Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells

Abstract

A phosphoryl protein tyrosine phosphatase (PTPase) activity has been characterized in rat pancreatic acinar membranes using 32P-labeled poly(Glu,Tyr) as substrate. Acinar membranes exhibited a high affinity for the substrate, with an apparent K , of 0.46 pM and an apparent V,,, of 0.9 nmol . mg protein-. min-Acinar membrane PTPase activity displayed specific characteristics of other PTPases; it was inhibited by the inhibitors Zn2+, orthovanadate and by the divalent cations Mn2+ and Mg2+, and was stimulated by the reducing-agent dithiothreitol. It was also inhibited by soybean trypsin inhibitor and stimulated by trypsin. Gel permeation of pancreatic acinar membranes gave a single peak of enzyme activity with an apparent molecular mass of 70000 Da. Further purification by HPLC on DEAE revealed two peaks of PTPase activity at 120 mM and 180 mM NaCl. These two peaks reacted in a Western-blot procedure with anti-(peptide) serum directed towards conserved domain of PTPase as a common 67-kDa form associated with lower-molecular-mass proteolytic fragments (31 -56 kDa). Incubation of pancreatic acini with somatostatin analogues, SMS 201-995 or BIM 23014, resulted in a stimulation of membrane PTPase activity. The stimulation was rapid and transient, with a maximal level reached within 15 min of addition. The two analogs stimulated PTPase activity in a dose-dependent manner with half-maximal activation occurring at 7 pM and 37 pM and maximal activation at 0.1 nM and 0.1 -1 nM for SMS 201-995 and BIM 23014, respectively. The stimulated-membrane PTPase activity also eluted at an apparent molecular mass of 70 kDa in gel-permeation chromatography. The two analogs inhibited the binding of [1251-Tyr3]SMS 201-995 to pancreatic acinar membranes with similar relative potencies to that observed on stimulation of PTPase activity. We conclude that pancreatic acinar membranes possess a low-molecular-mass PTPase which is stimulated by somatostatin analogs at concentrations involving activation of membrane somatostatin receptors.Protein tyrosine phosphorylation plays a crucial role in cellular regulation of various functions such as proliferation, differentiation and transformation and is controlled by two sets of enzymes; protein tyrosine kinases (PTK) and protein tyrosine phosphatase (PTPase). Whereas the field of PTK has grown explosively over the last 10 years, less is known about PTPase.Recently, a cytosolic PTPase from human placenta (PTPIB) was purified as a 37-kDa soluble protein [l, 21 and its amino acid sequence showed an unexpected similarity to cytoplasmic domains I and I1 of human leukocyte commun antigen CD45 [3], and leukocyte-commun-antigen-related molecule (LAR) [4]. CD45 and LAR have been subsequently shown to have intrinsic PTPase activity in their cytoplasmic domain [5, 61. The two proteins resemble receptor molecules since they have a transmembrane domain, a cytoplasmic domain composed of two repeated PTPase domains and contain different extracellular domains. More recently, a novel recep-

The Tyrosine Phosphatase SHP-1 Associates with the sst2 Somatostatin Receptor and Is an Essential Component of sst2-mediated Inhibitory Growth Signaling

“…We and others have demonstrated that somatostatin and analogues induce the stimulation of a membrane PTPase, which may be involved in the inhibitory effect of these peptides on cell proliferation (9,11,12,38). More recently, we have established the role of the somatostatin receptor sst2 in mediating the stimulatory effect of somatostatin on PTPase activity and its negative effect on cell growth (13,14).…”

“…In pancreatic tumor cells, we and others have previously shown that somatostatin and analogues antagonize the mitogenic effect of growth factors acting on tyrosine kinase receptors such as epidermal growth factor (9,10). Although the molecular events leading to the inhibition of cell proliferation are still poorly understood, it has been shown that, after binding to somatostatin receptors, somatostatin analogues cause a rapid stimulation of a membrane proteintyrosine phosphatase (PTPase) 1 activity and dephosphorylate phosphorylated epidermal growth factor receptors (9,11,12) suggesting that a PTPase may participate in the somatostatininduced inhibition of growth factor-mediated mitogenic signal. Recently, the expression of the sst2 somatostatin receptor subtype in NIH3T3 and Chinese hamster ovary (CHO) cells led us to the demonstration of the direct involvement of sst2 in both the antiproliferative effect of somatostatin and its stimulatory effect on PTPase activity (13,14).…”

Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and SSTR5 through different mechanisms.

“…We report evidence that only SSTR2 and -5 mediate the inhibition of cell proliferation induced by RC-160 and that these two receptor subtypes are coupled to distinct signal transduction pathways. The activation of a tyrosine phosphatase and the inhibition of calcium mobilization mediated, respectively, by SSTR2 and Abbreviations: S-14, somatostatin-14; SSTR1 to -5, somatostatin receptor subtypes 1 (22). After the CHO cells were grown in 75-cm2 flasks for 48 h, the cells were washed with ice-cold aMEM, scraped off, and resuspended in aMEM containing bovine serum albumin (1 mg/ml) and soybean trypsin inhibitor (0.3 mg/ml).…”

“…After centrifugation at 10,000 x g for 30 sec, aMEM was removed and cells were quickly lysed in liquid nitrogen in 50 mM Tris-HCl (pH 7.4) containing soybean trypsin inhibitor (0.3 mg/ml) and 0.1 mM phenylmethylsulfonyl fluoride. After thawing, the homogenate was assayed for the tyrosine phosphatase activity in 100 ,ul containing 50 mM Tris HCl (pH 7.0), bovine serum albumin (1 mg/ml), 30,000 cpm of 33P-labeled poly(Glu, Tyr), 1 gg of protein, and 5 mM dithiothreitol as described (10,22 …”

Bombesin induces a reduction of somatostatin inhibition of adenylyl cyclase activity, Gi function, and somatostatin receptors in rat exocrine pancreas

“…Binding of [ 125 I-Tyr 11 ]SS was performed on rat pancreatic acinar membranes by a modification of the method of Colás et al [7]. Tyr 11 -SS was radioiodinated by the chloramine-T method as described by Greenwood et al [15].…”