Biomedical subjects

C E Rudd

Publications and source records attributed to C E Rudd.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1994

Two-step TCR zeta/CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains, protein-tyrosine and lipid kinases.

A central question in T-cell immunity concerns the nature of intracellular signaling from the antigen receptor, the CD4/CD8 co-receptors and the CD28 antigen. Since the original discovery that T-cell receptors such as CD4 can interact with intracellular protein-tyrosine kinases such as p56lck, remarkable progress has been made in deciphering the signaling pathways that control T-cell growth and immune function. Here, Christopher Rudd and colleagues examine the role of protein-tyrosine kinases, SH2/SH3 domains and lipid kinases in the generation of signals from the TCR zeta/CD3 complex and the CD28 antigen.

Amino Acid Sequence↗

PubMed1994

Decreased CD8-p56lck activity in peripheral blood T-lymphocytes from patients with hereditary haemochromatosis.

Hereditary haemochromatosis (HH) is an autosomal recessive disease linked to certain MHC class-I specificities. The disease is characterized by increased iron absorption and, in some patients, abnormally low numbers of CD8+ T cells in the periphery. We were interested in whether CD4- and CD8-associated p56lck kinase activities were altered in patients with HH. In a study of 18 patients with HH (with and without low numbers of CD8+ cells), the level of autophosphorylation of the CD8-associated p56lck as well as its phosphotransferase activity, as determined by phosphorylation of an exogenous substrate, was significantly reduced by two- to three-fold relative to a control population of 23 healthy blood donors (P < 6 x 10(-7). CD8-p56lck activity was decreased in 16 out of 18 patients (ranging from 1.5- to 10-fold decrease). By contrast, the level of CD4-p56lck activity did not show an overall decrease relative to controls. In addition to an occasional decrease in the amount of CD8-associated lck, HH patient-derived T cells showed a consistent decrease in the relative CD8-p56lck specific activity. Immunofluorescence staining showed further that the difference could not be accounted by a discrepancy in the expression of CD8 alpha alpha or CD8 alpha beta complexes or MHC class I molecules. Decreased CD8-p56lck activity was seen both in patients undergoing intensive phlebotomy treatment and in patients in maintenance therapy (i.e. patients who had reached normal levels of iron stores), indicating that this abnormality does not appear to be corrected by iron depletion. To our knowledge, this is the first demonstration of an abnormality in a src-like receptor associated kinase in a human disease state linked to MHC class-I antigens.

Adult↗

PubMed1994

The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration.

Amino Acid Sequence↗

PubMed1993

Fc gamma RIIIA-mediated signaling involves src-family lck in human natural killer cells.

The cell surface receptor for IgG Fc domain (Fc gamma RIIIA, CD16) is present as a multimeric complex on human NK cells and is essential for antibody-dependent cellular cytotoxicity. Although CD16 engagement induces tyrosine phosphorylation of several substrates, the intracytoplasmic domains of the CD16 molecule lack intrinsic protein tyrosine kinase consensus sequences. We report here that, in human NK cells, engagement of the CD16 receptor induces tyrosine phosphorylation of p56lck tyrosine kinase. CD16 triggering through anti-receptor antibody enhances in vitro kinase activity of phosphotyrosyl proteins and autophosphorylation activity of anti-lck immunoprecipitates. Similar results were obtained after stimulation with IL-12. In vivo labeling experiments showed that CD16 cross-linking primarily induces in vivo phosphorylation of p56lck at the tyr-394 autophosphorylation site. Using in vitro kinase assay of CD16 immunoprecipitates from lysates of human NK cells, we demonstrated that a phosphorylated protein of 62 to 65 kDa was co-precipitated with CD16. Reprecipitation experiments also suggested that a small amount of p56lck may interact with this complex. These results indicate that CD16 triggering involves p56lck and that this kinase is therefore involved in the signaling pathways regulating NK cell cytolytic functions. However, additional kinases not yet identified are also likely to play a role in the early signaling events through CD16.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1993

T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

Animals↗

PubMed1993

Regulation of CD4-p56lck-associated phosphatidylinositol 3-kinase (PI 3-kinase) and phosphatidylinositol 4-kinase (PI 4-kinase).

