Phosphotyrosine-independent binding of a 62-kDa protein to the src homology 2 (SH2) domain of p56lck and its regulation by phosphorylation of Ser-59 in the lck unique N-terminal region.

A previously undescribed 62-kDa protein (p62) that does not contain phosphotyrosine but, nevertheless, binds specifically to the isolated src homology 2 (SH2) domain of p56lck has been identified. The additional presence of the unique N-terminal region of p56lck prevents p62 binding to the SH2 domain. However, phosphorylation at Ser-59 (or alternatively, its mutation to Glu) reverses the inhibition and allows interaction of the p56lck SH2 domain with p62. Moreover, p62 is associated with a serine/threonine kinase activity and also binds to ras GTPase-activating protein, a negative regulator of the ras signaling pathway. Thus, phosphotyrosine-independent binding of p62 to the p56lck SH2 domain appears to provide an alternative pathway for p56lck signaling that is regulated by Ser-59 phosphorylation.     

Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein.

Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.     

Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain.

A novel human cDNA encoding a cytosolic 62-kDa protein (p62) that binds to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner has been cloned. The cDNA is composed of 2074 nucleotides with an open reading frame encoding 440 amino acids. Northern analysis suggests that p62 is expressed ubiquitously in all tissues examined. p62 is not homologous to any known protein in the data base. However, it contains a cysteine-rich region resembling a zinc finger motif, a potential G-protein-binding region, a PEST motif, and several potential phosphorylation sites. Using T7-epitope tagged p62 expression in HeLa cells, the expressed protein was shown to bind to the lck SH2 domain. Deletion of the N-terminal 50 amino acids abolished binding, but mutagenesis of the single tyrosine residue in this region had no effect on binding. Thus, the cloned cDNA indeed encodes the p62 protein, which is a phosphotyrosine-independent ligand for the lck SH2 domain. Its binding mechanism is unique with respect to binding modes of other known ligands for SH2 domains.     

The Src homology 2 domain of the protein-tyrosine kinase p56lck mediates both intermolecular and intramolecular interactions.

A key event in signaling by many cell surface receptors is the activation of Src-like protein-tyrosine kinases and the assembly of protein complexes at the plasma membrane mediated by Src homology 2 and 3 (SH2 and SH3) domains. p56lck is a Src-related protein-tyrosine kinase which has SH2 and SH3 domains and is involved in T-cell signaling and oncogenic transformation. Here we demonstrate that purified recombinant SH2 and HSH3/SH2 domains of p56lck can mediate intermolecular interactions with a number of tyrosine-phosphorylated proteins present in lysates of NIH 3T3 cells transformed by a constitutively activated form of p56lck (p56lckF505). Two of the interacting tyrosine-phosphorylated proteins were identified as the p85 subunit of phosphatidylinositol 3-kinase and the GTPase-activating protein of p21ras. Using a synthetic phosphopeptide corresponding to the tyrosine-phosphorylated carboxyl terminus of p56lck (amino acids 494-509), purified recombinant Lck SH2 domain, and differentially phosphorylated forms of p56lck we provide evidence that the SH2 domain of p56lck can also mediate intramolecular interactions with the phosphorylated carboxyl terminus. Together these results suggest that the SH2 domain of p56lck has a dual function: (i) it can mediate intermolecular interactions with cellular proteins phosphorylated on tyrosine and thus might be involved in building up signaling complexes at the plasma membrane and (ii) it can bind to the tyrosine-phosphorylated carboxyl terminus of p56lck in an intramolecular fashion and thereby might be involved in the regulation of its intrinsic protein-tyrosine kinase activity. Phosphorylation/dephosphorylation of the regulatory tyrosine residue 505 might serve as a switch between these two functions.     

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.     

CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn.

