Calponin 1 Explained

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

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.[2] Human calponin 1 is a 33.2-KDa protein consists of 297 amino acids with an isoelectric point of 9.1,[3] thus calponin 1 is also known as basic calponin.

Evolution

Three homologous genes, Cnn1, Cnn2 and Cnn3, have evolved in vertebrates, encoding three isoforms of calponin: calponin 1,[4] calponin 2,[5] calponin 3,[6] 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[7] with a higher content in phasic smooth muscle of the digestive tract.[8]

Structure-function relationship

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.[9] However, the CH domain in calponin is not the binding site for actin nor does it regulate the modes of calponin-F-actin binding.[10] 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.[11]

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.[12] [13] [14] 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.[15] [16] [17]

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.[18] [19] The C-terminal tail regulates the interaction with F-actin by altering the function of the second actin-bing site of calponin.[20]

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.[2] [21] [22] In this regulation, binding of Ca2+-calmodulin and PKC phosphorylation dissociate calponin 1 from the actin filament and facilitate smooth muscle contraction.[23]

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.[24] [25] Matrix metalloproteinase-2 proteolysis of calponin 1 resulted in vascular hypocontractility to phenylephrine.[26] Vas deferens smooth muscle from calponin 1 knockout mice showed faster maximum shortening velocity.[27] Calponin 1 knockout mice exhibited blunted MAP response to phenylephrine administration.[28]

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.[23] Direct association was found between calponin 1 and PKCα[29] and PKCε.[11] Calmodulin-dependent kinase II and Rho-kinase are also found to phosphorylate calponin at Ser175 and Thr184 in vitro.[30] [31] 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[32] [33]

Unphosphorylated calponin binds to actin and inhibits actomyosin MgATPase. Ser175 phosphorylation alters the molecular conformation of calponin and dissociates calponin from F-actin.[34] The consequence is to release the inhibition of actomyosin MgATPase and increase the production of force.[14] [35] [36]

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.[37] [38] Based on the observation that PKC phosphorylation of calponin 1 weakens the binding affinity for the actin filaments,[34] the phosphorylated calponin may not be stable in the actin cytoskeleton thus be degraded in the cell.

