13 research outputs found

    Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

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    <div><p>Receptor tyrosine phosphatase gamma (PTPRG, or RPTPĪ³) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98ā€“99% reduction of RPTPĪ³ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPĪ³ C1060S, replaced the wild type. We showed that human wild type RPTPĪ³ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPĪ³ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPĪ³ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPĪ³ for use in an assay for identifying inhibitors, ā€œsubstrate trappingā€ mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPĪ³s retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPĪ³ did not. This suggested that wild type RPTPĪ³ might auto-dephosphorylate which was confirmed by an <em>in vitro</em> dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPĪ³. Additional investigation of RPTPĪ³ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.</p> </div

    Determination of Km of phosphatase RPTPĪ³ on peptide substrate ATQDD(pY)VLEVR (derived from peptide adjacent to Y1307).

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    <p>Figure here shows Pi released (microM per minute) was plotted against phospho-peptide in a reaction in which Vmax was 2.1 ĀµM/mim/14 nM RPTPĪ³ and Km is 49.6 ĀµM, using one site nonlinear binding algorithm in Graphpad Prism.</p

    RPTPĪ³ dephosphorylated itself in vitro.

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    <p>Wild type RPTPĪ³ or RPTPĪ³ C1060S plasmids were transiently transfected into HEK293F and isolated from lysates with anti c-myc/protein G sepharoses. RPTPĪ³ wild type or C1060S on the protein G sepharose was denatured in 8 M urea to linearized protein as substrates for reaction following. The Purified RPTPĪ³ wild type, C1060s sepharose beads was then incubated with recombinant, active purified RPTPĪ³ cyto enzymes in assay buffer with urea at a final concentration of 0.375 M. Lanes 1 and 2 are RPTPĪ³ WT and RPTPĪ³ C1060S on protein G sepharose in a mock reaction without phosphatase in the same buffer, and lanes 1ā€²and 2ā€² are RPTPĪ³ wild type and RPTPĪ³ C1060S reacted with RPTPĪ³ cytoplasmic region as phosphatase. Samples were subjected to western blot analysis using anti-phosphotyrosine 4G10 mAb. The densitometry of each band was determined to estimate the extent of removal of phosphotyrosine on the protein. The reacted IgG heavy chain bands were used as internal control to ensure equal loading of samples. It is estimated by densitometry that 75% of phosphotyrosine from RPTPC1060S was removed by addition of RPTPĪ³ enzyme.</p

    RPTPĪ³ substrate-trapping mutants in HEK293 cells.

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    <p>Phosphotyrosine-containing protein of about 190 kDa size ā€œtrappedā€ by RPTPĪ³ C1060S or RPTPĪ³ D1028A is RPTPĪ³. Recombinant wild type RPTPĪ³ and substrate-trapped mutants were immunoprecipitated from transiently transfected HEK293F lysate with anti-myc mAb. Lanes 1 and 1ā€²were IP product from pCDNA3 transfected cells, 2 and 2ā€² were from wild type RPTPĪ³ transfected cells, 3 and 3ā€² were from RPTPĪ³ C1060S transfected cells and 4 and 4ā€² were from RPTPĪ³ D1060A transfected cells. Lane 1ā€“4 were western reacted with anti-phosphotyrosine 4G10 mAb and lanes 1ā€²-4ā€² were with anti-myc mAb.</p

    Behavioral analysis on the wild type (WT) and knockdown mutant mice (MT).

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    <p>See details in the text. A and B. Repeated open field (A) and tail suspension tests (B) on the same animals. * - P<0.05, *** - P<0.001, compared to WT on the same day. C. Immobility time in the forced swim test. * - P<0.05, *** - P<0.001, compared to WT for the same measure.</p

    Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

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    <div><p>Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4<sup>+</sup> T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1<sup>āˆ’/āˆ’</sup> B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1<sup>āˆ’/āˆ’</sup> CD4<sup>+</sup> T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4<sup>+</sup> T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.</p></div

    Analysis of T cell apoptosis and functional immune responses in Mst1<sup>āˆ’/āˆ’</sup> mice.

