Yasuhiro Saito

Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity

Helicobacter pylori cagA-positive strains are associated with gastritis, ulcerations and gastric adenocarcinoma. CagA is delivered into gastric epithelial cells and, on tyrosine phosphorylation, specifically binds and activates the SHP2 oncoprotein, thereby inducing the formation of an elongated cell shape known as the ‘hummingbird’ phenotype. In polarized epithelial cells, CagA also disrupts the tight junction and causes loss of apical–basolateral polarity. We show here that H. pylori CagA specifically interacts with PAR1/MARK kinase, which has an essential role in epithelial cell polarity. Association of CagA inhibits PAR1 kinase activity and prevents atypical protein kinase C (aPKC)-mediated PAR1 phosphorylation, which dissociates PAR1 from the membrane, collectively causing junctional and polarity defects. Because of the multimeric nature of PAR1 (ref. 14), PAR1 also promotes CagA multimerization, which stabilizes the CagA–SHP2 interaction. Furthermore, induction of the hummingbird phenotype by CagA-activated SHP2 requires simultaneous inhibition of PAR1 kinase activity by CagA. Thus, the CagA–PAR1 interaction not only elicits the junctional and polarity defects but also promotes the morphogenetic activity of CagA. Our findings revealed that PAR1 is a key target of H. pylori CagA in the disorganization of gastric epithelial architecture underlying mucosal damage, inflammation and carcinogenesis.

Helicobacter pylori CagA interacts with E-cadherin and deregulates the β -catenin signal that promotes intestinal transdifferentiation in gastric epithelial cells

Infection with Helicobacter pylori cagA-positive strains is associated with gastric adenocarcinoma. Intestinal metaplasia is a precancerous lesion of the stomach characterized by transdifferentiation of the gastric mucosa to an intestinal phenotype. The H. pylori cagA gene product, CagA, is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Tyrosine-phosphorylated CagA specifically binds to and activates SHP-2 phosphatase, thereby inducing cell-morphological transformation. We report here that CagA physically interacts with E-cadherin independently of CagA tyrosine phosphorylation. The CagA/E-cadherin interaction impairs the complex formation between E-cadherin and β-catenin, causing cytoplasmic and nuclear accumulation of β-catenin. CagA-deregulated β-catenin then transactivates β-catenin-dependent genes such as cdx1, which encodes intestinal specific CDX1 transcription factor. In addition to β-catenin signal, CagA also transactivates p21WAF1/Cip1, again, in a phosphorylation-independent manner. Consequently, CagA induces aberrant expression of an intestinal-differentiation marker, goblet-cell mucin MUC2, in gastric epithelial cells that have been arrested in G1 by p21WAF1/Cip1. These results indicate that perturbation of the E-cadherin/β-catenin complex by H. pylori CagA plays an important role in the development of intestinal metaplasia, a premalignant transdifferentiation of gastric epithelial cells from which intestinal-type gastric adenocarcinoma arises.

SHP2 Tyrosine Phosphatase Converts Parafibromin/Cdc73 from a Tumor Suppressor to an Oncogenic Driver

Deregulation of SHP2 is associated with malignant diseases as well as developmental disorders. Although SHP2 is required for full activation of RAS signaling, other potential roles in cell physiology have not been elucidated. Here we show that SHP2 dephosphorylates parafibromin/Cdc73, a core component of the RNA polymerase II-associated factor (PAF) complex. Parafibromin is known to act as a tumor suppressor that inhibits cyclin D1 and c-myc by recruiting SUV39H1 histone methyltransferase. However, parafibromin can also act in the opposing direction by binding β-catenin, thereby activating promitogenic/oncogenic Wnt signaling. We found that, on tyrosine dephosphorylation by SHP2, parafibromin acquires the ability to stably bind β-catenin. The parafibromin/β-catenin interaction overrides parafibromin/SUV39H1-mediated transrepression and induces expression of Wnt target genes, including cyclin D1 and c-myc. Hence, SHP2 governs the opposing functions of parafibromin, deregulation of which may cause the development of tumors or developmental malformations.

Helicobacter pylori CagA causes mitotic impairment and induces chromosomal instability.

Infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric carcinoma. The cagA gene product CagA, which is delivered into gastric epithelial cells, specifically binds to and aberrantly activates SHP-2 oncoprotein. CagA also interacts with and inhibits partitioning-defective 1 (PAR1)/MARK kinase, which phosphorylates microtubule-associated proteins to destabilize microtubules and thereby causes epithelial polarity defects. In light of the notion that microtubules are not only required for polarity regulation but also essential for the formation of mitotic spindles, we hypothesized that CagA-mediated PAR1 inhibition also influences mitosis. Here, we investigated the effect of CagA on the progression of mitosis. In the presence of CagA, cells displayed a delay in the transition from prophase to metaphase. Furthermore, a fraction of the CagA-expressing cells showed spindle misorientation at the onset of anaphase, followed by chromosomal segregation with abnormal division axis. The effect of CagA on mitosis was abolished by elevated PAR1 expression. Conversely, inhibition of PAR1 kinase elicited mitotic delay similar to that induced by CagA. Thus, CagA-mediated inhibition of PAR1, which perturbs microtubule stability and thereby causes microtubule-based spindle dysfunction, is involved in the prophase/metaphase delay and subsequent spindle misorientation. Consequently, chronic exposure of cells to CagA induces chromosomal instability. Our findings reveal a bifunctional role of CagA as an oncoprotein: CagA elicits uncontrolled cell proliferation by aberrantly activating SHP-2 and at the same time induces chromosomal instability by perturbing the microtubule-based mitotic spindle. The dual function of CagA may cooperatively contribute to the progression of multistep gastric carcinogenesis.

Structural and functional diversity in the PAR1b/ MARK2-binding region of Helicobacter pylori CagA

Helicobacter pylori (H. pylori) cagA‐positive strains are associated with gastritis, peptic ulcerations, and gastric adenocarcinoma. Upon delivery into gastric epithelial cells, the cagA‐encoded CagA protein specifically binds and aberrantly activates SHP‐2 oncoprotein in a manner that is dependent on CagA tyrosine phosphorylation. CagA‐deregulated SHP‐2 then elicits aberrant Erk activation while causing an elongated cell shape known as the hummingbird phenotype. In polarized epithelial cells, CagA also binds to PAR1b/MARK2 and inhibits the PAR1b kinase activity, thereby disrupting tight junctions and epithelial cell polarity independent of CagA tyrosine phosphorylation. We show here that the CagA‐multimerization (CM) sequence that mediates interaction of CagA with PAR1b is not only essential for the CagA‐triggered junctional defects but also plays an important role in induction of the hummingbird phenotype by potentiating CagA‐SHP‐2 complex formation. We also show that the CM sequence of CagA isolated from East Asian H. pylori (referred to as the E‐CM sequence) binds PAR1b more strongly than that of CagA isolated from Western H. pylori (referred to as the W‐CM sequence). Within Western CagA species, the ability to bind PAR1b is proportional to the number of W‐CM sequences. Furthermore, the level of PAR1b‐binding activity of CagA correlates with the magnitude of junctional defects and the degree of hummingbird phenotype induction. Our findings reveal that structural diversity in the CM sequence is an important determinant for the degree of virulence of CagA, a bacterial oncoprotein that is associated with gastric carcinogenesis. (Cancer Sci 2008; 99: 2004–2011)

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.

Polarity-regulating Kinase Partitioning-defective 1b (PAR1b) Phosphorylates Guanine Nucleotide Exchange Factor H1 (GEF-H1) to Regulate RhoA-dependent Actin Cytoskeletal Reorganization

