Novel Isoform of Lymphoid Adaptor FYN-T-binding Protein (FYB-130) Interacts with SLP-76 and Up-regulates Interleukin 2 Production*

T-cell activation involves the participation of protein-tyrosine kinases p56 lck and ZAP-70/SYK as well as lymphoid proteins such as SLP-76 and FYB/SLAP. FYB/SLAP has the hallmarks of an adaptor protein that binds to the SH2 domains of the Src kinase FYN-T and SLP-76. Whereas two forms of FYB at 120 and 130 kDa have been identified biochemically, a cDNA encoding only the lower molecular weight isoform has been cloned (termed FYB-120 or SLAP-130). In this study, we report the isolation of an alternative isoform of FYB with a molecular mass of 130 kDa (FYB-130) that has the same structure as FYB-120 except for an insertion of 46 amino acids toward the carboxyl-terminal region of the protein. FYB-120 and FYB-130 share an ability to bind to the SH2 domains of FYN-T and SLP-76, to act as substrates for p59FYN-T, and to be expressed in the cytoplasm and nucleus of T-cells. Differences were noted between the isoforms in the efficiency of binding to SLP-76 and in the preferential expression of FYB-130 in mature T-cells. When co-expressed together with FYN-T and SLP-76, FYB-130 caused a significant increase in anti-CD3-driven NF-AT transcription. Finally, fluorescence in situ hybridization analysis localized the FYB gene to human chromosome 5 at position p13.1. FYB-130 therefore represents a novel variant of FYB protein that can up-regulate T-cell receptor-driven interleukin 2 production in mature T-cells.

[1]  S. Weremowicz,et al.  RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation , 1995, Molecular and cellular biology.

[2]  Dan R. Littman,et al.  Signal transduction by lymphocyte antigen receptors , 1994, Cell.

[3]  C. Turck,et al.  Molecular Cloning of SLAP-130, an SLP-76-associated Substrate of the T Cell Antigen Receptor-stimulated Protein Tyrosine Kinases* , 1997, The Journal of Biological Chemistry.

[4]  D. Littman,et al.  The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits , 1992, The Journal of cell biology.

[5]  S. Rogers,et al.  Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. , 1986, Science.

[6]  Arthur Weiss,et al.  ZAP-70: A 70 kd protein-tyrosine kinase that associates with the TCR ζ chain , 1992, Cell.

[7]  A. Weiss,et al.  Uncoupling of nonreceptor tyrosine kinases from PLC-gamma1 in an SLP-76-deficient T cell. , 1998, Science.

[8]  T. Mustelin.  T cell antigen receptor signaling: three families of tyrosine kinases and a phosphatase. , 1994, Immunity.

[9]  S. Orkin,et al.  Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner , 1993, Molecular and cellular biology.

[10]  A. Bairoch.  PROSITE: a dictionary of sites and patterns in proteins. , 1991, Nucleic acids research.

[11]  L. Samelson,et al.  LAT The ZAP-70 Tyrosine Kinase Substrate that Links T Cell Receptor to Cellular Activation , 1998, Cell.

[12]  A. Weiss,et al.  LAT Is Required for TCR-Mediated Activation of PLCγ1 and the Ras Pathway , 1998 .

[13]  G. Koretzky,et al.  Differential Regulation of Activation-induced Tyrosine Phosphorylation and Recruitment of SLP-76 to Vav by Distinct Isoforms of the CD45 Protein-tyrosine Phosphatase* , 1996, The Journal of Biological Chemistry.

[14]  G. Koretzky,et al.  Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production , 1996, The Journal of experimental medicine.

[15]  U. Francke,et al.  Diverse growth hormone receptor gene mutations in Laron syndrome. , 1993, American journal of human genetics.

[16]  C. Rudd,et al.  Biochemical analysis of p120/130: a protein-tyrosine kinase substrate restricted to T and myeloid cells. , 1997, Journal of immunology.

