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Biomedical subjects

C E Rudd

Publications and source records attributed to C E Rudd.

At least 19 recordsLinked to original sources
PubMed1998

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.

Adaptor Proteins, Signal Transducing↗
PubMed1998

CD28 receptor endocytosis is targeted by mutations that disrupt phosphatidylinositol 3-kinase binding and costimulation.

Although the lipid kinase phosphatidylinositol 3-kinase (PI-3K) binds at high levels to the cytoplasmic tail of CD28, controversy exists regarding its role in CD28 costimulation. Potentially, the kinase could be linked to a signaling cascade or be needed indirectly in events such as receptor endocytosis. Indeed, little is known regarding both the fate of CD28 following receptor ligation and the events that control the process. In this study, we help to resolve this issue by providing evidence that PI-3K plays a role in regulating CD28 endocytosis. We show that approximately 25 to 35% of wild-type CD28 becomes endocytosed following Ab binding (t1/2 = 10 min), followed by segregation into two pools; one pool is destined for degradation in lysosomal compartments and is blocked by chloroquine, and another pool that is recycled to the cell surface (t1/2 = 2.5 h). Recycling of CD28 could have an important impact on CD80/86-mediated costimulation by replenishing functionally active receptors on the cell surface. Several findings implicate PI-3K in the control of endocytosis. Modulation experiments indicate that CD28-PI-3K complexes are preferentially endocytosed, and mutations that alter PI-3K binding concordantly affect the efficacy of endocytosis. Importantly, mutations that inhibit receptor internalization also block cosignaling. Therefore, previous results documenting a requirement for PI-3K may be explained by a blockage of receptor internalization.

Binding Sites↗
PubMed1998

Growth factor receptor-bound protein 2 SH2/SH3 domain binding to CD28 and its role in co-signaling.

The co-stimulatory antigen CD28 has been shown to bind to several intracellular proteins including phosphatidylinositol 3-kinase, growth factor receptor-bound protein 2 (Grb2), and ITK. Paradoxically, Grb2 and phosphatidylinositol 3-kinase binding has been mapped to a similar pYMNM motif within the CD28 cytoplasmic tail. Given the importance of CD28 co-signaling to T cell function, questions exist regarding the mechanism by which Grb2 binds to CD28, and whether the interaction plays a role in co-stimulation. To biochemically characterize Grb2/CD28 binding, we initially utilized glutathione S-transferase-Grb2 fusion proteins carrying inactivating mutations within the SH2 and SH3 domains of Grb2, and assessed their ability to bind to CD28. In vitro binding experiments indicated that the Grb2 SH2 domain is critical for the association, while the SH3 domain plays an additional role in facilitating optimal binding. Enhanced binding via the SH3 domains was not observed when the C-terminal PXXP motif within CD28 was disrupted, thereby indicating that both SH2 and SH3 domains contribute to CD28 binding. Mutations that alter Grb2 binding were found to block the CD28-dependent interleukin-2 production. Further, tyrosine phosphorylation of Vav and the costimulation-dependent activation of Jun N-terminal kinase was blocked in cells defective in CD28/Grb2 binding. These results provide evidence for an alternate CD28-mediated signaling process involving Grb2 binding to the co-receptor.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production.

T cell receptor zeta (TcRzeta)/CD3 ligation initiates a signaling cascade that involves src kinases p56(lck) and zeta-associated protein 70, leading to the phosphorylation of substrates such as TcRzeta, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59(fyn), the TcRzeta/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59(fyn), FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRzeta/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Biochemical analysis of p120/130: a protein-tyrosine kinase substrate restricted to T and myeloid cells.

