Biomedical subjects

C E Rudd

Publications and source records attributed to C E Rudd.

At least 19 recordsLinked to original sources

PubMed2004

Phosphatidylinositol-3 kinase inhibitors reproduce the selective antiproliferative effects of imatinib on chronic myeloid leukaemia progenitor cells.

We investigated the role of the phosphatidylinositol-3 kinase (PI-3K) pathway in regulating the proliferation of primary chronic myeloid leukaemia (CML) progenitor cells by using imatinib to inhibit the activity of p210(Bcr-Abl). The effect of imatinib on the expression of PI-3K pathway proteins was investigated by kinase assays and Western blotting; PI-3K was inhibited by wortmannin or LY294002, Jak2 by AG490 and farnesylation by FTI II; progenitor cell proliferation (self-renewal) was measured by growing myeloid colonies in vitro, then replating them to observe secondary colony formation. Suppression of p210(Bcr-Abl) with imatinib indirectly suppressed the activity of PI-3K and its downstream targets (Erk, Akt and p70S6 kinase), thereby implicating the PI-3K pathway in p210(Bcr-Abl)-mediated signalling in primary CML progenitor cells. The PI-3K inhibitors, wortmannin and LY294002 reproduced the differential effects of imatinib on normal and CML progenitor cell proliferation in vitro by increasing normal cell (P = 0.001) and reducing CML cell proliferation (P = 0.0003). This differential effect was attributable to dysregulated signalling by granulocyte colony-stimulating factor in CML. The responses of individual patient's cells to wortmannin correlated with their responses to imatinib (P = 0.004) but not their responses to AG490 (Jak2 kinase inhibitor) or FTI II (farnesyltransferase inhibitor). Individual responses to wortmannin also correlated with responses to interferon alpha (IFNalpha) (P = 0.016). Imatinib-resistant K562 cells were sensitive to LY294002. Inhibition of the PI-3K pathway may be common to imatinib and IFNalpha and reflect dysregulated cytokine signalling. As imatinib-resistant cells remained sensitive to wortmannin and LY294002, targeting the PI-3K pathway may provide an alternative therapy for imatinib-resistant patients.

Alkyl and Aryl Transferases↗

PubMed2001

Cytotoxic T lymphocyte antigen 4 and CD28 modulate cell surface raft expression in their regulation of T cell function.

Coreceptors CD28 and cytotoxic T lymphocyte antigen (CTLA)-4 have opposing effects on TcR/CD3 activation of T cells. While CD28 enhances and CTLA-4 inhibits activation, the underlying molecular basis of these effects has yet to be established. In this context, ganglioside and cholesterol enriched membrane microdomains (rafts, GEMs) serve as centers of signaling in T cells. Although CD28 can promote TcR/raft colocalization, evidence is lacking on whether the surface expression of membrane rafts can be targeted by CTLA-4 in its modulation of T cell responses. In this study, we demonstrate that both CD28 and CTLA-4 profoundly alter the surface expression of membrane rafts during T cell activation. While CD28 increased expression and the number of peripheral T cells induced to express surface rafts in response to TcR ligation, CTLA-4 potently inhibited both TcR and TcR x CD28 induced raft expression on the surface of T cells. Consistent with this, CD28 increased the presence of the linker of activated T cells (LAT) in purified membrane rafts, while CTLA-4 coligation effectively blocked this increase. Further, the reversal of the CTLA-4 block with CD3/CD28 ligation was accompanied by an increase in surface raft expression and associated LAT. Our observations demonstrate for the first time that CTLA-4 targets the release of rafts to the surface of T cells, and provides a mechanism for the opposing effects of CD28 and CTLA-4 on costimulation.

Abatacept↗

PubMed2001

Src kinases Fyn and Lck facilitate the accumulation of phosphorylated CTLA-4 and its association with PI-3 kinase in intracellular compartments of T-cells.

Src kinases bind to surface receptors and mediate signaling events at the surface of cells. Little is known regarding whether these kinases can mediate events within intracellular compartments. The T-cell antigen CTLA-4 resides primarily in the trans-Golgi network (TGN), and as such could serve as a model to study the intracellular function of src kinases in their ability to phosphorylate the receptor. In this study, we show that tyrosine kinases p56lck and p59fyn phosphorylate the cytoplasmic domain of CTLA-4 in T-cells. Most interestingly, these kinases are also found in the Golgi apparatus, the intracellular compartment where most of CTLA-4 is localized. Transfection of Lck or Fyn resulted in increased phosphorylation of intracellular CTLA-4 and recruitment of PI-3 kinase. By contrast, phosphorylation did not influence the presence of the receptor in the TGN. These data demonstrate that src kinases operate to modulate receptor binding to intracellular signaling proteins introducing the possibility that intracellular forms of receptors may generate growth signals.