T-cell antigens including CD2, CD4, CD6, CD8, and CD28 serve as coreceptors with the T-cell receptor (TCR)/CD3 complex in control of T-cell growth. The molecular basis by which these antigens fulfill this role has remained a major issue. An initial clue to this question came with our finding that the sensitivity of in vitro kinase labeling (specifically using protein-tyrosine kinase p56lck) allowed detection of a physical association between CD4-p56lck and the TCR/CD3 complexes. Another T-cell antigen, CD5, is structurally related to the macrophage scavenger receptor family and, as such, can directly stimulate and/or potentiate T-cell proliferation. In this study, we reveal that in Brij 96-based cell lysates, anti-CD5 antibodies coprecipitated TCR zeta chain (TCR zeta)/CD3 subunits as well as the protein-tyrosine kinases p56lck and p59fyn. Conversely, anti-CD3 antibody coprecipitated CD5, p56lck, and p59fyn. Indeed, anti-CD5 and anti-CD3 gel patterns were virtually identical, except for a difference in relative intensity of polypeptides. Anti-CD4 coprecipitated p56lck, p32, and CD3/TCR zeta subunits but precipitated less CD5, suggesting the existence of CD4-TCR zeta/CD3 complexes distinct from the CD5-TCR zeta/CD3 complexes. Consistent with the formation of a multimeric CD5-TCR zeta/CD3 complex, anti-CD5 crosslinking induced tyrosine phosphorylation of numerous T-cell substrates, similar to those phosphorylated by TCR zeta/CD3 ligation. Significantly, as for TCR zeta, CD5 was found to act as a tyrosine kinase substrate induced by TCR/CD3 ligation. The kinetics of phosphorylation of CD5 (t1/2 = 20 sec) was among the earliest of activation events, more rapid than seen for TCR zeta (t1/2 = 1 min). CD5 represents a likely TCR/CD3-associated substrate for protein-tyrosine kinases (p56lck or p59fyn) and an alternative signaling pathway within a multimeric TCR complex.     

Phosphorylation of Ser-42 and Ser-59 in the N-terminal region of the tyrosine kinase p56lck.

Ser-42 and Ser-59 in the N-terminal region have been identified as the major phorbol ester-induced phosphorylation sites of p56lck. Phosphorylation of Ser-59 results in a gel shift from 56 kDa to 61 kDa. Simultaneous phosphorylation of Ser-42 and Ser-59 results in a further gel shift to 63 kDa. In vitro kinase assays show that Ser-59 can be uniquely phosphorylated by mitogen-activated protein kinase and that Ser-42 can be phosphorylated by either protein kinase A or protein kinase C.     

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 ζ (TcRζ)/CD3 ligation initiates a signaling cascade that involves src kinases p56^lck and ζ-associated protein 70, leading to the phosphorylation of substrates such as TcRζ, 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 TcRζ/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 TcRζ/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.     

Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine protein kinase, p56lck, reveals its oncogenic potential in fibroblasts.

p56lck, a cellular tyrosine protein kinase (EC 2.7.1.112) of the src family, is expressed in essentially all T cells and in some B cells. Expression in nonlymphoid cells is observed only rarely. We have found that mutation of a carboxyl-terminal phosphorylation site, tyrosine-505, reveals an oncogenic activity of this protein. Infection of fibroblasts with a retrovirus encoding wild-type p56lck is without consequence. In contrast, infection with a virus encoding the mutant protein leads to greatly increased phosphorylation of cellular proteins on tyrosine, morphological transformation, and anchorage-independent growth. This suggests that the tyrosine protein kinase activity and the oncogenic potential of p56lck are normally suppressed in vivo by phosphorylation of tyrosine-505. Since similar results were obtained previously with an analogous mutant of c-src, our results suggest that the protein kinase activity of all members of the src family of cytoplasmic tyrosine protein kinases will prove to be regulated by tyrosine phosphorylation at a conserved residue near the carboxyl terminus. Because p56lck is normally expressed only in lymphoid cells, it was possible that p56lck would be without effect in other tissues. The transformation of fibroblasts by mutant p56lck shows that this lymphoid protein can interact productively with nonlymphoid polypeptide substrates.     

Disruption of the CD4-p56lck complex is required for rapid internalization of CD4.

CD4 is a cell surface glycoprotein expressed by a subset of T lymphocytes and functions to enhance T-cell activation. CD4 is noncovalently associated via the cytoplasmic domain with the protein-tyrosine kinase p56lck, a member of the src protein-tyrosine kinase family. Upon activation of protein kinase C by phorbol ester, CD4 is phosphorylated on cytoplasmic serine residues and internalized from the cell surface, and disruption of the CD4-p56lck complex occurs. The exact relationship between these events is likely to be functionally significant, as cytoplasmic-domain serine phosphorylation and internalization have been shown to regulate the function of receptors that possess intrinsic protein-tyrosine kinase activity. Here we demonstrate that p56lck slows the rate of phorbol 12-myristate 13-acetate-induced internalization of CD4 in a manner that depends on a physical association between p56lck and CD4. This decreased rate is due at least in part to a requirement for disruption of the CD4-p56lck complex prior to internalization of CD4. Furthermore, disruption of the CD4-p56lck complex appears to depend on the integrity of the cytoplasmic-domain serine at position 408, probably due to a requirement for phosphorylation.     