T cell activation is mediated by a cascade of intracellular events involving protein-tyrosine kinases and their substrates. p56(lck) and p59(fyn) are protein-tyrosine kinases that associate with CD4/CD8 and the TCRzeta/CD3 complex, respectively. We previously reported the appearance of a protein doublet at 120 and 130 kDa that preferentially associates with p59(fyn) and undergoes tyrosine phosphorylation upon receptor ligation. In this paper, we demonstrate that p120/130 is a novel protein that is restricted in expression to T cells, thymocytes and myeloid cells. Internal peptide sequencing and immunoblotting using an anti-p120/130 antisera showed that p120/130 is a unique protein that is distinct from p130(cas) and p125(cbl). By contrast, p120 and p130 shared similar peptide patterns and are structurally related. Alkaline phosphatase digestion of precipitates showed that they are not related due to phosphorylation. p120/130 was found to associate constitutively with a 55-kDa protein of unknown identity, but which is distinct from p56(lck) and Shc. p120/130 also undergoes a unique kinetics of phosphorylation and associates with the Ag receptor in response to TCR ligation. In keeping with the association with p59(fyn), T cells from p59(fyn)-negative mice exhibit reduced phosphorylation of the protein. p120/130 therefore represents a novel TCR associated intracellular molecule with potential to play a role in T cell signaling.

Amino Acid Sequence↗
PubMed1997

The Fes protein-tyrosine kinase phosphorylates a subset of macrophage proteins that are involved in cell adhesion and cell-cell signaling.

The c-fps/fes proto-oncogene encodes a 92-kDa protein-tyrosine kinase that is expressed at high levels in macrophages. We have previously shown that overexpression of c-fps/fes in a CSF-1-dependent macrophage cell line (BAC1.2F5) partially released these cells from their factor dependence and that this correlated with the tyrosine phosphorylation of a subset of proteins in a tissue-specific manner. We have now identified one of the macrophage substrates of Fes as the crk-associated substrate (Cas) and a second substrate as a 130-kDa protein that has been previously described as a T cell activation-dependent substrate and is unrelated to Cas. Both of these proteins, which have optimal consensus sequences for phosphorylation by Fes, were tightly associated with this kinase through its SH2 domain, suggesting that they were direct substrates of Fes. Remarkably, when the Fes SH2 domain was used as an affinity reagent to identify potential substrates of endogenous Fes in control BAC1.2F5 cells, the phosphotyrosyl proteins that were recognized were the same as those that were specifically phosphorylated when Fes was overexpressed in the same cells. We conclude that the substrates we identified may be structurally related or identical to the physiological targets of this kinase in macrophages. The known functions of Cas and p130 suggest that Fes kinase may play a role in signaling triggered by cell adhesion and cell-cell interactions during immune responses of macrophages.

Animals↗
PubMed1997

Regulation of Vav-SLP-76 binding by ZAP-70 and its relevance to TCR zeta/CD3 induction of interleukin-2.

T cell activation stimulates p56(lck), p59(fyn), ZAP-70, Vav-SLP-76 binding, and IL-2 transcription. Major questions concern the tyrosine-kinase and relevant site(s) needed for Vav-SLP-76 complex formation and its role in IL-2 production. Here, we show that of the three kinases, only ZAP-70 phosphorylates SLP-76 at specific sites that allow Vav SH2 domain binding. Therefore, while p56(lck) regulates proximal events, ZAP-70 acts downstream on targets such as SLP-76. We also show by in vitro and in vivo analysis that two SLP-76 pYESP motifs (Y113 and Y128) mediate binding, the first being more efficient. A third pYEPP motif (Y145) failed to bind. Finally, TCR zeta CD3 ligation of T cell hybridoma DC27.10 induces IL-2 production without detectable Vav-SLP-76 binding. Therefore, despite effects of Vav-SLP-76 on IL-2 expression, Vav-SLP-76 binding per se is not essential for IL-2 production in all T cells.

Adaptor Proteins, Signal Transducing↗
PubMed1996

The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human.

Amino Acid Sequence↗
PubMed1996

Hematopoietic cell phosphatase (HCP) regulates p56LCK phosphorylation and ZAP-70 binding to T cell receptor zeta chain.

Ligation of the T cell receptor complex and CD4 leads to activation of the protein tyrosine kinases p56lck and p59fyn resulting in phosphorylation of TcR zeta chain and the recruitment of ZAP-70. In this study, we have reconstituted p56lck phosphorylation of TcR zeta and ZAP-70 recruitment in heterologous cells and examined the role of the tyrosine phosphatase HCP in regulating the process. Both p56lck and p59fyn induce significant phosphorylation of TcR zeta. However, under conditions of comparable p56lck and p59fyn expression, p56lck was found to induce three to four fold greater in vivo phosphorylation of TcR zeta. HCP dephosphorylated p56lck, ZAP-70 and the TcR zeta chain. Further, dephosphorylation of the different TcR zeta isoforms results in disruption of the interaction between TcR zeta and ZAP-70. These results indicate that HCP acts to negatively regulate signal transduction pathways in T cells.