Abatacept↗

PubMed2001

Positive regulation of T cell activation and integrin adhesion by the adapter Fyb/Slap.

The molecular adapter Fyb/Slap regulates signaling downstream of the T cell receptor (TCR), but whether it plays a positive or negative role is controversial. We demonstrate that Fyb/Slap-deficient T cells exhibit defective proliferation and cytokine production in response to TCR stimulation. Fyb/Slap is also required in vivo for T cell-dependent immune responses. Functionally, Fyb/Slap has no apparent role in the activation of known TCR signaling pathways, F-actin polymerization, or TCR clustering. Rather, Fyb/Slap regulates TCR-induced integrin clustering and adhesion. Thus, Fyb/Slap is the first molecular adapter to be identified that couples TCR stimulation to the avidity modulation of integrins governing T cell adhesion.

Actins↗

PubMed2001

Adaptor FYB (Fyn-binding protein) regulates integrin-mediated adhesion and mediator release: differential involvement of the FYB SH3 domain.

Aggregation of the high-affinity IgE receptor (FcepsilonRI) on mast cells activates a tyrosine phosphorylation cascade that is required for adhesion and degranulation events leading to the release of histamine and other inflammatory mediators. The full range of intracellular mediators that regulate this process is unknown. Recent studies have identified a group of immune cell-specific adaptor proteins that include linker for activation of T-cell (LAT), SH2-domain-containing leukocyte protein (SLP-76), and Fyn-T-binding protein (FYB)/SLP-76-associated protein (SLAP). In this study, we demonstrate that FYB can up-regulate integrin-mediated adhesion to fibronectin and mediator release in RBL-2H3 mast cells. The regulation of these two events could be distinguished from each other by the requirement of the FYB SH3 domain in beta-hexosaminidase release, but not adhesion, and the up-regulation of mediator release by FYB in nonadherent cells. FcepsilonRI aggregation increased FYB tyrosine phosphorylation, whereas confocal immunofluorescence microscopy showed that FYB colocalizes with F-actin in membrane ruffles and plaques. Our findings identify FYB as a regulator of integrin-mediated adhesion and degranulation events, which, in the case of mast cells, has potential applications to inflammatory and allergic responses.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Lnk adaptor: novel negative regulator of B cell lymphopoiesis.

Originally thought to have the functions now ascribed to the linker for activation of T cells protein (LAT), Lnk is coming into its own as an adaptor protein that mediates signaling through several receptor pathways. An essential role for Lnk in B cell development and maturation was recently uncovered by Perlmutter and colleagues. Rudd discusses the role of Lnk in B cells and hypothesizes a mechanism whereby Lnk, and its closely related protein family members, the adaptor molecules containing pleckstrin homology (PH) and Src-homology 2 (SH2) domains (APS), and Src-homology 2-B protein (SH2-B), may mediate signal promotion or attenuation.

Adaptor Proteins, Signal Transducing↗

PubMed2001

CTLA-4 blockade of antigen-induced cell death.

While cytotoxic T lymphocyte antigen-4 (CTLA-4) negatively regulates T-cell receptor (TCR)-driven interleukin (IL)-2 production and proliferation, little is known regarding whether the coreceptor has the capacity to inhibit other events, such as Fas ligand (FasL) expression and antigen-induced cell death (AICD). In this study, it is shown that CTLA-4 expressed in a T-cell hybridoma can elicit a potent block of FasL expression and AICD. Inhibition occurred independently of CTLA-4 blockage of IL-2 production and was partially reversed by a single mutation in the cytoplasmic YVKM motif. These findings indicate that CTLA-4 can block TCR signaling prior to bifurcation of signals leading to IL-2 production and apoptosis.

Abatacept↗

PubMed2001

A regulatory role for cytoplasmic YVKM motif in CTLA-4 inhibition of TCR signaling.

CTLA-4 negatively regulates TCR signaling, although the molecular basis for this effect has yet to be elucidated. The cytoplasmic YVKM motif, while binding to phosphatidylinositol 3-kinase, SHP-2 and the AP-1/AP-2 clathrin adaptor complexes, has been reported to play no role in CTLA-4 function. In contrast, in this study, we demonstrate that, although not essential, the YVKM motif contributes to optimal CTLA-4 blockage of TCRzeta or combined TCRzeta/CD28 signaling. Significantly, dependency on the YVKM motif varied with the mode of anti-receptor presentation, where soluble antibody ligation was more dependent on the presence of the motif than immobilized antibody. Previous studies have mainly relied on the use of immobilized antibody. Neither SHP-2 binding, alterations in TCRzeta chain phosphorylation, nor ZAP-70 recruitment was involved in CTLA-4 wild-type or mutant inhibition. Overall, our findings clearly implicate the YVKM motif in optimal CTLA-4 function.

