p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4-p56lck association-dependent manner

We previously showed that the association of CD4 and G(M3) ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the G(M1) ganglioside that partially co-localized with G(M3) in these domains. In the present study, we show that CD4-p56(lck) association in CD4 signalling is required for the redistribution of p56(lck), PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56(lck). In addition, we show that although these proteins associated in different ways with G(M1) and G(M3), all of the associations were dependent on CD4-p56(lck) association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56(lck) in detergent-insoluble membranes without association with G(M1) or G(M3). We propose that CD4 ligation and binding with p56(lck) and their interaction with G(M3) and/or G(M1) gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.     

The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3''-kinase from T lymphocytes.

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.     

A Raf-1-related p110 polypeptide associates with the CD4-p56lck complex in T cells.

The CD4 and CD8 antigens on T cells have been shown to associate with the Src family member p56lck and a GTP-binding protein, p32. The identification of receptor interactions with intracellular mediators is essential in the elucidation of downstream signals mediated by engagement of these receptor complexes. In this study, we report the detection of an additional 110-kDa polypeptide (p110) associated with the CD4-p56lck complex in human peripheral blood T lymphocytes and leukemic T-cell lines. p110 bound preferentially to CD4-p56lck as an assembled complex and poorly, if at all, to the individual components. p110 was recognized directly by an antiserum to the C-terminal region of the serine/threonine kinase Raf-1 and is related to a p110 polypeptide detected in anti-Raf-1 immunoprecipitates. Despite its association with the CD4-p56lck complex, p110 was found to be phosphorylated predominantly on serine residues. Furthermore, phorbol ester treatment of cells resulted in a transient increase in the detection of p110 associated with CD4-p56lck, concomitant with the modulation of CD4-p56lck from the cell surface. This Raf-1-related p110 is therefore likely to play a role in signals generated from the CD4-p56lck complex. p110 may serve as a bridge between the CD4-p56lck complex and the serine/threonine kinase pathways of T-cell activation.     

Structure-based design of peptidomimetic antagonists of p56(lck) SH2 domain

Starting from the tetrapeptide Ac-pYEEI-NHMe and using a structure-based approach, we have designed and synthesised a peptidomimetic ligand for p56(lck) SH2 domain containing a conformationally restricted replacement for the two glutamate residues. We have explored replacments for the isoleucine residue in the pY+3 pocket and thus identified 1-(R)-amino-3-(S)-indaneacetic acid as the most potent replacement. We also report the X-ray crystal structures of two of the antagonists.     

Thermodynamics of phosphotyrosine peptide–peptoid hybrids binding to the p56lck SH2 domain

A frequently used approach to transform peptides into more drug‐like compounds is preparation of the corresponding peptoids or peptide–peptoid hybrids. Although peptoids have advantages, there may also be some disadvantages such as their increased flexibility and the reduced ability for hydrogen bond formation due to alkylation of the backbone amide nitrogen, which might affect the free Gibbs energy (ΔG). To obtain more insight into these contributions to ΔG, we performed thermodynamic analyses on the interaction between peptide–peptoid hybrids, based on the sequence ‐pTyr‐Glu‐Glu‐Ile‐, and the p56^lck (Lck) Src homology 2 domain. van't Hoff analysis was performed on binding data obtained from surface plasmon resonance competition experiments in a temperature range of 10–40 °C. It is observed that amino acid–peptoid substitutions do not have a systemic negative effect on the entropic contributions to ΔG. However, loss in hydrogen‐bonding capacity of the backbone may strongly reduce the binding enthalpy and contribute to the observed lower binding affinity. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Experimental and calculated shift in pK(a) upon binding of phosphotyrosine peptide to the SH2 domain of p56(lck)

The pH dependence of the affinity of a 11-mer phosphotyrosine (pY) peptide (EPQpYEEIPIYL-NH2) for the SH2 domain of the tyrosine kinase p56(lck) was investigated with surface plasmon resonance (SPR). From SPR competition experiments the affinity in solution was obtained. The pH dependence of the affinity in solution can be well described by a proton linkage model with a single pK(a) shift upon binding, from 6.1 to 4.7. This shift is ascribed to the transition from the -2 to the -1 ionisation state of the tyrosine phosphate group. Based on the X-ray structure for the complex with Lck SH2, a pK(a) value of 5.3 for the bound pY peptide was computed, modelling the solvated protein as a system of point charges in a continuum. With the phosphate in the -2 state the binding energy is 1.8 kcal/mol more favourable than for the -1 state, corresponding to a 20-fold higher affinity. A proper charge is relevant in the design of potential therapeutic Lck SH2 ligands with mimics for the metabolically unstable tyrosine phosphate group.     