Correlation between Src family member regulation by the protein-tyrosine-phosphatase CD45 and transmembrane signaling through the T-cell receptor.

Stimulation of tyrosine phosphorylation is an early and important event in antigen-induced T-cell activation. T-cell clones deficient in expression of CD45, a transmembrane protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), are impaired in their ability to respond to either antigen or T-cell receptor cross-linking. Analysis of the CD45-deficient CD8+ T-cell clone L3M-93 demonstrates that the Src family members p56lck and p59fyn show increased immunoreactivity with anti-phosphotyrosine antibody and exhibit decreased kinase activity. The site of increased tyrosine phosphorylation in Src family members was identified by comparison of cyanogen bromide peptide maps. Phosphorylation of the C-terminal phosphopeptide, containing the negative regulatory site of tyrosine phosphorylation, from the CD45-deficient cells was increased 8-fold for p56lck and 2-fold for p59fyn. These data suggest that CD45 dephosphorylates the negative regulatory site of multiple Src family members in the cytotoxic T-lymphocyte clone L3 and show a correlation between the ability to respond efficiently to antigen and the dephosphorylation of Src family members by CD45.     

En bloc substitution of the Src homology region 2 domain activates the transforming potential of the c-Abl protein tyrosine kinase.

Src homology region 2 (SH2) domains are present in many proteins involved in signal transduction. In nonreceptor protein tyrosine kinases the SH2 domain has been implicated in regulation of tyrosine kinase activity and in mediating interactions involved in downstream signaling. Different SH2 domains exhibit distinct binding specificities for both phosphotyrosine- and phosphoserine/phosphothreonine-containing proteins. We show that different SH2 domains are not functionally equivalent within the context of the c-ABL1b protooncogene. c-ABL1b, altered by replacement of its SH2 domain with the N-terminal SH2 domain of Ras GTPase-activating protein, exhibited activated transforming capability, caused intracellular tyrosine phosphorylation of p62, and was relocalized from nucleus to cytoplasm. This en bloc substitution apparently uncouples two distinct functions of the SH2 domain so that c-ABL escapes normal regulatory control while it retains the capability to elicit signals that promote transformation. The SH2 domain of the ARG protein tyrosine kinase, which shares high amino acid-sequence homology with the SH2 domain of ABL, was less effective in activating the oncogenic potential of c-ABL. The effects that the N-terminal SH2 domain of Ras GTPase-activating protein has in the context of c-ABL resemble the effects of deleting the SH3 domain. Thus, the SH2 and SH3 domains may have coordinate roles as regulatory control elements within the context of c-ABL.     

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.     

p56lck-independent activation and tyrosine phosphorylation of p72syk by T-cell antigen receptor/CD3 stimulation.

Activation of resting T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex is initiated by rapid tyrosine phosphorylation of cellular proteins. Protein-tyrosine kinases (PTKs) of the src family are known to be important, but the mechanism of their recruitment and their interactions with PTKs of other families are incompletely understood. We show that a member of another family of PTKs, the p72syk kinase, is constitutively bound to the TCR/CD3 complex and becomes tyrosine phosphorylated and activated within 1 min after TCR/CD3 stimulation. This activation did not depend on the presence of p56lck in T cells and in transfected COS cells. In both cases, however, the phosphorylation of cellular substrates was augmented by src family PTKs. We propose that p72syk may act as an immediate receptor-activated kinase upstream of the related p70zap PTK and the src family PTKs p56lck and p59fyn in T cells and that these src family PTKs act as signal amplifiers.     

Src homology region 2 domains direct protein-protein interactions in signal transduction.

Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21ras GTPase-activating protein (GAP), phospholipase C gamma, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set of phosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.     

Direct analysis of the binding of Src-homology 2 domains of phospholipase C to the activated epidermal growth factor receptor.

