Recombinant Human PTPN11, GST-tagged, Active

• Cat.No.: PTPN11-461H
• Product Overview: Recombinant human PTPN11 (246-593) was expressed inE.colicells using a N-terminal GST tag. MW = 69 kDa.

Specification

• Species: Human
• Source: E.coli
• Tag: GST
• Protein Length: 246-593 a.a.
• Description: Mammalian PTPases can be subdivided into 1 of 2 broad categories: transmembrane receptor PTPases and intracellular PTPases. PTPN11 is one of the 2 closely related mammalian intracellular PTPases whose sequences encode 2 tandem SRC homology 2 (SH2) domains that are located at the amino-terminal side of a single PTPase catalytic domain. This PTP is widely expressed in most tissues and plays a regulatory role in various cell signaling events that are important for a diversity of cell functions, such as mitogenic activation, metabolic control, transcription regulation, and cell migration.
• Sequence: 246-593.
• Applications: Phosphatase Assay, Western Blot.
• Storage And Stability: Store product at –70°C. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles.
• Publications:
  Decline in arylsulfatase B expression increases EGFR expression by inhibiting the protein-tyrosine phosphatase SHP2 and activating JNK in prostate cells (2018)

Gene Information

• Gene Name: PTPN11 protein tyrosine phosphatase, non-receptor type 11 [ Homo sapiens ]
• Synonyms: protein tyrosine phosphatase, non-receptor type 11; CFC; NS1; SHP2; BPTP3; PTP2C; PTP-1D; SH-PTP2; SH-PTP3; MGC14433; PTPN11; protein tyrosine phosphatase-2; protein-tyrosine phosphatase 2C; EC 3.1.3.48; PTP-2C; SHP-2; SHPTP2; Shp2; Noonan syndrome 1; Protein-tyrosine phosphatase 2C
• Gene ID: 5781
• mRNA Refseq: NM_002834
• Protein Refseq: NP_002825
• MIM: 176876
• UniProt ID: Q06124
• Chromosome Location: 12q24
• Pathway: Adipocytokine signaling pathway; Chronic myeloid leukemia; Epithelial cell signaling in Helicobacter pylori infection; Jak-STAT signaling pathway; Leukocyte transendothelial migration; Natural killer cell mediated cytotoxicity; Neurotrophin signaling pathway; Renal cell carcinoma
• Function: hydrolase activity; insulin receptor binding; insulin receptor substrate binding; non-membrane spanning protein tyrosine phosphatase activity; peptide hormone receptor binding; phospholipase binding; protein domain specific binding

Citation

Decline in arylsulfatase B expression increases EGFR expression by inhibiting the protein-tyrosine phosphatase SHP2 and activating JNK in prostate cells

Journal: The Journal of Biological Chemistry    PubMed ID: 29794138    Data: 2018/7/13

Authors: Sumit Bhattacharyya, Leo Feferman, Joanne K. Tobacman

Article Snippet:Then preparations were blocked for 1 h at RT with blocking buffer (PBS, pH 7.4, with 1% BSA).Then preparations were blocked for 1 h at RT with blocking buffer (PBS, pH 7.4, with 1% BSA).. After blocking, 100 μl of (0.5 μg/ml) PTPN11-GST (Creative BioMart, Shirley, NY) in PBS was added to each well.. Plates were incubated at RT for 2 h and then washed three times with wash buffer.Plates were incubated at RT for 2 h and then washed three times with wash buffer.