CD4 serves as a receptor for MHC class II antigens and as a receptor for the human immunodeficiency virus (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. Although anti-CD4 crosslinking may increase lck activity, the effects of HIV-1 gp120 have been controversial. Activated protein-tyrosine kinases are known to associate with certain intracellular proteins possessing src-homology regions (SH-2 domains) such as phosphatidylinositol 3-kinase (PI 3-kinase). In this paper, we demonstrate that the CD4:p56lck complex associates with significant amounts of phosphatidylinositol (PI) kinase activity. High pressure liquid chromatographic (HPLC) analysis of the reaction products demonstrated the presence of phosphatidylinositol 3-phosphate (PI 3-P) and phosphatidylinositol 4-phosphate (PI 4-P), thus indicating that PI 3 and PI 4 kinases associate with CD4-p56lck. The p85 subunit of PI 3-kinase was also detected in anti-CD4 immunoprecipitates by immunoblotting with anti-p85 antiserum. Significantly, p56lck binding to CD4 appears to be necessary for the detection of lipid kinase activity associated with p56lck. Also, anti-HIV gp120 and anti-CD4 crosslinking induced a 10-15-fold increase in levels of both PI 3- and PI 4-kinase activity in anti-CD4 precipitates. Stimulation of CD4-p56lck-linked PI kinases by crosslinked HIV-1 gp120 may play a role in HIV-1-induced immune defects.

1-Phosphatidylinositol 4-Kinase↗

PubMed1993

src-related protein tyrosine kinases and their surface receptors.

The CD4-p56lck and CD8-p56lck complexes have served as a paradym for an expanding number of interactions between src-family members (p56lck, p59fyn, p56lyn, p55blk) and surface receptors. These interactions implicate src-related kinases in the regulation of a variety of intracellular events, from lymphokine production and cytotoxicity to the expression of specific nuclear binding proteins. Different molecular mechanisms appear to have evolved to facilitate the receptor-kinase interactions, including the use of N-terminal regions, SH2 regions and kinase domains. Variation exists in stoichiometry, affinity and the nature of signals generated by these complexes in cells. The CD4-p56lck complex differs from receptor-tyrosine kinases in a number of important ways, including mechanisms of kinase domain regulation and recruitment of substrates such as PI 3-kinase. Furthermore, they may have a special affinity for receptor-substrates such as the TcR zeta, MB1/B29 or CD5 receptors, and act to recruit other SH2-carrying proteins, such as ZAP-70 to the receptor complexes. Receptor-src kinase interactions represent the first step in a cascade of intracellular events within the protein-tyrosine kinase/phosphatase cascade.

Amino Acid Sequence↗

PubMed1993

A 72-kilodalton fyn-related polypeptide (p72fyn-R) binds to the antigen-receptor/CD3 (TcR/CD3) complex.

Protein-tyrosine kinases play crucial roles in the activation and transformation of T lymphocytes. In this study, we have identified a variant of the fyn kinase at 70-72 kDa (termed p72fyn-R) that can preferentially associate with the TcR/CD3 complex in certain T cells. Phosphoamine acid analysis revealed that the CD3-associated p72fyn-R is labeled on both tyrosine and serine/threonine residues. TcR/CD3-associated p72fyn-R could be specifically reprecipitated using anti-fyn antisera to both the N and C terminus of p59fyn. In addition, two-dimensional phosphotryptic peptide map patterns of TcR/CD3-associated p72fyn and anti-fyn-precipitable p72 were identical. By contrast, a comparison of p72fyn-R and p62fyn showed similarities and differences. p72fyn-R possesses a peptide corresponding to the autophosphorylation site that migrates in the same position as found for p59/62fyn. However, p72fyn-R possessed at least four novel phosphorylated sites labeled on serine and threonine residues that are absent in the p62fyn pattern. Phosphatase digestion experiments indicated that p72fyn-R is more resistant to dephosphorylation than p59/62fyn. Two-dimensional phosphotryptic analysis indicated that the novel serine/threonine phosphorylation sites were responsible for the resistance to phosphatase digestion. Although the exact nature of the relationship between p72fyn-R and p59/62fyn remains undetermined, these data indicate that TcR/CD3 may utilize novel variants of src-related kinases in the generation of signals which regulate T-cell growth.