A number of proteins involved in intracellular signaling contain regions of homology to the product of the src oncogene that are termed Src-homology (SH) 2 domains. SH2 domains are believed to mediate the association of these proteins with various tyrosine-phosphorylated receptors in a growth factor-dependent manner. We have examined the kinetic characteristics of one of these interactions, the binding of the SH2 domains of phospholipase C gamma 1 with the receptor for epidermal growth factor (EGF). Bacterial fusion proteins were prepared containing the two SH2 domains of PLC gamma 1 and labeled metabolically with [35S]methionine/cysteine. A fusion protein containing both SH2 domains bound to the purified EGF receptor from EGF-treated cells, whereas no binding to receptors from control cells was detected. Binding was rapid, reaching apparent equilibrium by 10 min. Dissociation of the complex occurred only in the presence of excess unlabeled SH2 protein and exhibited two kinetic components. Similarly, analysis of apparent equilibrium binding revealed a nonlinear Scatchard plot, further indicating complex binding kinetics that may reflect cooperative behavior. The binding of the fusion protein containing both SH2 domains was inhibited by a fusion protein containing only the amino-terminal SH2 domain, although at concentrations an order of magnitude higher than that observed with the complete fusion protein. Fusion proteins containing SH2 domains from the GTPase-activating protein, the p85 regulatory subunit of phosphatidylinositol 3'-kinase, or the Abl oncoprotein competed less effectively. Binding of the PLC gamma 1 SH2 fusion protein to a mutant EGF receptor lacking the two carboxyl-terminal tyrosine phosphorylation sites exhibited a significantly lower affinity than that observed with the wild type, suggesting that this region of the receptor may play an important role. This binding assay represents a means with which to evaluate the pleiotropic nature of growth factor action.     

T-lymphocyte interleukin 2-dependent tyrosine protein kinase signal transduction involves the activation of p56lck.

Addition of interleukin 2 (IL-2) to IL-2-dependent T cells results in tyrosine protein kinase signal transduction events even though the IL-2 receptor alpha and beta chains lack intrinsic enzymatic activity. Here we report that addition of IL-2 to IL-2-dependent human T cells transiently stimulates the specific activity of p56lck, a member of the src family of nonreceptor tyrosine protein kinases expressed at high levels in T lymphocytes. The ability of IL-2 to induce p56lck activation was found to be independent of the capacity of p56lck to associate with either CD4 or CD8. Following IL-2 treatment, p56lck was found to undergo serine/threonine phosphorylation modifications that resulted in altered mobility of the lck gene product on polyacrylamide gels. These observations raise the possibility that p56lck participates in IL-2-mediated signal transduction events in T cells.     

A novel SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells

Because of the probable causal relationship between constitutive p210(bcr/abl) protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210(bcr/abl)-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain-containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210(bcr/abl)-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4, 5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210(bcr/abl)-mediated myeloid expansion.     

Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.

Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.     

The adapter protein CrkL associates with CD34

CD34 is a cell-surface transmembrane protein expressed specifically at the stem/progenitor stage of lymphohematopoietic development that appears to regulate adhesion. To elucidate intracellular signals modified by CD34, we designed and constructed glutathione-S-transferase (GST)- fusion proteins of the intracellular domain of full-length CD34 (GST-CD34i(full)). Precipitation of cell lysates using GST-CD34i(full) identified proteins of molecular mass 39, 36, and 33 kd that constitutively associated with CD34 and a 45-kd protein that associated with CD34 after adhesion. By Western analysis, we identified the 39-kd protein as CrkL. In vivo, CrkL was coimmunoprecipitated with CD34 using CD34 antibodies, confirming the association between CrkL and CD34. CD34 peptide inhibition assays demonstrated that CrkL interacts at a membrane-proximal region of the CD34 tail. To identify the CrkL domain responsible for interaction with CD34, we generated GST-fusion constructs of adapter proteins including GST-CrkL3' (C-terminal SH3) and GST-CrkL5' (N-terminal SH2SH3). Of these fusion proteins, only GST-CrkL3' could precipitate endogenously expressed CD34, suggesting that CD34 binds the C-terminal SH3 domain of CrkL. Interestingly, there appears to be differential specificity between CrkL and CrkII for CD34, because GST-CD34i(full) did not precipitate CrkII, a highly homologous Crk family member. Furthermore, GST-CD34i(full) did not bind c-Abl, c-Cbl, C3G, or paxillin proteins that are known to associate with CrkL, suggesting that CD34 directly interacts with the CrkL protein. CD34i(full) association with Grb or Shc adapter proteins was not detected. Our investigations shed new light on signaling pathways of CD34 by demonstrating that CD34 couples to the hematopoietic adapter protein CrkL. (Blood. 2001;97:3768-3775)