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.

TcRzeta/CD3 ligation initiates a signaling cascade involving CD4/CD8-p56(lck), p59(fyn), and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB/SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunofluorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.

[1]  S. Meuer,et al.  Molecular Cloning of SKAP55, a Novel Protein That Associates with the Protein Tyrosine Kinase p59 fyn in Human T-lymphocytes* , 1997, The Journal of Biological Chemistry.

[2]  A. Lupas,et al.  Predicting coiled coils from protein sequences , 1991, Science.

[3]  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.

[4]  C. Rudd,et al.  The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck , 1994, Molecular and cellular biology.

[5]  T Pawson,et al.  SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. , 1991, Science.

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

[7]  F. Crick,et al.  The packing of α‐helices: simple coiled‐coils , 1953 .

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

[9]  S. Ley,et al.  Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes , 1994, The Journal of cell biology.

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

[11]  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.

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

[13]  C. Rudd,et al.  p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  H. Schneider,et al.  Selective CD28pYMNM mutations implicate phosphatidylinositol 3-kinase in CD86-CD28-mediated costimulation. , 1995, Immunity.

[15]  T. Pawson,et al.  SH2 domains recognize specific phosphopeptide sequences , 1993, Cell.

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

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

[18]  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.

[19]  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.

[20]  D. Parry,et al.  α-Helical coiled coils — a widespread motif in proteins , 1986 .

[21]  C. Rudd,et al.  A 72-kilodalton fyn-related polypeptide (p72fyn-R) binds to the antigen-receptor/CD3 (TcR/CD3) complex. , 1993, Journal of Biological Chemistry.

[22]  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.

[23]  M. Fournel,et al.  Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn , 1992, The Journal of experimental medicine.

[24]  S. D. Gorman,et al.  Inability of CD8α′polypeptides to associate with p56 lck correlates with impaired function in vitro and lack of expression in vivo , 1989, Nature.

[25]  John Calvin Reed,et al.  Anergic Th1 cells express altered levels of the protein tyrosine kinases p56lck and p59fyn. , 1992, Journal of immunology.

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

[27]  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.

[28]  L. Pauling,et al.  Compound Helical Configurations of Polypeptide Chains: Structure of Proteins of the α-Keratin Type , 1953, Nature.

[29]  R. Abraham,et al.  Genetic Evidence for Differential Coupling of Syk Family Kinases to the T-Cell Receptor: Reconstitution Studies in a ZAP-70-Deficient Jurkat T-Cell Line , 1998, Molecular and Cellular Biology.

[30]  O. Janssen,et al.  Two-step TCR zeta/CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains, protein-tyrosine and lipid kinases. , 1994, Immunology today.

[31]  C. Rudd,et al.  CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

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

[33]  B. Mayer,et al.  SH3 Domains: Minding your p's and q's , 1995, Current Biology.

[34]  P. Anderson,et al.  Biochemical identification of a direct physical interaction between the CD4: p56lck and Ti(TcR)/CD3 complexes , 1991, European journal of immunology.

[35]  T. L. Collins,et al.  Views on Vav. , 1997, Immunology today.

[36]  L. Ivashkiv.  Cytokines and STATs: how can signals achieve specificity? , 1995, Immunity.

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

[38]  Mary J. Tharayil,et al.  Growth Factor Receptor-bound Protein 2 SH2/SH3 Domain Binding to CD28 and Its Role in Co-signaling* , 1998, The Journal of Biological Chemistry.

[39]  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.

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

[41]  J. Gribben,et al.  Differential association of protein tyrosine kinases with the T cell receptor is linked to the induction of anergy and its prevention by B7 family-mediated costimulation , 1996, The Journal of experimental medicine.