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    <p>(<b>A</b>) Increased apoptosis of activated Mst1<sup>āˆ’/āˆ’</sup> splenic T cells in vitro. Early apoptotic T cells were quantitated by flow cytometry in after stimulation with the indicated mAbs for 48 hrs (nā€Š=ā€Š5 per genotype). (<b>B</b>) Analysis of apoptosis in unstimulated MST<sup>āˆ’/āˆ’</sup> T cells. Early apoptotic T cells were quantitated by flow cytometry in CD44<sup>low</sup> and CD44<sup>high</sup> subsets of freshly isolated splenocytes (nā€Š=ā€Š5 per genotype). (<b>C</b>) The percentages of naĆÆve (CD62L<sup>high</sup>CD44<sup>low</sup>), effector memory (CD62L<sup>low</sup>CD44<sup>high</sup>), and CD279-, CD25-, and CD69-positive splenic CD4<sup>+</sup> T cells were examined by flow cytometry (nā€Š=ā€Š5 per genotype). (<b>D</b>) Cytokine production by splenic CD62L<sup>high</sup>CD44<sup>āˆ’</sup> CD4<sup>+</sup> T cells from WT and Mst1<sup>āˆ’/āˆ’</sup> mice (nā€Š=ā€Š5 per genotype) was examined 48 hrs after stimulation with mAbs to CD3 and CD28 (both at 1 Āµg/ml). (<b>E</b>) Th1 and Th2 polarized cells were generated from naĆÆve splenic CD62L<sup>high</sup>CD44<sup>āˆ’</sup> CD4<sup>+</sup> T cells after in vitro culture in polarizing conditions for 5 days. The percentages of effector memory (CD62L<sup>low</sup>CD44<sup>high</sup>) CD4<sup>+</sup> T cells were examined by flow cytometry (nā€Š=ā€Š5 per genotype). The percentage of sub-G0/G1 apoptotic cells was determined by the BrdU/7-AAD Flow kit and flow cytometry (nā€Š=ā€Š5 per genotype) following restimulation in vitro with plate-bound CD3 mAb (5 Āµg/ml) for 48 hrs. (<b>F</b>) Mst1<sup>āˆ’/āˆ’</sup> deficiency leads to decreased Ag-specific adaptive immune responses in vivo. Mst1<sup>āˆ’/āˆ’</sup> and WT mice (nā€Š=ā€Š6 and 9, respectively) were immunized with OVA in CFA. On day 14, serum samples were analyzed for OVA-specific IgG1 and IgG2a concentrations. Values and statistical significance are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098151#pone-0098151-g002" target="_blank">Fig. 2</a> and are representative of at least two independent experiments. Pre, preimmune serum.</p

    Mst1<sup>āˆ’/āˆ’</sup> mice exhibit decreased incidence and severity of CIA.

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    <p>(<b>A</b>) Mice of indicated genotype (nā€Š=ā€Š11ā€“13) were immunized with CII in CFA and observed for clinical signs of arthritis at the time points depicted on the X axis. Histological scores of synovial inflammation and cartilage/bone erosion were analyzed on day 45 after immunization. Data are expressed as mean (Ā± SEM); * (p<0.05) indicates significant differences in comparison to WT littermates (Mannā€“Whitney U test). Similar data were obtained in two additional independent experiments. (<b>B</b>) Representative pictures of histological and radiographic signs of arthritis in the same mice as in (A), obtained on day 45 after immunization with CII (left and middle panels). Arrows of the H&E-stained sections of paw joints point to severe synovial inflammation and cartilage erosion in the Mst1<sup>āˆ’/āˆ’</sup> animals. Representative ĀµCT images of subchondral bone changes characteristic of arthritis were taken on day 45 after immunization (right panels).</p

    Delayed cell cycle progression of Mst1<sup>āˆ’/āˆ’</sup> T cells in vitro.

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    <p>(<b>A</b>) Mst1<sup>āˆ’/āˆ’</sup> and WT T cells were activated for 36 hrs with the indicated stimuli, pulsed with BrdU, and analyzed by flow cytometry (nā€Š=ā€Š5 mice per genotype). The representative dot plots and fractional values show cell subsets residing in the indicated phases of cell cycle. (<b>B</b>) Nuclear DNA content at different stages of the cell cycle was determined by FACS analysis of splenic T cells stimulated for 48 hrs with plate-bound CD3 mAb (1 Āµg/ml) and stained with propidium iodide. Histograms are representative of 5 samples per genotype. Values on bar graphs and statistical significance are expressed as in Fig. 2. Similar data were obtained in two additional independent experiments.</p

    Deficient proliferation of Mst1<sup>āˆ’/āˆ’</sup> T and B cells in vitro.

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    <p>Single cell suspensions of splenic T and B lymphocytes were stimulated with various T and B cell-specific stimuli at the indicated concentrations. Cell proliferation was assessed by [<sup>3</sup>H]-thymidine incorporation after 64ā€“66 hrs of stimulation. Data are expressed as the mean Ā± SEM of counts per minute (cpm) in triplicate cultures (nā€Š=ā€Š5 per genotype). Values are expressed as mean Ā± SEM. * (p<0.05) and ** (p<0.01) indicate significant differences in comparison to WT animals (<i>t</i> test).</p
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