In order to express and purify the catalytic domain of SHP-1/SHP-2 (named as D1C and D2C respectively) and determine their kinetics, the constructed D1C and D2C plasmids were transformed into Escherichia coli BL21 and the expression was induced with IPTG. The harvested cells were suspended in extraction buffer. After sonication, the solution was applied to HPLC and the results were confirmed by SDS-PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic phosphotyrosine (pY) as substrate by malachite green method and analyzed by Lineweaver-Burk plot calculation. From this study, we found D1C and D2C were expressed successfully in soluble state in Escherichia coli BL21 and purified efficiently with HPLC system. The molecular weight of D1C was 34.6 kD, and its Michaelis constant (K(m)) was 2.04 mmol catalytic constant (K(cat)) was 44.98 s(-1), specific constant (K(cat)/K(m)) was 22.05 L/(mmol x s); the molecular weigh of D2C was 35.3 kD, and its Michaelis constant (K(m)) was 2.47 mmol, catalytic constant (K(cat)) was 27.45 s(-1), specific constant (K(cat)/K(m)) was L/(mmol x s). The enzyme activity of D1C is stronger than that of D2C.
Catalytic Domain ; Chromatography, High Pressure Liquid ; Escherichia coli ; genetics ; metabolism ; Kinetics ; Plasmids ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

Diabetes mellitus is caused by deficiency of insulin secretion from the pancreatic islet beta cells and/or insulin resistance in liver, muscle and adipocytes, resulting in glucose intolerance and hyperglycemia. Several protein tyrosine phosphatases, such as PTP1B (PTPN1), TCPTP (PTPN2), LYP (PTPN22), PTPIA-2, PTPMEG2 (PTPN9) or OSTPTP are involved in insulin signaling pathway, insulin secretion and autoreactive attack to pancreatic beta cells. Genetic mutation or overexpression of these phosphotases has been found to cause or increase the risk of diabetes mellitus. Some population with high risk for type 2 diabetes has overexpressed PTP1B, a prototypical tyrosine phosphatase which down-regulates insulin and leptin signal transduction. Animal PTP1B knockout model and PTP1B specific inhibitor cellular studies indicate PTP1B may serve as a therapeutic target for type 2 diabetes. TCPTP shares more than 70% sequence identity with PTP1B in their catalytic domain. TCPTP dephosphorylates tyrosine phosphorylated substrates overlapping with PTP1B but also has its own distinct dephosphorylation sites and functions. Recent research indicates TCPTP may have role in type 1 diabetes via dysregultaion of cytokine-mediated immune responses or pancreatic beta cell apoptosis. The tyrosine phosphatase LYP, which down-regulates LCK activity in T cell response, can become mutated as R620W which is highly correlated to type 1 diabetes. LYP R620W may be a gain of function mutation which suppresses TCR signaling. Patients bearing the R620W mutant have impaired T cell responses and increased populations of (CD45RO+CD45RA-) CD4+ T cells. A detailed elucidation of mechanism of R620W in type 1 diabetes and specific LYP inhibitor development will help characterize LYP R620W as a therapeutic target. A receptor tyrosine phosphatase, PTPIA-2/beta is a major autoantigen of type 1 diabetes. A diagnosis kit identifying PTPIA-2/beta autoantibodies is valuable in early detection and prevention of type 1 diabetes. In addition, other phosphatase like OSTPTP and PTPMEG2 are involved in type 2 diabetes via regulation of insulin production, beta cell growth or insulin signaling. Research into understanding the mechanism of these tyrosine phosphatases in diabetes, such as their precise functions in the regulation of insulin secretion, the insulin response and the immune response will strengthen our knowledge of diabetes pathophysiology which may result in new diagnostic and therapeutic strategies for diabetes.
Animals ; Diabetes Mellitus ; enzymology ; Diabetes Mellitus, Type 1 ; enzymology ; Diabetes Mellitus, Type 2 ; enzymology ; Humans ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; genetics ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 2 ; genetics ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 22 ; genetics ; metabolism ; Protein Tyrosine Phosphatases, Non-Receptor ; classification ; genetics ; metabolism

CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kappa Da in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60v-src in vivo and CPTP1 also can associate with p60v-src in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60v-src was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60v-src in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kappa Da protein which has a possibility to be tyrosine- phosphorylated by p60v-src in v-src-transformed Rat- 1 fibroblasts. These results suggest that HPTP1B- type PTPs may play an important role in p60src dependent signal pathway in eucaryotic cells.
Animals ; Blotting, Western ; Cell Line, Transformed ; Chickens ; Female ; Fibroblasts ; Oncogene Protein pp60(v-src)/*metabolism ; Phosphoprotein Phosphatase/genetics/*metabolism ; Precipitin Tests ; Protein Binding ; Protein-Tyrosine-Phosphatase/genetics/*metabolism ; Rabbits ; Rats ; Recombinant Fusion Proteins/genetics/metabolism

Catalytic Domain ; genetics ; Dual Specificity Phosphatase 3 ; chemistry ; genetics ; metabolism ; Genetic Code ; Humans ; Mutation ; Phosphorylation ; Protein Conformation ; Signal Transduction ; genetics ; Structure-Activity Relationship ; Tyrosine ; analogs & derivatives ; chemistry ; genetics

OBJECTIVETo investigate the role of cytoplasmic domain of integrin alpha IIb in platelet signal transduction.

METHODSBinding capacity of integrin alpha IIb(R995A) to antibody platelet activation complex-1 (PAC-1) and pp125 focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting.

RESULTSWithout activation, wild-type alpha IIb beta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alpha IIb(R995A)beta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK) in wild-type alpha IIb beta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions.

CONCLUSIONThe mutation in integrin alpha IIb(R995A) alters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.

Animals ; Blood Platelets ; metabolism ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; Cytoplasm ; metabolism ; Dual Specificity Phosphatase 2 ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Phosphorylation ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; physiology ; Point Mutation ; Protein Phosphatase 2 ; Protein Tyrosine Phosphatases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Transfection

PTPL1 is a protein with a predicted MW of 270 kD, and plays a major role in many cellular functions, including cell survival, proliferation, differentiation and motility. Evidence has demonstrated that PTPL1 is associated with tumor. Although many conflicting results suggested that PTPL1 has two contradictory effects (supressing or promoting ) on tumor, the real effect depends on the involved substrate and the cellular context. Expression of PTPL1 is low in lymphoma, while it is high in myeloid leukemia. PTPL1 has been regarded as a tumor suppressor in lymphoma, the methylation of PTPL1 promoter leads to gene expression reduced or disappeared, playing a lymphoma tumor suppressor role. This review focuses on PTPL1 domain and its interacting proteins, the relationship between PTPL1 and hematological malignancies.
Cell Movement ; Cell Survival ; Genes, Tumor Suppressor ; Hematologic Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Protein Tyrosine Phosphatase, Non-Receptor Type 13 ; genetics ; metabolism

OBJECTIVETo observe SHP-1 protein expression in breast cancer cell line MDA-MB-231 before and after SHP-1 gene transfer and its effect on the proliferation of MDA-MB-231 cells.

METHODSThe eukaryotic expression vector pEGFP-C3-SHP-1 was constructed and transfected into breast cancer cell line MDA-MB-231 via Lipofectamine 2000, and the positive clones were selected using G418. SHP-1 expression in MDA-MB-231 cells was detected with immunocytochemistry and Western blotting, and the cell growth curve was observed using MTT assay.

RESULTSSHP-1 was highly expressed in transfected MDA-MB-231 cells, whose proliferation was significantly inhibited (P<0.05).

CONCLUSIONSHP-1 gene transfer into MDA-MB-231 cells results in inhibition of the cell proliferation.

Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Transfer Techniques ; Humans ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism

OBJECTIVETo explore the effect of overexpression of SH2-containing tyrosine phosphatase 1 (SHP-1) on sensitivity of chronic myelogenous 1eukemia (CML) K562 cell line to imatinib and its related mechamism.