Alkaline Phosphatase↗

PubMed1993

Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex.

Amino Acid Sequence↗

PubMed1993

Association of a 70-kDa tyrosine phosphoprotein with the CD16: zeta: gamma complex expressed in human natural killer cells.

The CD16: zeta: gamma receptor complex allows natural killer (NK) cells to recognize and eliminate antibody-coated target cells. Whereas the ectodomain of CD16 is the receptor for Fc gamma domains of immunoglobulins, disulfide-linked homo- and heterodimers composed of zeta and gamma are required for the cell surface expression, and signal transduction properties of the complex. Engagement of CD16 activates the tyrosine kinase pathway, which induces the tyrosine phosphorylation of several substrates, including the zeta subunit and the phospholipase C gamma-1 and gamma-2 isoforms. Here we show that CD16 stimulation of either peripheral blood NK cells, leukemic NK cells, or Jurkat transformants expressing a CD16: zeta: gamma receptor complex, results in the tyrosine phosphorylation of a 70 kDa zeta-associated protein (pp70). Similarly, a 70-kDa zeta-associated phosphoprotein in T cells has been shown to be a tyrosine kinase (ZAP-70). Peptide mapping analysis indicates that the 70-kDa zeta-associated phosphoproteins from T cells and NK cells are structurally indistinguishable. We conclude that the CD16: zeta: gamma complex may use a ZAP-70-related non-receptor tyrosine kinase, in the CD16 signaling cascade leading to NK cell activation.

Humans↗

PubMed1993

Human CD4/CD45RA+ and CD4/CD45RA- T cell subsets express CD4-p56lck complexes, CD4-associated lipid kinases, TCR/CD3-p59fyn complexes, and share similar tyrosine kinase substrates.

T cell activation appears to be regulated by an interplay between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). p56lck and p59fyn have been found to associate with CD4 and TCR-CD3 respectively. The CD45 family of transmembrane PTPases has been shown to be able to regulate the activities of these receptor-associated PTKs in vitro. In man, CD45 contains five different isoforms whose distribution defines subsets of T cells having distinct activation requirements and in vitro functions. Several groups have reported a physical interaction between distinct isoforms of CD45 and CD2, CD4, and the TCR-CD3 complex. Given the potential regulatory interaction between CD45 and PTKs in CD4+ subsets expressing different CD45 isoforms, we have examined CD4 associated and TCR-CD3- associated PTK activities, associated phosphatidyl inositol (PI) kinases and substrates of tyrosine phosphorylation in CD45RA+ and CD45RA- CD4+ T cell lines derived from peripheral blood. Both subsets express CD4-associated p56lck and TCR-CD3-associated p59fyn kinases which exhibit identical in vitro phosphorylation at the Y-394 and Y-420 autophosphorylation sites respectively. Further, both subsets exhibited PI kinases activity associated with CD4-p56lck. Consistent with these observations, anti-CD3 crosslinking induced the phosphorylation of a similar spectrum of intracellular substrates in these CD45RA+ and CD45RA- CD4+ T cell lines. These observations indicate that despite the possible interaction between CD45 isoforms and CD4 or TCR-CD3, the mere expression of the CD45RA isoform does not in and of itself alter the presence of receptor-associated kinases or their intracellular targets.

1-Phosphatidylinositol 4-Kinase↗

PubMed1993

Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase.

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.

1-Phosphatidylinositol 4-Kinase↗