[17]  A. Lanzavecchia,et al.  From TCR Engagement to T Cell Activation A Kinetic View of T Cell Behavior , 1999, Cell.

[18]  C. Turck,et al.  Molecular Cloning of SLP-76, a 76-kDa Tyrosine Phosphoprotein Associated with Grb2 in T Cells (*) , 1995, The Journal of Biological Chemistry.

[19]  J. Rast,et al.  α, β, γ, and δ T Cell Antigen Receptor Genes Arose Early in Vertebrate Phylogeny , 1997 .

[20]  L. Samelson,et al.  Complex complexes: signaling at the TCR. , 1996, Immunity.

[21]  L. Lai,et al.  Identification of an IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). II. PPBSF is a covalently linked heterodimer of IL-7 and a Mr 30,000 cofactor. , 1998, Journal of immunology.

[22]  G. Koretzky,et al.  Polymorphism in and localization of the gene LCP2 (SLP-76) to chromosome 5q33.1-qter. , 1996, Genomics.

[23]  A. Lupas,et al.  Predicting coiled-coil regions in proteins. , 1997, Current opinion in structural biology.

[24]  G. Koretzky,et al.  SLP-76 and Vav Function in Separate, but Overlapping Pathways to Augment Interleukin-2 Promoter Activity* , 1999, The Journal of Biological Chemistry.

[25]  J. Wu,et al.  Vav and SLP-76 interact and functionally cooperate in IL-2 gene activation. , 1996, Immunity.

[26]  O. Janssen,et al.  T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T) , 1993, The Journal of experimental medicine.

[27]  B. Guy,et al.  HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product , 1987, Nature.

[28]  G. Koretzky,et al.  Adaptor proteins in lymphocyte antigen-receptor signaling. , 1998, Current opinion in immunology.

[29]  A. Weiss,et al.  Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. , 1994, Science.

[30]  S. Eliason,et al.  Requirement for the leukocyte-specific adapter protein SLP-76 for normal T cell development. , 1998, Science.

[31]  Z. Li,et al.  Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[32]  F. Alt,et al.  Impaired Viability and Profound Block in Thymocyte Development in Mice Lacking the Adaptor Protein SLP-76 , 1998, Cell.

[33]  C. Rudd,et al.  FYN-T-FYB-SLP-76 Interactions Define a T-cell Receptor ζ/CD3-mediated Tyrosine Phosphorylation Pathway That Up-regulates Interleukin 2 Transcription in T-cells* , 1999, The Journal of Biological Chemistry.

[34]  R. Johnson,et al.  Activation of ZAP‐70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function. , 1995, The EMBO journal.

[35]  K. Schuebel,et al.  Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product , 1997, Nature.

[36]  O. Acuto,et al.  p56lck interacts via its src homology 2 domain with the ZAP-70 kinase , 1994, The Journal of experimental medicine.

[37]  David H. Williams,et al.  Phosphorylation of SLP-76 by the ZAP-70 Protein-tyrosine Kinase Is Required for T-cell Receptor Function* , 1996, The Journal of Biological Chemistry.

[38]  R. F. Smith,et al.  BEAUTY-X: enhanced BLAST searches for DNA queries , 1998, Bioinform..

[39]  B. Mayer,et al.  Regulation of PAK activation and the T cell cytoskeleton by the linker protein SLP-76. , 1998, Immunity.

[40]  S. Pelech,et al.  Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. , 1991, The Journal of biological chemistry.

[41]  G. Koretzky,et al.  Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity. , 1996, Journal of immunology.

[42]  M. Lenburg,et al.  Nef induces CD4 endocytosis: Requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain , 1994, Cell.

[43]  C. Rudd,et al.  FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[44]  I. Weissman,et al.  Two distinct pathways of positive selection for thymocytes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  H. Boehmer.  The developmental biology of T lymphocytes. , 1988 .