title: Once phosphorylated, tyrosines in carboxyl terminus of protein-tyrosine kinase Syk interact with signaling proteins, including TULA-2, a negative regulator of mast cell degranulation.
abstract: Activation of the high affinity IgE-binding receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the COOH terminus of the protein-tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcεRI signaling in mast cells. Therefore, once phosphorylated, the terminal tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.
authors.affiliation: Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Rodrigo Orlandini
authors.initials: RO
authors.lastname: de Castro
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.forename: Jacqueline R
authors.initials: JR
authors.lastname: Groves
authors.forename: Emilia Alina
authors.initials: EA
authors.lastname: Barbu
authors.forename: Reuben P
authors.initials: RP
authors.lastname: Siraganian
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015550:Cell Degranulation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008407:Mast Cells / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D017455:Receptors, IgE / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D000072377:Syk Kinase
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D010455:Peptides; D034741:RNA, Small Interfering; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D017027:Protein Tyrosine Phosphatases; C553497:TULA-2 protein, mouse