Negative autoregulation of Src homology region 2-domain-containing phosphatase-1 in rat basophilic leukemia-2H3 cells.
Src homology region 2-domain-containing phosphatase-1 (SHP-1) plays an important role in the regulation of signaling from various receptors in hematopoietic cells. In mast cells, SHP-1 has been shown to negatively regulate the initial signaling triggered by high-affinity receptor for IgE (FcepsilonRI) and positively regulate downstream outputs. To clarify the molecular mechanisms of SHP-1 in mast cells, we determined substrates for SHP-1 by using the substrate-trapping approach. When phosphatase-inactive SHP-1 was over-expressed in rat basophilic leukemia (RBL)-2H3 cells, tyrosine phosphorylation of a 68-kDa protein was enhanced before and after FcepsilonRI aggregation. Immunoprecipitation and western blot analyses revealed that this protein is SHP-1, either endogenous or ectopically expressed. FcepsilonRI-induced activation of Lyn and Syk was comparable between cells expressing wild-type (wt) and phosphatase-inactive SHP-1. In vitro phosphatase assay and combined transfection, immunoprecipitation and immunoblot analyses showed that tyrosine 536 of SHP-1 was potent phosphorylation site and that SHP-1 could dephosphorylate this site that had been phosphorylated by Lyn. Furthermore, the phosphatase activity of SHP-1 immunoprecipitated from cells expressing a phosphatase-inactive SHP-1 was increased compared with that from vector-transfected or wt SHP-1-expressing cells. Finally, expression of phosphatase-inactive SHP-1 resulted in decreased activation of mitogen-activated protein kinases and suppressed transcription of cytokine genes, whereas wt SHP-1 enhanced these processes. Taken collectively, these results suggest that SHP-1 may be a physiological substrate of SHP-1 in RBL-2H3 cells and that dephosphorylation of SHP-1 leads to a decrease in its catalytic activity and an enhancement of downstream signaling. A negative autoregulatory circuit of SHP-1 may contribute to mast cell regulation.
Department of Immunology and Signal Transduction, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Science, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan.
Tomoko
T
Ozawa
Kazuko
K
Nakata
Kazuya
K
Mizuno
Hidetaka
H
Yakura
D000818:Animals; D045744:Cell Line, Tumor; D016207:Cytokines / Q000235:genetics / Q000276:immunology; D015536:Down-Regulation / Q000276:immunology; D020869:Gene Expression Profiling; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology; D015471:Leukemia, Basophilic, Acute / Q000276:immunology*; D008407:Mast Cells / Q000276:immunology; D020928:Mitogen-Activated Protein Kinases / Q000276:immunology; D010766:Phosphorylation; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000235:genetics / Q000276:immunology*; D011505:Protein-Tyrosine Kinases / Q000276:immunology; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000276:immunology; D000072377:Syk Kinase; D014158:Transcription, Genetic; D019061:src-Family Kinases / Q000276:immunology
D016207:Cytokines; D047908:Intracellular Signaling Peptides and Proteins; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; C509250:Ptpn6 protein, rat