EGFR is increased following decline in ARB or SHP2 and reduced by AP-1 inhibition but unaffected by galectin-3 silencing. A, in human prostate stem cells, ARSB siRNA increased EGFR to ～2.8 times the baseline level of 35.2 ± 1.1 ng/mg of protein (p < 0.001, one-way analysis of variance with Tukey–Kramer post-test; n = 3). Inversely, ARSB overexpression reduced the EGFR to 19.0 ± 1.9 ng/mg of protein (p < 0.001; n = 3). B, representative Western blotting shows the increase in density of EGFR in the prostate stem cells following ARSB silencing. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a control. Minimal EGFR degradation is evident. C, the increase in EGFR protein caused by ARSB silencing was blocked by inhibition of AP-1 (SR11302; 5 μm for a 2-h pretreatment and then 24 h). In contrast, galectin-3 silencing by siRNA or inhibition of SP1 nuclear binding by mithramycin (250 nm for 24 h) had no effect on the ARSB siRNA- induced increase in EGFR. D, EGFR expression was increased by ARSB siRNA or by the SHP2 inhibitor PHPS1 (30 μm for 24 h) in prostate epithelial cells and prostate stem cells (p < 0.001; n = 3). The increase following PHPS1 was greater than the increase following ARSB silencing (p < 0.001). cJMP (400 μm for a 2-h pretreatment and then 24 h) inhibited the ARSB silencing-induced increase, consistent with the observed effect of AP-1 inhibition shown in C. E, EGFR expression was increased by ARSB siRNA and further increased by the dominant negative SHP2 DNA construct (p < 0.001). The constitutively active SHP2 construct significantly inhibited the EGFR, compared with the vector control (p < 0.001; n = 3). Findings indicate that the decline in SHP2 mediated the effect of ARSB knockdown on EGFR. Bars, mean ± S.D. (error bars). consi, control siRNA; PEC, prostate epithelial cells; si, siRNA. ***, p ≤ 0.001. N.D., no difference.

SHP2 activity is reduced following ARSB silencing and is attributable to binding with C4S. A, active SHP2 was measured using an exogenous phosphorylated substrate in the prostate stem and epithelial cells. ARSB silencing significantly reduced the SHP2 activity (p < 0.001, n = 3), and the active SHP2 was further reduced by PHPS1 (p < 0.001, n = 3). B, binding of biotinylated, predominantly C4S chondroitin sulfate to GST-tagged SHP2 (amino acids 246–593), which was coated on the wells of a microtiter plate, peaked at a concentration of 625 ng/ml, based on a standard curve of optical density versus concentration of biotinylated, predominantly C4S chondroitin sulfate. C, the linear range of the biotinylated C4S (log2) with GST-tagged SHP2 had slope m = 0.25 and y intercept = ?0.74. D, the binding of the biotinylated chondroitin sulfate preparations to GST-tagged SHP2 coated wells was determined using the saturating concentration for C4S of 625 ng/ml. 529.0 ± 52.5 ng/ml of C4S bound to the plate (p < 0.001, n = 3). In contrast, of 625 ng/ml of predominantly C6S 24.2 ± 1.4 ng/ml bound to the SHP2. These findings demonstrate that the C4S preparation had much higher affinity to bind with SHP2 than C6S. Bars, mean value ± S.D. (error bars). ***, p ≤ 0.001.

Phospho-JNK is increased in prostate stem cells and prostate tissue following ARSB silencing or SHP2 inhibition. A, in prostate stem cells, ARSB silencing significantly increased (p < 0.001, n = 3) and ARSB overexpression significantly reduced (p < 0.001, n = 3) the phospho-JNK concentration. B, ARSB siRNA significantly increased the phospho-JNK in the prostate epithelial and stem cells (p < 0.001, n = 3). The SHP2 inhibitor PHPS1 further increased the phospho-JNK levels (p < 0.001). JIP-1 significantly reduced phospho-JNK (p < 0.001) in the cells. C, phospho-JNK was significantly increased by the DN SHP2 construct (p < 0.001, n = 3) and significantly inhibited by the CA SHP2 in the prostate stem cells (p < 0.001, n = 3). D, in malignant prostate tissue, the phospho-JNK was significantly higher than in the normal prostate tissue (p < 0.001, unpaired t test, two-tailed, n = 6). Bars, mean value ± S.D. (error bars). ***, p ≤ 0.001.