METHODSK562 cells were infected with the lentiviral plasmids containing the specified retroviral vector (pEX-SHP-1-puro-Lv105) or the mock vector (pEX-EGFP-puro-Lv105). The expression of SHP-1 in K562 cells treated with 0.2 µmol/L imatinib (IM) for 72 h was determined by Western blot. After transfection the CCK-8 assay was used to determine the proliferation of the tramfected K562 cells (K562(SHP-1) and K562(EGFP) cells) at 72 h after exposure to different doses of IM, the half inhibitary concentration (IC50) was calculated. The mechanisms of the overexpression effects of SHP-1 and IM on the proliferation in K562 cells was investigated, the BCR-ABL1 activity and the level of tyrosine phosphorylation of CrkL (pCrkL) was measured by flow cytometry; the Western blot was used to detect the expression and activity of these molecules controlling cell growth, including MAPK, AKT, STAT5 and JAK2.

RESULTSAfter exposure of K562 cells to 0.08 µmol/L IM for 72 h, there was no significant change of SHP-1 expression in K562 cells. After exposure to 0.2 µmol/L of IM for 72 h, the inhibitory rate of K562(SHP-1) group was higher than that of K562(EGFP) group (P < 0.05), indicating that overexpression of SHP-1 in K562 cells could enhance the proliferation inhtibition effect of IM on K562 cells. The IC50 of IM in K562(SHP-1) cells was the lower as compared with that of K562(EGFP) cells (P < 0.05) after exposure to different concentrations of IM for 72 h. The slope of K562(SHP-1) cells was the largest ranging 0.02 - 0.16 µmol/L of IM. Overexpression of SHP-1 and IM could inhibit the activity BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in the K562 cell line and displayed a synergistic effect.

CONCLUSIONSHP-1 inhibits BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in K562 cells, the overexpression of SHP-1 can enhance the sensitivity of K562 cells to IM.

Cell Proliferation ; Drug Resistance, Neoplasm ; Genetic Vectors ; Humans ; Imatinib Mesylate ; pharmacology ; K562 Cells ; drug effects ; Phosphorylation ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; Signal Transduction ; Transfection

The amino acid sequence (1-301aa) coding the human PTP1B catalytic domain (PTP1Bc) was obtained from the GenBank. The PTP1Bc gene was constructed by overlapping PCR, then was inserted into vector pET-22b(+) and expressed efficiently in E. coli BL21(DE3) under optimum condition after IPTG induction. The proteins were expressed mainly as inclusion bodies with the yield of more than 30% of total bacterial proteins. The expressed products were purified through Ni(2+)-affinity chromatographic column. After purification, the purity of the proteins was more than 95%. Western blotting analysis suggested that the purified proteins could specially combine with anti-PTP1B antibody. Enzyme activity assay showed that the protein has phosphatase activities.
Catalysis ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Inclusion Bodies ; metabolism ; Polymerase Chain Reaction ; methods ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

OBJECTIVETo study the effect of Panax notoginseng saponins (PNS) on (synaptophysin, syp) and tau gene expression in the brain tissue in senescence accelerated mouse prone 8 (SAMP 8).

METHODSAMP8 were randomly divided into 4 groups: PNS 23.38, 93.50 mg x kg(-1) group, huperzin A 0.038 6 mg x kg(-1) x d(-1) group and blank control group; the drug groups were treated with the designed drugs respectively per day by intragastric administration for 4 consecutive weeks, and double distilled water was given to blank control group. After treatment, the mRNA content of tau and syp were assayed by reverse transcription (RT) and real-time polymerase chain reaction (real-time PCR).

RESULTCompared with blank control group, the syp mRNA contents were increased in PNS groups (P < 0.05 or P < 0.01), and the tau mRNA content were not significant difference in all groups.

CONCLUSIONThis study suggests that PNS can up-regulate syp gene expression at transcriptional level in the brain of SAMP 8.

Aging ; drug effects ; metabolism ; Animals ; Brain ; drug effects ; metabolism ; Gene Expression ; drug effects ; genetics ; Mice ; Panax notoginseng ; chemistry ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; metabolism ; Saponins ; pharmacology ; tau Proteins ; genetics ; metabolism