Ezrin-calpain I interactions in gastric parietal cells

Yao, Xuebiao; Thibodeau, Alain; Forte, John G.

doi:10.1152/ajpcell.1993.265.1.c36

Cited by 90 publications

(75 citation statements)

References 27 publications

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“…These 80-kDa spots clearly represent ezrin in the typical 2-D separation profile that is characteristic of phosphorylated isotypes of ezrin (Hanzel et al, 1991). Moreover, the 6H11-antibody-positive spots, migrating at 55 kDa in the range of pH 6.9 to 6.7, represent proteolytic products of ezrin as reported by Yao et al (1993). The portion of the blot probed for y-actin shows a major spot with an Mr-43 kDa in the pH range of -5.2, and a minor slightly more acidic spot.…”

Section: Co-immunoprecipitation Of Actin Isoforms Together With Ezrinsupporting

confidence: 61%

“…Western blots of these same fractions were probed for ezrin and actin (Figure 8, middle and lower panels). The 6H11 antibody ( Figure 8, middle panel) was used to verify the 80-kDa band as authentic ezrin and that the band(s) at -55 kDa represents a proteolytic product of ezrin as reported by Yao et al (1993). The lower panel of Figure 8 shows that the 43-kDa band in the HCl-eluted fractions clearly cross-reacted with the C4 antibody, confirming this peptide as actin.…”

Section: Co-immunoprecipitation Of Actin Isoforms Together With Ezrinmentioning

confidence: 59%

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Polarized distribution of actin isoforms in gastric parietal cells.

Yao

Chaponnier

Gabbiani

et al. 1995

MBoC

109

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The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic ,B-actin and -y-actin. Densitometry revealed a ratio for f3-actin/ y-actin that equaled 0.73 + 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the ,B-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of ,B-actin were also identified near the basolateral membrane. The y-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of ,B-actin, but not y-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of 13-actin/ y-actin in the immunoprecipitate (3/,y = 2.14 + 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that ,B-and 'y-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between 1B-actin and ezrin in gastric parietal cells.Finally, our results suggest that the ,B-and y-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion. INTRODUCTION essential role in a variety of cellular processes such as maintaining cell shape, cell motility, and cytokinesis.

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Section: Co-immunoprecipitation Of Actin Isoforms Together With Ezrinsupporting

confidence: 61%

Section: Co-immunoprecipitation Of Actin Isoforms Together With Ezrinmentioning

confidence: 59%

Polarized distribution of actin isoforms in gastric parietal cells.

Yao

Chaponnier

Gabbiani

et al. 1995

MBoC

109

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“…Calpain proteolysis of ezrin with associated cytoskeletal changes has been demonstrated in endothelial cells, lymphocytes, neutrophils, NIH-3T3 cells, renal proximal tubule cells and gastric parietal cells Ariyoshi et al, 2001;Chen et al, 1994;Potter et al, 1998;Shcherbina et al, 1999;Yao et al, 1993). Ezrin co-localized with acetylated α-tubulin, a marker of ciliated cells, in the pulmonary epithelium.…”

Section: Discussionmentioning

confidence: 98%

“…One of the cytoskeletal proteins sensitive to calpain proteolysis is the linking protein ezrin (Yao et al, 1993). Ezrin is an 80/81 kDa protein originally described as a substrate for receptor protein tyrosine kinases (Crepaldi et al, 1997;Gould et al, 1986).…”

Section: Calpastatin Is Decreased In the Pulmonary Epithelium In The mentioning

confidence: 99%

Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells

Gomperts

Gong-Cooper

Hackett

2004

Journal of Cell Science

122

102

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The forkhead box transcription factor Foxj1 is required for cilia formation and left-right axis determination. To define the role of Foxj1 in ciliogenesis, microarray analysis was performed to identify differentially expressed genes in the pulmonary epithelium of foxj1 +/+ and foxj1 -/-mice. In the absence of Foxj1, the expression of calpastatin, an inhibitor of the protease calpain, decreased. RNase protection confirmed the decrease in calpastatin expression and decreased calpastatin was detected in the proximal pulmonary epithelium of foxj1 -/-mice by immunohistochemistry. No change was detected in the expression of calpain 2 in the pulmonary epithelium by western blot or immunohistochemistry. By western blot and immunofluorescence, ezrin, a substrate for calpain, was also found to decrease in the pulmonary epithelium of foxj1 -/-mice. No change in ezrin gene expression was found by RT-PCR. A decrease in ezrin binding phosphoprotein-50 (EBP-50) was also detected by immunofluorescence in the foxj1 -/-mouse pulmonary epithelium. Immunoelectron microscopy demonstrated ezrin associated with the basal bodies of cilia in the pulmonary epithelium. Treatment of tracheal explants from foxj1 -/-mice with a calpain inhibitor resulted in a partial reappearance of cilia observed in these mice. Additionally, following treatment of foxj1 -/-tracheal explants with calpain inhibitor, basal bodies were observed in an apical location along with relocalization of ezrin and EBP-50. Regulation of calpain activity by calpastatin thus provides a mechanism for regulating the anchoring of basal bodies to the apical cytoskeleton in ciliated cells. In the absence of Foxj1, decreased calpastatin expression with decreased ezrin and EBP-50 results in an inability of basal bodies to anchor to the apical cytoskeleton and subsequent failure of axonemal formation.