Animals↗
PubMed1995

Phosphorylation of src family lck tyrosine kinase following interleukin-12 activation of human natural killer cells.

Interleukin-12 (IL-12) is a heterodimeric cytokine that augments the cytolytic activity of human NK cells and T cells but has little mitogenic activity on resting lymphocytes. The intracellular signaling pathways involved in NK cell activation by IL-12 have not been fully characterized. In the present studies we found that IL-12 induces tyrosine phosphorylation of a 56-kDa protein, identified in Western blot experiments as p56lck, in resting NK cells. IL-12 was active in the range of 0.1 to 1000 U/ml, with maximal activity between 10 and 100 U/ml (30 and 300 pM). The maximal effect was noted 5 min after stimulation and was almost completely inhibited by genistein. IL-12 induced similar effects in resting and IL-2 activated NK cells. Following IL-12 stimulation of resting NK cells, immunoprecipitated lck kinase exhibited increased in vitro autophosphorylation activity 5 and 10 min after activation. Several additional substrates were phosphorylated in vitro following IL-12 stimulation, including proteins of 70 and 110 kDa. These studies indicate that lck tyrosine kinase is involved in the IL-12 signaling pathway in human NK cells. As the primary functional effect of IL-12 on resting NK cells is the enhancement of cytolytic activity and secretory function rather than cell proliferation, these findings suggest that lck tyrosine kinase is involved in these signaling pathways in human NK cells.

Animals↗
PubMed1995

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.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1995

CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells.

CTLA-4 is a T cell antigen that is structurally related to CD28 and serves as a high affinity ligand for the B cell antigen B7-1/2. Unlike CD28, the function of CTLA-4 is unclear, although reports have implicated the antigen in the costimulation of T cells. Recently, phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the costimulatory function of CD28 by virtue of its ability to bind to a pYMNM motif within the cytoplasmic tail of the antigen. In this study, we show that CTLA-4 can also associate with PI 3-kinase as detected by lipid kinase analysis and immunoblotting with anti-p85 antiserum. High pressure liquid chromatographic separation of deacylated lipids showed the presence of a peak corresponding to PI-3-P. Anti-CTLA-4 ligation of the receptor induced a significant increase in the levels of precipitable PI 3-kinase activity. Peptide binding studies revealed that the NH2- and COOH-terminal SH2 domains of p85 bind the CTLA-4 cytoplasmic pYVKM motif with an affinity (ID50: 0.6 and 0.04 microM), that is similar to CD28. CTLA-4 binding to PI 3-kinase provides further evidence that CTLA-4 is not an inert counterreceptor, but rather is coupled to an intracellular signaling molecule with the capacity to regulate cell growth.

Abatacept↗
PubMed1995

T cell antigen CD28 binds to the GRB-2/SOS complex, regulators of p21ras.

The T cell molecule CD28 provides a co-stimulatory signal that is required for T cell proliferation, and has been implicated in the control of T cell anergy. An important clue to the signaling mechanism of CD28 is the finding that CD28 can bind to phosphatidylinositol 3-kinase (PI 3-kinase) by means of a cytoplasmic phospho-YMNM (pYMNM) motif. A remaining issue concerns whether CD28 can recruit other intracellular signaling molecules. In this study, we show that CD28 uses the same pYMNM motif to recruit a second intracellular protein, GRB-2. CD28-associated GRB-2, as detected by anti-GRB-2 immunoblotting, was found in human peripheral T cells, HPB-ALL and Jurkat cells. As in the case of PI3-kinase, antibody-induced cross-linking of CD28 induces a time-dependent recruitment of GRB-2. Likewise, mutation of the pY-191 residue within the pYMNM motif reduces GRB-2 binding. Peptide binding studies show that the SH2 domain of GRB-2 binds to the pYMNM motif with an affinity comparable to GRB-2/SHC, but some 10- to 100-fold lower than the CD28/PI 3-kinase. Despite this, CD28/GRB-2 and CD28/PI 3-kinase complexes are found to co-exist in peripheral T cells. Finally, immunoblotting shows that CD28 also associates with the gene product of the human homolog of the Drosophila Son of sevenless gene (SOS), a GRB-2-complexed guanine nucleotide exchange factor responsible for converting p21ras to a GTP-bound active state. CD28-associated GRB2/SOS is likely to serve an important link in the regulation of p21ras and lymphokine expression mediated by CD28.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Selective CD28pYMNM mutations implicate phosphatidylinositol 3-kinase in CD86-CD28-mediated costimulation.