SLP-76 binding to p56lck: a role for SLP-76 in CD4-induced desensitization of the TCR/CD3 signaling complex.

Nonreceptor protein tyrosine kinases and associated substrates play a pivotal role in Ag receptor stimulation of resting cells and in the initiation of activation-induced cell death (AICD) of preactivated T cells. CD4-associated p56lck has been implicated not only in the activation of primary T cells, but also in the inhibition of T cell responses. We have previously shown that CD4+ T cell clones can be rescued from AICD when surface CD4 is engaged before the TCR stimulus. In this study, we show that prevention of AICD is associated with a CD4-dependent inhibition of TCR-triggered tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and Vav. We provide evidence for a SLP-76 interaction with Src homology 3 domains of p56lck and identify amino acids 185-194 of SLP-76 as relevant docking site. In view of the multiple functions of p56lck and SLP-76/Vav in the initiation of TCR/CD3/CD4 signaling, we propose a model for the CD4-dependent inhibition of TCR signaling and AICD of preactivated T cells. Our data suggest that preformed activation complexes of adapter proteins and enzymes in the vicinity of the CD4/p56lck complex are no longer available for the TCR signal when CD4 receptors are engaged before TCR stimulation.     

Human immunodeficiency virus type 1 glycoprotein precursor retains a CD4-p56lck complex in the endoplasmic reticulum.

The cell surface glycoprotein, CD4, is the receptor for human immunodeficiency virus (HIV) in T lymphocytes. Following HIV infection, there is reduced expression of CD4 on the cell surface, and this downregulation probably results, at least in part, from the formation of complexes containing the HIV type 1 (HIV-1) glycoprotein precursor (gp160) and CD4 that are not transported from the endoplasmic reticulum (ER). At the plasma membrane of T cells, CD4 is tightly associated with a cytoplasmic tyrosine kinase (p56lck) that is involved in T-cell activation. Using a transient expression system with HeLa cells, we show by pulse-labeling and immunoprecipitation that newly synthesized CD4 can associate with p56lck before CD4 is transported from the ER. In the presence of HIV-1 gp160, a ternary complex of gp160-CD4 and p56lck forms in the ER. Using confocal immunofluorescence microscopy, we observed complete retention of p56lck in the ER. Such mislocation of a tyrosine kinase to the cytoplasmic face of the ER could play a role in lymphocyte killing caused by HIV infection or expression of gp160 alone.     

Short related sequences in the cytoplasmic domains of CD4 and CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase.

We report that the cytoplasmic domains of the T-lymphocyte glycoproteins CD4 and CD8 alpha contain short related amino acid sequences that are involved in binding the amino-terminal domain of the intracellular tyrosine protein kinase, p56lck. Transfer of as few as six amino acid residues from the cytoplasmic domain of the CD8 alpha protein to the cytoplasmic domain of an unrelated protein conferred p56lck binding to the hybrid protein in HeLa cells. The common sequence motif shared by CD4 and CD8 alpha contains two cysteines, and mutation of either cysteine in the CD4 sequence eliminated binding of p56lck.p56lck also contains two cysteine residues within its CD4-CD8 alpha-binding domain, and both are critical to the interaction with CD4 or CD8 alpha. Because the interaction does not involve disulfide bond formation, a metal ion could stabilize the complex.     

Role of solution conformation and flexibility of short peptide ligands that bind to the p56(lck) SH2 domain

A general approach in drug design is making ligands more rigid in order to avoid loss in conformational entropy (deltaS(conf)) upon receptor binding. We hypothesized that in the high affinity binding of pYEEI peptide ligands to the p56(lck) SH2 domain this loss in deltaS(conf) might be diminished due to preorganization of the fourfold negatively charged pYEEI peptide in the bound, extended, conformation. A thermodynamic analysis was performed on the peptides Ac-pYEEI-NH(2), Ac-pYAAI-NH(2) and Ac-pYGGI-NH(2) using surface plasmon resonance (SPR) competition experiments to assay affinity constants at different temperatures. To study the effect of solution conformation and flexibility a computational conformation analysis was performed from which low energy conformations in solution were calculated, and S(conf) estimated. It was found that the calculated low energy conformations for especially the pYE moiety in solution resemble that in the bound state. In the calculated minimum energy conformation in solution isoleucine is bent towards the pY aromatic ring, the occurrence of such conformation is experimentally confirmed by NMR. The estimated values for S(conf) of the EE- and AA-peptide were similar, suggesting no predominant role of preorganization of the solution conformation due to electrostatic repulsion. Apparently the thermodynamics obey the same entropy-enthalpy compensation relationship, which also was found to hold for other peptides and peptidomimetics binding to p60(src) family SH2 domains. The implications of the results for drug design are discussed.     

p56lck association with CD4 is required for the interaction between CD4 and the TCR/CD3 complex and for optimal antigen stimulation.

