Fukashi Ochi

PI 3-KINASE GAMMA AND PROTEIN KINASE C-ZETA MEDIATE RAS-INDEPENDENT ACTIVATION OF MAP KINASE BY A GI PROTEIN-COUPLED RECEPTOR

Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS‐dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS‐independent manner in CHO cells that overexpress a dominant‐negative mutant of the guanine nucleotide exchange protein SOS (CHO‐ΔSOS cells). LPA also induced the activation of MAP kinase kinase (MEK), but not that of RAF1, in CHO‐ΔSOS cells. The RAS‐independent activation of MAP kinase by LPA was blocked by inhibitors of phosphatidylinositol 3‐kinase (PI3K) or by overexpression of a dominant‐negative mutant of the γ isoform of PI3K. Furthermore, LPA induced the activation of the atypical ζ isoform of protein kinase C (PKC‐ζ) in CHO‐ΔSOS cells in a manner that was sensitive to wortmannin or to the dominant‐negative mutant of PI3Kγ, and overexpression of a dominant‐negative mutant of PKC‐ζ inhibited LPA‐induced activation of MAP kinase. These observations indicate that Gi protein‐coupled receptors induce activation of MEK and MAP kinase through a RAS‐independent pathway that involves PI3Kγ‐dependent activation of atypical PKC‐ζ.

Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Roles of Fak and Src family kinases

SHPS-1 is a receptor-like glycoprotein that undergoes tyrosine phosphorylation and binds SHP-2, an Src homology 2 domain containing protein tyrosine phosphatase, in response to various mitogens. Cell adhesion to extracellular matrix proteins such as fibronectin and laminin also induced the tyrosine phosphorylation of SHPS-1 and its association with SHP-2. These responses were markedly reduced in cells overexpressing the Csk kinase or in cells that lack focal adhesion kinase or the Src family kinases Src or Fyn. However, unlike Src, focal adhesion kinase did not catalyze phosphorylation of the cytoplasmic domain of SHPS-1 in vitro. Overexpression of a catalytically inactive SHP-2 markedly inhibited activation of mitogen-activated protein (MAP) kinase in response to fibronectin stimulation without affecting the extent of tyrosine phosphorylation of focal adhesion kinase or its interaction with the docking protein Grb2. Overexpression of wild-type SHPS-1 did not enhance fibronectin-induced activation of MAP kinase. These results indicate that the binding of integrins to the extracellular matrix induces tyrosine phosphorylation of SHPS-1 and its association with SHP-2, and that such phosphorylation of SHPS-1 requires both focal adhesion kinase and an Src family kinase. In addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, SHP-2 may play an important role, partly through its interaction with SHPS-1, in the activation of MAP kinase in response to the engagement of integrins by the extracellular matrix.

Roles of the Complex Formation of SHPS-1 with SHP-2 in Insulin-stimulated Mitogen-activated Protein Kinase Activation

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathioneS-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.

Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: Roles of RHO, FAK, and a SRC family kinase

SHPS-1 is an ∼120 kDa glycosylated receptor like protein that contains three immunoglobulin-like domains in its extracellular region as well as four potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites in its cytoplasmic region. Lysophosphatidic acid (LPA) stimulated the rapid tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, a protein tyrosine phosphatase containing SH2 domains in Rat-1 fibroblasts. LAP-induced tyrosine phosphorylation of SHPS-1 was inhibited by Clostridium botulinum C3 exoenzyme (which inactivates RHO) but not by pertussis toxin. The protein kinase C activator phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulated tyrosine phosphorylation of SHPS-1; however, down-regulation of protein kinase C by prolonged exposure of cells to TPA did not affect LAP-induced tyrosine phosphorylation of SHPS-1. LPA-induced tyrosine phosphorylation of SHPS-1 was markedly reduced in either focal adhesion kinase (FAK)-deficient mouse cells or CHO cells overexpressing the tyrosine kinase CSK. Overexpression of a catalytically inactivate SHP-2 markedly inhibited MAP kinase activation in response to low concentrations of LPA in CHO cells, whereas overexpression of a wild-type SHPS-1 did enhance this effect of LPA. Furthermore, MAP kinase activation in response to a low concentration of LPA was inhibited by botulinum C3 exoenzyme. These results indicate that LPA-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2 may be mediated by a RHO-dependent pathway that includes FAK and a SRC family kinase. Thus, in addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, the formation of a complex between SHPS-1 and SHP-2 may, in part, play an important role in the activation of MAP kinase in response to low concentrations of LPA.

A case of ischemic colitis during pregnancy

mural inflammation, including degeneration of the muscularis mucosae and submucosa. The crypts were partly destroyed, with a ghost-like appearance characteristic of ischemic colitis (Fig. 2a). In the small capillaries in the submucosal layer, some fibrin thrombi were observed, showing a blue color with phosphotungstic acid-hematoxylin (PTAH) staining (Fig. 2b). Having ruled out an infectious cause, we diagnosed her illness as ischemic colitis. She was treated conservatively by fasting and intravenous hydration. She recovered and was discharged on the seventh hospital day. After a normal delivery, she remains asymptomatic, at 11 months after discharge. Ischemic colitis is a disorder of older adults, who frequently show generalized atherosclerosis as a predisposing condition. However, it is being recognized more frequently in young healthy adults, in whom transient ischemic colitis typically presents with an abrupt onset of left-side abdominal pain, occasional nausea and vomiting, and bloody diarrhea.3 In a young adult thought to have transient ischemic colitis, the illness is generally benign and self-limited.3 Most patients have only a single episode of transient Fig. 1. Endoscopic appearance of the stomach. Six type II c (superficial depressed type) lesions (1–3,5–7), one type II a lesion (superficial elevated type) (4), and an adenoma (lesion A) were observed

Pedunculated early carcinoma of supra-ampullary duodenum presenting as acute pancreatitis.

SummaryBackground. A 43-yr-old man was admitted to our hospital after sudden onset of epigastric pain. He was diagnosed with acute pancreatitis by clinical, laboratory, and radiographic signs. Examinations for the etiology of acute pancreatitis revealed a duodenal tumor arising at the proximal portion of the descending limb, extending by a long stalk, and coming into contact with Vater’s papilla. The tumor was snare-resected endoscopically. Histological examination showed an early carcinoma. Extra-ampullary carcinoma of the duodenum should be considered an unusual cause of acute pancreatitis secondary to obstruction of the major duodenal papilla. Endoscopic polypectomy is effective because of the difficulty in making a precise diagnosis by endoscopic biopsy.