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“…It is reported to be down-regulated in a subset of gastric cancer patients and is involved in the suppression of tumorigenesis (20 -22). -Calpain is also involved in the acid secretory process by cleaving ezrin, a membrane cytoskeletal protein, in parietal cells (23). How these data relate to the physiological functions of nCL-2/-2Ј has yet to be elucidated.…”

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confidence: 99%

Stomach-specific Calpain, nCL-2, Localizes in Mucus Cells and Proteolyzes the β-Subunit of Coatomer Complex, β-COP

Hata

Koyama

Kawahara

et al. 2006

Journal of Biological Chemistry

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Calpain is a Ca2؉ -regulated cytosolic protease. Mammals have 14 calpain genes, half of which are predominantly expressed in specific organ(s); the rest are expressed ubiquitously. A defect in calpains causes lethality/pathogenicity, indicating their physiological indispensability. nCL-2/calpain-8a was identified as a stomach-specific calpain, whose physiological functions are unclear. To elucidate these, we characterized nCL-2 in detail. Unexpectedly, nCL-2 was localized strictly to the surface mucus cells in the gastric epithelium and the mucus-secreting goblet cells in the duodenum. Yeast twohybrid screening identified several nCL-2-intracting molecules. Of these, the ␤-subunit of coatomer complex (␤-COP) occurs in the stomach pit cells and is proteolyzed by nCL-2 in vitro. Furthermore, ␤-COP and nCL-2 co-expressed in COS7 cells co-localized in the Golgi, and Ca 2؉ -ionophore stimulation caused the proteolysis of ␤-COP near the linker region, resulting in the dissociation of ␤-COP from the Golgi. These results strongly suggest novel functions for nCL-2 that involve the membrane trafficking of mucus cells via interactions with coat protein.Calpain (EC 3.4.22.17) is an intracellular Ca 2ϩ -regulated cysteine protease, comprising a superfamily in most organisms, including some bacteria, budding yeasts, fungi, plants, and animals. Calpains proteolyze specific substrates at very limited sites to irreversibly modify/modulate their functions, structures, and activities, and are thus called "modulator proteases." Fourteen calpain genes have been identified in humans and mice and can be classified into two categories according to their expression, i.e. ubiquitous or tissue-specific (1-3). Ubiquitous -and m-calpains, two major calpains in mammals, form a heterodimer composed of a distinct 80-kDa catalytic large subunit (abbreviated to "CL" and "mCL," respectively) 2 and a common 30-kDa regulatory small subunit (30K). Calpain three-dimensional structures define four (I-IV) and two (V and VI) domains in the large and small subunits, respectively: the regulatory N-terminal domain (I), the protease domain (II), the C2-domain-like Ca 2ϩ /phospholipid-binding domain (III), the penta-EF-hand domains (IV and VI), and the Glyclustering domain (V) (4, 5). In the absence of Ca 2ϩ , the protease domain (II) is further divided into two subdomains, IIa and IIb, which form a single active domain upon binding to Ca 2ϩ (6, 7). Genetic studies in mammals and other organisms have clearly shown the physiological indispensability of the calpains. A gene knock-out mouse with a mutation in the gene for 30K, Capn4, exhibits embryonic lethality (8); mutations in the human gene for skeletal-muscle-specific p94/calpain 3, CAPN3, are responsible for limb-girdle muscular dystrophy type 2A (9); and a single nucleotide polymorphism in intron 3 of the human gene for calpain 10, CAPN10, is associated with type 2 diabetes (10). Mutations in the calpain genes of various other organisms, including yeasts, plants, nematodes, and Drosophila, result in sign...

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Ezrin-calpain I interactions in gastric parietal cells

Cited by 90 publications

References 27 publications

Polarized distribution of actin isoforms in gastric parietal cells.

Polarized distribution of actin isoforms in gastric parietal cells.

Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells

Stomach-specific Calpain, nCL-2, Localizes in Mucus Cells and Proteolyzes the β-Subunit of Coatomer Complex, β-COP

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