Background: Polarity-regulating kinase PAR1b is also involved in regulation of the actin cytoskeleton. Results: PAR1b inhibits RhoA activator GEF-H1 by inducing phosphorylation on S885 and S959. Conclusion: PAR1b controls RhoA activity through phosphorylation-dependent regulation of GEF-H1. Significance: PAR1b coordinates the microtubule- and actin-cytoskeletal systems in cell regulation. Partitioning-defective 1b (PAR1b), also known as microtubule affinity-regulating kinase 2 (MARK2), is a member of evolutionally conserved PAR1/MARK serine/threonine kinase family, which plays a key role in the establishment and maintenance of cell polarity at least partly by phosphorylating microtubule-associated proteins (MAPs) that regulate microtubule stability. PAR1b has also been reported to influence actin cytoskeletal organization, raising the possibility that PAR1b functionally interacts with the Rho family of small GTPases, central regulators of the actin cytoskeletal system. Consistent with this notion, PAR1 was recently found to be physically associated with a RhoA-specific guanine nucleotide exchange factor H1 (GEF-H1). This observation suggests a functional link between PAR1b and GEF-H1. Here we show that PAR1b induces phosphorylation of GEF-H1 on serine 885 and serine 959. We also show that PAR1b-induced serine 885/serine 959 phosphorylation inhibits RhoA-specific GEF activity of GEF-H1. As a consequence, GEF-H1 phosphorylated on both of the serine residues loses the ability to stimulate RhoA and thereby fails to induce RhoA-dependent stress fiber formation. These findings indicate that PAR1b not only regulates microtubule stability through phosphorylation of MAPs but also influences actin stress fiber formation by inducing GEF-H1 phosphorylation. The dual function of PAR1b in the microtubule-based cytoskeletal system and the actin-based cytoskeletal system in the coordinated regulation of cell polarity, cell morphology, and cell movement.

Mammalian Pragmin regulates Src family kinases via the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif that is exploited by bacterial effectors.

Several pathogenic bacteria have adopted effector proteins that, upon delivery into mammalian cells, undergo tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) or EPIYA-like sequence motif by host kinases such as Src family kinases (SFKs). This EPIYA phosphorylation triggers complex formation of bacterial effectors with SH2 domain-containing proteins that results in perturbation of host cell signaling and subsequent pathogenesis. Although the presence of such an anomalous protein interaction suggests the existence of a mammalian EPIYA-containing protein whose function is mimicked or subverted by bacterial EPIYA effectors, no molecule that uses the EPIYA motif for biological function has so far been reported in mammals. Here we show that mammalian Pragmin/SgK223 undergoes tyrosine phosphorylation at the EPIYA motif by SFKs and thereby acquires the ability to interact with the SH2 domain of the C-terminal Src kinase (Csk), a negative regulator of SFKs. The Pragmin–Csk interaction prevents translocalization of Csk from the cytoplasm to the membrane and subsequent inactivation of membrane-associated SFKs. As a result, SFK activity is sustained in cells where Pragmin is phosphorylated at the EPIYA motif. Because EPIYA phosphorylation of Pragmin is mediated by SFKs, cytoplasmic sequestration of Csk by Pragmin establishes a positive feedback regulation of SFK activation. Remarkably, the Helicobacter pylori EPIYA effector CagA binds to the Csk SH2 domain in place of Pragmin and enforces membrane recruitment of Csk and subsequent inhibition of SFKs. This work identifies Pragmin as a mammalian EPIYA effector and suggests that bacterial EPIYA effectors target Pragmin to subvert SFKs for successful infection.

A study of acidic glycosaminoglycans in human gastric tissue.

The acidic glycosaminoglycans (AGAG) in normal portions of human gastric tissue were separated by electrophoresis in 3 buffer systems. Paper chromatographic separation of the constitutional disaccharide units by digestion of chondroitin sulfates (CS) with chondroitinase-ABC and chondroitinase-AC was carried out after fractionation of CS by ion-exchange resin column chromatography. Thin-layer chromatography of hexosamines and other biochemical analysis were also performed. The presence of hyaluronic acid in the gastric tissue was substantiated by the enzymatic susceptibility to streptomyces hyaluronidase. The results indicated that human gastric AGAG consisted of, in the order of amount, heparan sulfates, dermatan sulfate, hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate and presumably oversulfated chondroitin sulfate.

Roentgenologic studies on the small intestine (XV) Radiographic features of the small intestine after a barium-lactose mixture meal in japanese

ConclusionIn all of the healthy Japanese, radiographic studies of the small intestine examined with a barium suspension added 50 g or 25 g lactose displayed marked or moderate abnormalities comprising barium dilation and intestinal hurry. The results of observation after 10 g lactose load, showed moderate abnormalities in all of 4 milk-diarrhea. However, being examined by this method, about a half of healthy controls showed similar findings as well.