_001267033.1; Chimpanzee calponin 2, JAA13388.1; Chimpanzee calponin 3, JAA44470.1; Chinese hamster calponin 1, EGV96164.1; Chinese hamster calponin 2, EGV99480.1; Chinese hamster calponin 3, EGW11625.1; Croaker calponin 1, KKF32470.1; Croaker calponin 2, KKF29575.1; Croaker calponin 3, KKF23334.1; Damara mole calponin 1, KFO28782.1; Damara mole calponin 2, KFO28912.1; Damara mole calponin 3, KFO26533.1; Eastern diamondback rattlesnake calponin 2, AFJ49586.1; Ferret calponin 1, NP_001297140.1; Green turtle calponin 1, EMP37252.1; Green turtle calponin 3, EMP30806.1; Human calponin 1, NP_001290.2; Human calponin 2, AAI48265.1; Human calponin 3, AAB35752.1; King cobra calponin 3, ETE62831.1; Marmoset calponin 3, JAB48100.1; Mouse calponin 1, AAI38864.1; Mouse calponin 2, EDL31614.1; Mouse calponin 3, AAH85268.1; Mouse-eared bat calponin 1, ELK28732.1; Mouse-eared bat calponin 2, ELK29427.1; Mouse-eared bat calponin 3, ELK30701.1; Naked mole calponin 1, EHA97296.1; Naked mole calponin 2, EHB16944.1; Naked mole calponin 3, EHB05382.1; Orangutan calponin 2, NP_001124601.1; Rat calponin 1, NP_113935.1; Rat calponin 3, NP_062232.1; Rhesus macaque calponin 3, NP_001248738.1; Salmon calponin 1, NP_001139857.1; Salmon calponin 2, NP_001133873.1; Salmon calponin 3, NP_001133337.1; Sheep, calponin 1, NP_001009456.1; Snakehead calponin 2, AFJ79963.1; Timber rattlesnake calponin 2, JAG45504.1; Western clawed frog calponin 1, NP_001015796.1; NP_998841.1; Western clawed frog calponin 2, Western clawed frog calponin 3, NP_989257.1; Wild swine calponin 1, NP_999043.1; Yak calponin 1, ELR59973.1; Yak calponin 2, ELR60222.1; Yak calponin 3, ELR46902.1; Zebrafish calponin 2, NP_998514.1; Zebrafish calponin 3, NP_956047.1. *The aliases of Africa clawed frog Cnn1 and Cnn2 genes deposited in NCBI database are annotated as Cnn2 and Cnn1, respectively, which is inconsistent with the sequence homology analysis and requires further validation.

Figure 2. Linear structure and comparison of calponin isoforms

The illustrations summarize the primary structures and comparison of the three calponin isoforms. (A) The linear structural map primarily summarized from studies of chicken calponin 1 outlines the structural and functional domains of calponin. The N-terminal CH domain, the two actin-binding sites, the three repeating sequence motifs, and the C-terminal variable region are shown. The CH domain overlaps with the ERK 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 both repeats 2 and 3, whereas a Thr₁₈₄ equivalent is conserved in repeat 2. Different from calponin 1 and calponin 3, calponin 2 has an additional, potentially phosphorylatable, serine at position 177. (B) Each of the three calponin isoform genes contains seven exons. While the three isoforms are largely conserved in the N-terminal and middle regions, they have a C-terminal variable region encoded by exon 7 that is significantly diverged in length and amino acid sequences.

Figure 3. Proteins that interact with calponin

Multiple cytoskeleton and regulatory proteins have been reported to bind or interact with calponin. Summarized in this figure, calponin binds actin filaments through two actin-binding sites (ABS1 and ABS2) and other actin filaments-associated proteins tropomyosin via the N-terminal CH domain and gelsolin via the actin-binding sites. Calponin interacts with microtubules through the region including the actin-binding sites and the repeating motifs and with desmin through the region spanning from the CH domain to the actin-binding sites. The residues 144–182 of calponin interact with myosin. In the presence of Ca²⁺, calmodulin and S100 bind the region of the actin-binding sites of calponin and reverse the calponin inhibition of the myosin MgATPase. 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).