SHP-1 associates with both platelet-derived growth factor receptor and the p85 subunit of phosphatidylinositol 3-kinase.

The Src homology 2 (SH2)-containing protein tyrosine phosphatase 1, SHP-1, is highly expressed in all hematopoietic cells as well as in many non-hematopoietic cells, particularly in some malignant epithelial cell lines. In hematopoietic cells, SHP-1 negatively regulates multiple cytokine receptor pathways. The precise function and the targets of SHP-1 in non-hematopoietic cells, however, are largely unknown. Here we demonstrate that SHP-1 associates with both the tyrosine-phosphorylated platelet-derived growth factor (PDGF) receptor and the p85 subunit of phosphatidylinositol 3-kinase in MCF-7 and TRMP cells. Through the use of mutant PDGF receptors and performing peptide competition for immunoprecipitation, it was determined that SHP-1 independently associates with the PDGF receptor and p85 and that its N-terminal SH2 domain is directly responsible for the interactions. Overexpression of SHP-1 in TRMP cells transfected with the PDGF receptor markedly inhibited PDGF-induced c-fos promoter activation, whereas the expression of three catalytically inactive SHP-1 mutants increased the c-fos promoter activation in response to PDGF stimulation. These results indicate that SHP-1 might negatively regulate PDGF receptor-mediated signaling in these cells. Identification of the association of SHP-1 with the PDGF receptor and p85 in MCF-7 and TRMP cells furthers our understanding of the function of SHP-1 in non-hematopoietic cells.

Localization and down-regulating role of the protein tyrosine phosphatase PTP2C in membrane ruffles of PDGF-stimulated cells.

PTP2C (also known as Syp/SH-PTP2/PTPlD) is a soluble protein tyrosine phosphatase present in most cell types. It interacts directly with activated PDGF receptor via its SH2 domains, which results in its phosphorylation on tyrosine residue(s). The phosphorylated PTP2C in turn binds to the SH2 domain of GRB2, serving as an adaptor in the transduction of mitogenic signals from the growth factor receptor to the Ras and MAP kinase signaling pathways. We investigated the interaction of PTP2C with the PDGF receptor by examining the localization of both proteins after PDGF stimulation of 293 cells which stably express the human PDGF receptor. In resting cells, transiently expressed PTP2C was distributed throughout the cytoplasm. Upon stimulation with PDGF, PTP2C was translocated from the cytoplasm to membrane ruffles. Immunofluorescence examination revealed that PTP2C colocalized with actin, the PDGF receptors, and hyper-tyrosine- phosphorylated protein(s). Neither deletion of the SH2 domains nor point mutations at either the catalytic site or the major phosphorylation site affected membrane ruffling or the localization of PTP2C to the ruffles of PDGF-stimulated cells. However, the expression of a catalytically inactive mutant PTP2C substantially prolonged ruffling activity following PDGF stimulation. These results suggest that PTP2C is involved in the down-regulation of the membrane ruffling pathway, and in contrast to its positive function in the MAP kinase pathway, the phosphatase activity negatively regulates ruffling activity.

Beta 2-adrenoceptor density in fibroblast culture correlates with human NaCl sensitivity.

To study salt sensitivity in humans and its relation to expression of adrenoceptors, 20 male normotensive Caucasians were investigated on a diet of 180 mmol NaCl/day followed by 60 mmol NaCl/day over 2 wk and again by 180 mmol NaCl/day over 2 wk, and blood pressure changes were assessed by long-term oscillatory blood pressure monitoring under basal conditions. Individual cell cultures of skin fibroblasts from skin biopsies were also established, and alpha 2- and beta 2-adrenoceptors were measured. Seven subjects were salt sensitive, and the remainder were salt resistant. Cultured skin fibroblasts in salt-sensitive subjects express less than half the number of beta 2-adrenoceptors compared with salt-resistant subjects (65 +/- 12.7 vs. 173 +/- 14.8 fmol/mg, P less than 0.001), and there is a correlation between the absolute rise of blood pressure on a high-salt diet and the density of beta 2-adrenoceptors (r = -0.67, P less than 0.01). It remains to be established whether a reduced in vitro density of beta 2-adrenoceptors in cultured cells is causally related to salt sensitivity in normotensive humans.

Initial steps in the degradation of benzene sulfonic acid, 4-toluene sulfonic acids, and orthanilic acid in Alcaligenes sp. strain O-1.

Alcaligenes sp. strain O-1 grew with benzene sulfonate (BS) as sole carbon source for growth with either NH4+ or NH4+ plus orthanilate (2-aminobenzene sulfonate, OS) as the source(s) of nitrogen. The intracellular desulfonative enzyme did not degrade 3- or 4-aminobenzene sulfonates in the medium, although the enzyme in cell extracts degraded these compounds. We deduce the presence of a selective permeability barrier to sulfonates and conclude that the first step in sulfonate metabolism is transport into the cell. Cell-free desulfonation of BS in standard reaction mixtures required 2 mol of O2 per mol. One mol of O2 was required for a catechol 2,3-dioxygenase. When meta ring cleavage was inhibited with 3-chlorocatechol in desalted extracts, about 1 mol each of O2 and of NAD(P)H per mol of BS were required for the reaction, and SO3(2-) and catechol were recovered in high yield. Catechol was shown to be formed by dioxygenation in an experiment involving 18O2. 4-Toluene sulfonate was subject to NAD(P)H-dependent dioxygenation to yield SO3(2-) and 4-methylcatechol, which was subject to meta cleavage. OS also required 2 mol of O2 per mol and NAD(P)H for degradation, and SO3(2-) and NH4+ were recovered quantitatively. Inhibition of ring cleavage with 3-chlorocatechol reduced the oxygen requirement to 1 mol per mol of OS SO3(2-) (1 mol) and an unidentified organic intermediate, but no NH4+, were observed.