CD28 costimulatory signals are required for lymphokine production and T cell proliferation. CD28 signaling recruits the intracellular proteins PI 3-kinase, ITK, and GRB-2/SOS. PI 3-kinase and GRB-2/SOS bind the CD28 cytoplasmic pYMNM motif via SH2 domains. We generated CD28 pYMNM mutants and found that Y191 mutation (Y191CD28F) disrupted both PI 3-kinase and GRB-2 binding, while M194 mutation (M194CD28C) disrupted only PI 3-kinase binding. Both mutants still bound ITK. We have assessed the ability of these selective mutants to support IL-2 production upon TCR zeta/CD3 ligation in the presence of CHO-CD86 (B7-2) cells. Both Y191CD28F and M194CD28C mutants failed to generate IL-2. These data directly implicate PI 3-kinase in CD28-mediated costimulation leading to IL-2 secretion. Wortmannin, an inhibitor of PI 3-kinase, induced cell apoptosis and as such was unsuitable for use in this study.

Animals↗
PubMed1994

T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif.

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cell proliferation. T-cell receptor zeta/CD3 engagement without CD28 ligation leads to a state of nonresponsiveness/anergy, thereby implicating CD28 in the control of peripheral tolerance to foreign antigens or tumors. A key unresolved question has concerned the mechanism by which CD28 generates intracellular signals. Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase with Src-homology 2 (SH2) domain(s) that binds to the platelet-derived growth factor receptor (PDGF-R), an interaction that is essential for signaling by growth factor. In this study, we demonstrate that CD28 binds to PI 3-kinase by means of a Y(P)MXM motif within its cytoplasmic tail. CD28-associated PI 3-kinase was detected by lipid kinase and HPLC analysis as well as by reconstitution experiments with baculoviral-expressed p85 subunit of PI 3-kinase. CD28 bound directly to the p85 subunit without the need for the associated p110 subunit. Site-directed mutagenesis and peptide competition analysis using Y(P)-MXM-containing peptides showed that PI 3-kinase bound to a Y(P)MXM motif within the CD28 cytoplasmic tail (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss of binding, while mutation of M194 caused partial loss of binding. Binding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C- and N-terminal SH2 domains with an affinity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of signaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-kinase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.

Amino Acid Sequence↗
PubMed1994

Identification of two SH3-binding motifs in the regulatory subunit of phosphatidylinositol 3-kinase.

Src homology 3 (SH3) domains have been recently shown to bind to proline-rich sequences contained in 3BP1, 3BP2, and SOS. In a recent study we demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) associates with the Fyn SH3 domain. Here we show that p85, the regulatory subunit of PI 3-kinase, binds directly to the SH3 domains of Abl, Lck, Fyn, and p85 itself. An examination of p85 amino acid sequence revealed two proline-rich sequences in its N-terminal region similar to those present in 3BP1, 3BP2, and SOS. To test whether these sequences mediate the association of p85 with SH3 domains two peptides with amino acid composition corresponding to the p85 alpha proline-rich sequences were synthesized and used in competition assays. Both peptides worked equally well in inhibiting the binding of PI 3-kinase activity and p85 alpha to Fyn SH3 domain, whereas a control peptide had no effect. These results indicate that, as in 3BP1 and SOS, the proline-rich sequences in p85 mediate its interaction with SH3 domains. These results also suggest that the SH3 domain of p85 may "self-associate" with the proline-rich motifs of the same subunit as part of the PI 3-kinase regulatory mechanism.

Amino Acid Sequence↗