Notes and References

  1. Web site: Entrez Gene: calponin 1, basic, smooth muscle .
  2. Takahashi K, Abe M, Hiwada K, Kokubu T . A novel troponin T-like protein (calponin) in vascular smooth muscle: interaction with tropomyosin paracrystals . Journal of Hypertension Supplement . 6 . 4 . S40–3 . December 1988 . 3241227 . 10.1097/00004872-198812040-00008. 38679688 .
  3. Gao J, Hwang JM, Jin JP . Complete nucleotide sequence, structural organization, and an alternatively spliced exon of mouse h1-calponin gene . Biochemical and Biophysical Research Communications . 218 . 1 . 292–7 . January 1996 . 8573148 . 10.1006/bbrc.1996.0051 .
  4. Strasser P, Gimona M, Moessler H, Herzog M, Small JV . Mammalian calponin. Identification and expression of genetic variants . FEBS Letters . 330 . 1 . 13–8 . September 1993 . 8370452 . 10.1016/0014-5793(93)80909-e. 41687174 . free .
  5. Masuda H, Tanaka K, Takagi M, Ohgami K, Sakamaki T, Shibata N, Takahashi K . Molecular cloning and characterization of human non-smooth muscle calponin . Journal of Biochemistry . 120 . 2 . 415–24 . August 1996 . 8889829 . 10.1093/oxfordjournals.jbchem.a021428.
  6. Applegate D, Feng W, Green RS, Taubman MB . Cloning and expression of a novel acidic calponin isoform from rat aortic vascular smooth muscle . The Journal of Biological Chemistry . 269 . 14 . 10683–90 . April 1994 . 10.1016/S0021-9258(17)34113-3 . 8144658 . free .
  7. Hossain MM, Hwang DY, Huang QQ, Sasaki Y, Jin JP . Developmentally regulated expression of calponin isoforms and the effect of h2-calponin on cell proliferation . American Journal of Physiology. Cell Physiology . 284 . 1 . C156–67 . January 2003 . 12388067 . 10.1152/ajpcell.00233.2002 . 2107783 .
  8. Jin JP, Walsh MP, Resek ME, McMartin GA . Expression and epitopic conservation of calponin in different smooth muscles and during development . Biochemistry and Cell Biology . 74 . 2 . 187–96 . 9213427 . 10.1139/o96-019 . 1996.
  9. Gimona M, Djinovic-Carugo K, Kranewitter WJ, Winder SJ . Functional plasticity of CH domains . FEBS Letters . 513 . 1 . 98–106 . February 2002 . 11911887 . 10.1016/s0014-5793(01)03240-9. 2288740 . free .
  10. Galkin VE, Orlova A, Fattoum A, Walsh MP, Egelman EH . The CH-domain of calponin does not determine the modes of calponin binding to F-actin . Journal of Molecular Biology . 359 . 2 . 478–85 . June 2006 . 16626733 . 10.1016/j.jmb.2006.03.044 .
  11. Leinweber BD, Leavis PC, Grabarek Z, Wang CL, Morgan KG . Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actin-binding proteins . The Biochemical Journal . 344 . 117–23 . November 1999 . 10548541 . 10.1042/0264-6021:3440117 . 1 . 1220621.
  12. Abe M, Takahashi K, Hiwada K . Effect of calponin on actin-activated myosin ATPase activity . Journal of Biochemistry . 108 . 5 . 835–8 . November 1990 . 2150518 . 10.1093/oxfordjournals.jbchem.a123289.
  13. Mezgueldi M, Fattoum A, Derancourt J, Kassab R . Mapping of the functional domains in the amino-terminal region of calponin . The Journal of Biological Chemistry . 267 . 22 . 15943–51 . August 1992 . 10.1016/S0021-9258(19)49625-7 . 1639822 . free .
  14. Winder SJ, Walsh MP . Calponin: thin filament-linked regulation of smooth muscle contraction . Cellular Signalling . 5 . 6 . 677–86 . November 1993 . 8130072 . 10.1016/0898-6568(93)90029-l.
  15. Jin JP, Zhang Z, Bautista JA . Isoform diversity, regulation, and functional adaptation of troponin and calponin . Critical Reviews in Eukaryotic Gene Expression . 18 . 2 . 93–124 . 18304026 . 10.1615/critreveukargeneexpr.v18.i2.10 . 2008.
  16. Wu KC, Jin JP . Calponin in non-muscle cells . . 52 . 3 . 139–48 . 18946636 . 10.1007/s12013-008-9031-6 . 2008. 6365920 .
  17. Liu R, Jin JP . 2015 . Calponin: A mechanical tension-modulated regulator of cytoskeleton and cell motility. . Current Topics in Biochemical Research . 16 . 1–15 .
  18. Bartegi A, Roustan C, Kassab R, Fattoum A . Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin effects of F-actin and salts . European Journal of Biochemistry . 262 . 2 . 335–41 . June 1999 . 10336616 . 10.1046/j.1432-1327.1999.00390.x. free .
  19. Danninger C, Gimona M . Live dynamics of GFP-calponin: isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences . Journal of Cell Science . 113 . 3725–36 . November 2000 . 11034901 . 21. 10.1242/jcs.113.21.3725 .
  20. Burgstaller G, Kranewitter WJ, Gimona M . The molecular basis for the autoregulation of calponin by isoform-specific C-terminal tail sequences . Journal of Cell Science . 115 . Pt 10 . 2021–9 . May 2002 . 10.1242/jcs.115.10.2021 . 11973344 .