Abatacept↗

PubMed2001

CD28 signaling via VAV/SLP-76 adaptors: regulation of cytokine transcription independent of TCR ligation.

Since CD28 provides cosignals in T cell responses, a key question is whether the coreceptor operates exclusively via TCRzeta/CD3 or also operates as an independent signaling unit. In this study, we show that CD28 can cooperate with VAV/SLP-76 adaptors to upregulate interleukin 2/4 transcription independently of TCR ligation. CD28 signaling is dependent on VAV/SLP-76 complex formation and induces membrane localization of these complexes. CD28-VAV/SLP-76 also functions in nonlymphoid cells to promote nuclear entry of NFAT, indicating that these adaptors are the only lymphoid components needed for this pathway. Further downstream, CD28-VAV/SLP-76 synergizes with Rac1 and causes F-actin remodelling proximal to receptor. Autonomous CD28 signaling may account for the distinct nature of the second signal and in trans amplification of T cell responses.

Actins↗

PubMed2000

SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55.

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRzeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.

Amino Acids, Diamino↗

PubMed2000

Lymphocyte signaling: Cbl sets the threshold for autoimmunity.

Cbl, a negative regulator of immune signaling, has recently been shown to act as a ubiquitin-protein ligase. Further, two new papers describing Cbl-b-deficient mice suggest that Cbl-b sets the threshold of signaling in T and B cells and prevents the development of autoimmunity.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Tyrosine phosphatase SHP-2 binding to CTLA-4: absence of direct YVKM/YFIP motif recognition.

CTLA-4 is well documented in its negative regulation of T-cell proliferation. However, little is known regarding the signaling mechanisms induced by CTLA-4. CTLA-4 associates with the phosphatidylinositol 3-kinase, the phosphatase SHP-2 and the clathrin adaptor complexes AP-1 and AP-2. SHP-2 SH2 domain binding to CTLA-4 is unusual given the absence of a I/VxYxxI/V/L motif. Here, we demonstrate that the phosphorylation of CTLA-4 tyrosines (YVKM and YFIP) fails to allow for single or tandem SHP-2 SH2 domain binding. This was observed using wild-type and inactive SHP-2 as well as a construct with the isolated two SH2 domains. The phosphorylated YVKM and YFIP motifs therefore do not appear to represent novel binding motifs for SHP-2 SH2 domains. At the same time, we could confirm that SHP-2 can associate with CTLA-4 in murine T-cells indicating that the interaction between the phosphatase and CTLA-4 is an indirect event, possibly mediated by PI 3-kinase/SHP-2 binding.

Abatacept↗

PubMed2000

Resting lymphocyte kinase (Rlk/Txk) targets lymphoid adaptor SLP-76 in the cooperative activation of interleukin-2 transcription in T-cells.

Rlk/Txk is a T-cell-specific member of the Btk/Tec family of tyrosine kinases, whereas SLP-76 is a lymphoid adaptor that is essential for pre-TcR and mature TcR signaling. Although Rlk deficient T-cells show partial defects in T-cell proliferation, Rlk can complement ITK-/- cells with multiple defects in TcR initiated early events and interleukin (IL)-2 production. A key question is the nature of the target of Rlk responsible for bridging the TcR with the activation of IL-2 transcription. In this study, we identify a pathway in which Rlk phosphorylates SLP-76 leading to the phosphorylation of PLCgamma1, activation of ERKs, and the synergistic up-regulation of TcR-driven IL-2 NFAT/AP-1 transcription. Rlk phosphorylated the N-terminal region of SLP-76, a region that has been previously shown to serve as a target for ZAP-70. Loss of N-terminal YESP/YEPP sites of SLP-76 or the Rlk kinase activity attenuated cooperativity between Rlk and SLP-76. These observations support a model where the TcR can utilize Rlk (as well as ZAP-70) in the phosphorylation of key sites in SLP-76 leading to the up-regulation of Th1 preferred cytokine IL-2.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The CD28-related molecule ICOS is required for effective T cell-dependent immune responses.

While CD28 is critical for expansion of naive T cells, recent evidence suggests that the activation of effector T cells is largely independent of CD28/B7. We suggest that ICOS, the third member of the CD28/CTLA-4 family, plays an important role in production of IL-2, IL-4, IL-5, and IFNgamma from recently activated T cells and contributes to T cell-dependent B help in vivo. Inhibition of ICOS attenuates lung mucosal inflammation induced by Th2 but not Th1 effector populations. Our data indicate a critical function for the third member of the CD28 family in T cell-dependent immune responses.