By fluorescence resonance energy transfer, we have previously demonstrated that upon anti-CD3 mAb-mediated activation of a murine T cell hybridoma expressing human CD4, CD4 moves into close association with the TCR/CD3 complex. It was shown that this association between CD4 and the TCR/CD3 complex was dependent upon the presence of an intact CD4 cytoplasmic domain. We have now expressed, in a murine T cell hybridoma, mutated forms of CD4 containing cysteine to serine point mutations at positions 420, 422, or 430. The mutations at positions 420 and 422, but not 430, abolish association with p56lck. By using fluorescence resonance energy transfer, we demonstrate that mutations of CD4 which fail to interact with p56lck are unable to associate with the TCR/CD3 complex under conditions in which wild-type CD4 and the 430 mutant CD4 do associate with the TCR/CD3 complex. In addition, these mutants have a diminished response to CD4-dependent stimuli. We conclude that the association between CD4 and the TCR/CD3 complex during T cell activation plays an important role in CD4-dependent responsiveness and this association requires the interaction of CD4 with p56lck. These results also suggest that a substrate for p56lck may be expressed in the TCR/CD3 complex.     

Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns (3,4,5)-P(3)), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P(3) binds to alpha-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of alpha-actinin with the integrin beta subunit, and that PtdIns (3,4,5)-P(3) could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P(3), has important implications for the regulation of cellular adhesive strength and motility.     

The CD4/CD8:p56lck complex in T lymphocytes: a potential mechanism to regulate T-cell growth

The CD4 and CD8 antigens on the surface of T cells appear to bind to major histocompatibility complex (MHC) class II and I antigens, respectively. These receptors have also been found to regulate T cell growth in a manner independent of MHC recognition. In this report, we describe recent work showing that the CD4 and CD8 receptors are coupled to a protein-tyrosine kinase, p56lck, from T lymphocytes. The p56lck protein is a member of the src family, which plays a crucial role in the activation and transformation of various mammalian cells. The CD4/CD8:p56lck complex is catalytically active as shown by its ability to phosphorylate at 55-60 kDa. Two-dimensional, nonequilibrium gel electrophoresis demonstrated the similarity of p56lck associated with the CD4 and CD8 antigens. Detergents were found to vary in their ability to solubilize the CD4:p56lck complex in a catalytically active form. We further demonstrated by in vitro phosphorylation that members of the CD3 complex including the gamma, delta, and epsilon chains, as well as a putative zeta subunit can be phosphorylated at tyrosyl residues by the CD4/CD8:p56lck complex. Thus, this interaction may play an important role in the activation of T cells, and may mediate the cooperative interaction between the CD4/CD8 antigens and the Ti(TcR)/CD3 complex. This interaction also represents a possible precedent by which other members of the src family (c-src, c-yes, c-fgr, etc.) may be found to interact with mammalian growth receptors.     

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.     

A switch of G protein-coupled receptor binding preference from phosphoinositide 3-kinase (PI3K)-p85 to filamin A negatively controls the PI3K pathway

Frequent oncogenic alterations occur in the phosphoinositide 3-kinase (PI3K) pathway, urging identification of novel negative controls. We previously reported an original mechanism for restraining PI3K activity, controlled by the somatostatin G protein-coupled receptor (GPCR) sst2 and involving a ligand-regulated interaction between sst2 with the PI3K regulatory p85 subunit. We here identify the scaffolding protein filamin A (FLNA) as a critical player regulating the dynamic of this complex. A preexisting sst2-p85 complex, which was shown to account for a significant basal PI3K activity in the absence of ligand, is disrupted upon sst2 activation. FLNA was here identified as a competitor of p85 for direct binding to two juxtaposed sites on sst2. Switching of GPCR binding preference from p85 toward FLNA is determined by changes in the tyrosine phosphorylation of p85- and FLNA-binding sites on sst2 upon activation. It results in the disruption of the sst2-p85 complex and the subsequent inhibition of PI3K. Knocking down FLNA expression, or abrogating FLNA recruitment to sst2, reversed the inhibition of PI3K and of tumor growth induced by sst2. Importantly, we report that this FLNA inhibitory control on PI3K can be generalized to another GPCR, the mu opioid receptor, thereby providing an unprecedented mechanism underlying GPCR-negative control on PI3K.     

Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.     

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.