  21. Takahashi K, Hiwada K, Kokubu T . Isolation and characterization of a 34,000-dalton calmodulin- and F-actin-binding protein from chicken gizzard smooth muscle . Biochemical and Biophysical Research Communications . 141 . 1 . 20–6 . November 1986 . 3606745 . 10.1016/s0006-291x(86)80328-x.
  22. Allen BG, Walsh MP . The biochemical basis of the regulation of smooth-muscle contraction . Trends in Biochemical Sciences . 19 . 9 . 362–8 . September 1994 . 7985229 . 10.1016/0968-0004(94)90112-0.
  23. Naka M, Kureishi Y, Muroga Y, Takahashi K, Ito M, Tanaka T . Modulation of smooth muscle calponin by protein kinase C and calmodulin . Biochemical and Biophysical Research Communications . 171 . 3 . 933–7 . September 1990 . 2222454 . 10.1016/0006-291x(90)90773-g.
  24. Nigam R, Triggle CR, Jin JP . h1- and h2-calponins are not essential for norepinephrine- or sodium fluoride-induced contraction of rat aortic smooth muscle . Journal of Muscle Research and Cell Motility . 19 . 6 . 695–703 . August 1998 . 9742453 . 10.1023/a:1005389300151. 29905113 .
  25. Facemire C, Brozovich FV, Jin JP . The maximal velocity of vascular smooth muscle shortening is independent of the expression of calponin . Journal of Muscle Research and Cell Motility . 21 . 4 . 367–73 . May 2000 . 11032347 . 10.1023/a:1005680614296. 30450046 .
  26. Castro MM, Cena J, Cho WJ, Walsh MP, Schulz R . Matrix metalloproteinase-2 proteolysis of calponin-1 contributes to vascular hypocontractility in endotoxemic rats . Arteriosclerosis, Thrombosis, and Vascular Biology . 32 . 3 . 662–8 . March 2012 . 22199370 . 10.1161/ATVBAHA.111.242685 . free .
  27. Takahashi K, Yoshimoto R, Fuchibe K, Fujishige A, Mitsui-Saito M, Hori M, Ozaki H, Yamamura H, Awata N, Taniguchi S, Katsuki M, Tsuchiya T, Karaki H . Regulation of shortening velocity by calponin in intact contracting smooth muscles . Biochemical and Biophysical Research Communications . 279 . 1 . 150–7 . December 2000 . 11112431 . 10.1006/bbrc.2000.3909 .
  28. Masuki S, Takeoka M, Taniguchi S, Nose H . Enhanced baroreflex sensitivity in free-moving calponin knockout mice . American Journal of Physiology. Heart and Circulatory Physiology . 284 . 3 . H939–46 . March 2003 . 12433658 . 10.1152/ajpheart.00610.2002 .
  29. Somara S, Bitar KN . Direct association of calponin with specific domains of PKC-alpha . American Journal of Physiology. Gastrointestinal and Liver Physiology . 295 . 6 . G1246–54 . December 2008 . 18948438 . 10.1152/ajpgi.90461.2008 . 2604804.
  30. Walsh MP . The Ayerst Award Lecture 1990. Calcium-dependent mechanisms of regulation of smooth muscle contraction . Biochemistry and Cell Biology . 69 . 12 . 771–800 . December 1991 . 1818584 . 10.1139/o91-119.
  31. Kaneko T, Amano M, Maeda A, Goto H, Takahashi K, Ito M, Kaibuchi K . Identification of calponin as a novel substrate of Rho-kinase . Biochemical and Biophysical Research Communications . 273 . 1 . 110–6 . June 2000 . 10873572 . 10.1006/bbrc.2000.2901 .
  32. Fraser ED, Walsh MP . Dephosphorylation of calponin by type 2B protein phosphatase . Biochemistry . 34 . 28 . 9151–8 . July 1995 . 7619814 . 10.1021/bi00028a026.
  33. Ichikawa K, Ito M, Okubo S, Konishi T, Nakano T, Mino T, Nakamura F, Naka M, Tanaka T . Calponin phosphatase from smooth muscle: a possible role of type 1 protein phosphatase in smooth muscle relaxation . Biochemical and Biophysical Research Communications . 193 . 3 . 827–33 . June 1993 . 8391807 . 10.1006/bbrc.1993.1700 .
  34. Jin JP, Walsh MP, Sutherland C, Chen W . A role for serine-175 in modulating the molecular conformation of calponin . The Biochemical Journal . 350 . 579–88 . September 2000 . 10947974 . 10.1042/0264-6021:3500579 . 2 . 1221287.
  35. Tang DC, Kang HM, Jin JP, Fraser ED, Walsh MP . Structure-function relations of smooth muscle calponin. The critical role of serine 175 . The Journal of Biological Chemistry . 271 . 15 . 8605–11 . April 1996 . 8621490 . 10.1074/jbc.271.15.8605. free .
  36. Gerthoffer WT, Pohl J . Caldesmon and calponin phosphorylation in regulation of smooth muscle contraction . Canadian Journal of Physiology and Pharmacology . 72 . 11 . 1410–4 . November 1994 . 7767886 . 10.1139/y94-203.
  37. Bárány M, Bárány K . Calponin phosphorylation does not accompany contraction of various smooth muscles . Biochimica et Biophysica Acta (BBA) - Molecular Cell Research . 1179 . 2 . 229–33 . November 1993 . 8218366 . 10.1016/0167-4889(93)90146-g.
  38. Gimona M, Sparrow MP, Strasser P, Herzog M, Small JV . Calponin and SM 22 isoforms in avian and mammalian smooth muscle. Absence of phosphorylation in vivo . European Journal of Biochemistry . 205 . 3 . 1067–75 . May 1992 . 1576991 . 10.1111/j.1432-1033.1992.tb16875.x. free .

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