Abatacept↗

PubMed1999

Cutting edge: SLP-76 cooperativity with FYB/FYN-T in the Up-regulation of TCR-driven IL-2 transcription requires SLP-76 binding to FYB at Tyr595 and Tyr651.

SLP-76 (Src homology (SH) 2-domain-containing leukocyte protein of 76 kDa) and FYB/SLAP (FYN-T-binding protein/SLP-76-associated protein) are two hemopoietic cell-specific adaptor proteins downstream of TCR-activated protein tyrosine kinases. SLP-76 has been implicated as an essential component in T cell signaling. FYB is selectively phosphorylated by FYN-T, providing a template for the recruitment of FYN-T and SLP-76 SH2 domains. Coexpression of FYN-T, FYB, and SLP-76 can synergistically up-regulate IL-2 production in T cells upon TCR ligation. In this report, we show that two tyrosines, Tyr595 and Tyr651, of FYB are major sites of phosphorylation by FYN-T and mediate binding to SLP-76 in Jurkat T cells. Furthermore, the synergistic up-regulation of IL-2 promoter activity in the FYN-T-FYB-SLP-76 pathway is contingent upon the interaction between FYB and SLP-76, but not the interaction between FYB and FYN-T. These observations define a pathway by which SLP-76 interacts with downstream components in the up-regulation of T cell cytokine production.

Adaptor Proteins, Signal Transducing↗

PubMed1999

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 p59(FYN-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.

Adaptor Proteins, Signal Transducing↗

PubMed1999

SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages.

Inhibitory immunoreceptors downregulate signaling by recruiting Src homology 2 (SH2) domain-containing tyrosine and/or lipid phosphatases to activating receptor complexes [1]. There are indications that some inhibitory receptors might also perform other functions [2] [3]. In adherent macrophages, two inhibitory receptors, SHPS-1 and PIR-B, are the major proteins binding to the tyrosine phosphatase SHP-1. SHPS-1 also associates with two tyrosine-phosphorylated proteins (pp55 and pp130) and a protein tyrosine kinase [4]. Here, we have identified pp55 and pp130 as the adaptor molecules SKAP55hom/R (Src-kinase-associated protein of 55 kDa homologue) and FYB/SLAP-130 (Fyn-binding protein/SLP-76-associated protein of 130 kDa), respectively, and the tyrosine kinase activity as PYK2. Two distinct SHPS-1 complexes were formed, one containing SKAP55hom/R and FYB/SLAP-130, and the other containing PYK2. Recruitment of FYB/SLAP-130 to SHPS-1 required SKAP55hom/R, whereas PYK2 associated with SHPS-1 independently. Formation of both complexes was independent of SHP-1 and tyrosine phosphorylation of SHPS-1. Finally, tyrosine phosphorylation of members of the SHPS-1 complexes was regulated by integrin-mediated adhesion. Thus, SHPS-1 provides a scaffold for the assembly of multi-protein complexes that might both transmit adhesion-regulated signals and help terminate such signals through SHP-1-directed dephosphorylation. Other inhibitory immunoreceptors might have similar scaffold-like functions.

Animals↗

PubMed1999

Cytolytic T lymphocyte-associated antigen-4 and the TCR zeta/CD3 complex, but not CD28, interact with clathrin adaptor complexes AP-1 and AP-2.

The negative signaling receptor cytolytic T lymphocyte-associated Ag-4 (CTLA-4) resides primarily in intracellular compartments such as the Golgi apparatus of T cells. However, little is known regarding the molecular mechanisms that influence this accumulation. In this study, we demonstrate binding of the clathrin adaptor complex AP-1 with the GVYVKM motif of the cytoplasmic domain of CTLA-4. Binding occurred primarily in the Golgi compartment of T cells, unlike with AP-2 binding that occurs mostly with cell surface CTLA-4. Although evidence was not found to implicate AP-1 binding in the retention of CTLA-4 in the Golgi, AP-1 appears to play a role in shuttling of excess receptor from the Golgi to the lysosomal compartments for degradation. In support of this, increased CTLA-4 synthesis resulted in an increase in CTLA-4/AP-1 binding and a concomitant increase in the appearance of CTLA-4 in the lysosomal compartment. At the same time, the level of intracellular receptor was maintained at a constant level, suggesting that CTLA-4/AP-1 binding represents one mechanism to ensure steady state levels of intracellular CTLA-4 in T cells. Finally, we demonstrate that the TCR zeta/CD3 complex (but not CD28) also binds to AP-1 and AP-2 complexes, thus providing a possible link between these two receptors in the regulation of T cell function.

Abatacept↗