Formation of Shc/Grb2- and Crk adaptor complexes containing tyrosine phosphorylated Cbl upon stimulation of the B-cell antigen receptor

B-cell antigen receptor (BCR) stimulation induces tyrosine phosphorylation of the Shc adaptor protein and its association with Grb2. The Shc/Grb2 complex may be involved in Ras activation, since Grb2 interacts with the guanine nucleotide exchange factor Sos. We reveal here an additional complexity of the BCR-induced Shc/Grb2 complex: it contains tyrosine phosphorylated proteins of 130, 110 and 75 kDa. The 130 kDa molecule inducibly associates with Shc, while the 75 kDa protein interacts with the carboxy-terminal SH3 domain of Grb2. The 110 kDa molecule is defined as Cbl, the product of the c-cbl oncogene, which is strongly phosphorylated on tyrosine upon BCR stimulation. Cbl constitutively interacts with the SH3 domains of Grb2, with a preference for the amino-terminal domain, and is in this way recruited to Shc upon BCR stimulation. Immunodepletion studies showed that Grb2-associated Cbl can be phosphorylated by BCR-induced tyrosine kinases independent of a Shc/Grb2 interaction. This indicates that the BCR can also couple to a Grb2 complex without the involvement of Shc. Cbl not only interacts with Grb2, but also with the adaptor protein Crk. In contrast to its constitutive interaction with Grb2, tyrosine-phosphorylated Cbl only associates with Crk after BCR stimulation. In summary, we observe that the BCR activates Shc/Grb2-, Grb2- and Crk adaptor complexes of distinct composition, which may allow selective coupling to different signal transduction cascades. Cbl participates in all three adaptor complexes and is tyrosine phosphorylated upon BCR stimulation, pointing to a central role for this molecule in the regulation of antigen receptor-induced B cell responses.     

Interaction of hematopoietic progenitor kinase 1 with adapter proteins Crk and CrkL leads to synergistic activation of c-Jun N-terminal kinase

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In order to further characterize the HPK1-mediated JNK signaling cascade, we searched for HPK1-interacting proteins that could regulate HPK1. We found that HPK1 interacted with Crk and CrkL adaptor proteins in vitro and in vivo and that the proline-rich motifs within HPK1 were involved in the differential interaction of HPK1 with the Crk proteins and Grb2. Crk and CrkL not only activated HPK1 but also synergized with HPK1 in the activation of JNK. The HPK1 mutant (HPK1-PR), which encodes the proline-rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstream effectors, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JNK activation. These results suggest that the Crk proteins serve as upstream regulators of HPK1. We further observed that the HPK1 mutant HPK1-KD(M46), which encodes the kinase domain with a point mutation at lysine-46, and HPK1-PR blocked interleukin-2 (IL-2) induction in Jurkat T cells, suggesting that HPK1 signaling plays a critical role in IL-2 induction. Interestingly, HPK1 phosphorylated Crk and CrkL, mainly on serine and threonine residues in vitro. Taken together, our findings demonstrate the functional interaction of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and CrkL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.     

Networks of interaction of p120cbl and p130cas with Crk and Grb2 adaptor proteins

P120cbl, the product of the c-cbl proto-oncogene, has previously been shown to become tyrosine phosphorylated following EGF stimulation of cells, and to bind constitutively to the SH3 domain of the adaptor protein Grb2. Here we show that another adaptor protein, Crk, binds through its SH2 domain to tyrosine phosphorylated p120cbl. In addition, Crk becomes phosphorylated on tyrosine and serine following EGF treatment of PC12 and other cell lines. In unstimulated cells, while Grb2 is not bound to any tyrosine phosphoprotein, Crk is bound via its SH2 domain to tyrosine phosphorylated p130cas, the Crk-associated v-Src substrate. Following EGF treatment, Crk dissociates from p130cas, possibly due to a higher affinity of Crk SH2 for p120cbl compared with p130cas. Interaction between Grb2 and p120cbl increases threefold following EGF treatment of cells; in vitro, this induction of Grb2 association with unphosphorylated p120cbl can be mimicked by the addition of tyrosine phosphorylated Shc, suggesting a transfer of information between the SH2 and SH3 domains of Grb2. These data indicate that adaptor proteins can exchange binding partners in response to stimuli, and that different adaptor proteins can bind to the same partners by different mechanisms.     

Identification of two tyrosine phosphoproteins, pp70 and pp68, which interact with phospholipase Cgamma, Grb2, and Vav after B cell antigen receptor activation

Tyrosine phosphorylation of cellular proteins mediates the assembly and localization of effector proteins through interactions facilitated by modular Src homology 2 (SH2) and phosphotyrosine binding domains. We describe here two tyrosine-phosphorylated proteins with Mr values of 70,000 and 68,000 that interact with Grb2, phospholipase C (PLCgamma1 and PLCgamma2), and Vav after B cell receptor cross-linking. The interaction of pp70 and pp68 with PLC and Vav is mediated by the carboxyl-terminal SH2 domain of PLC and the SH2 domain of Vav. In contrast, the interaction of pp70 and pp68 with Grb2 requires cooperative binding of the SH2 and SH3 domains of Grb2. Western blot analysis demonstrated that neither pp70 nor pp68 represented the recently described linker protein SLP-76, which binds Grb2, PLC, and Vav in T cells after T cell receptor activation. Moreover, SLP-76 protein was not detected in a number of B cell lines or in normal mouse B cells. Hence, we propose that pp70 and pp68 likely represent B cell homologs of SLP-76 which facilitate and coordinate B cell activation.     

Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.     

Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases

Grb10 and its close homologues Grb7 and Grb14, belong to a family of adapter proteins characterized by a proline-rich region, a central PH domain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their interaction with a variety of activated tyrosine kinase receptors is well documented, but their actual function remains a mystery. The Grb10 SH2 domain was isolated from a two-hybrid screen using the MEK1 kinase as a bait. We show that this unusual SH2 domain interacts, in a phosphotyrosine-independent manner, with both the Raf1 and MEK1 kinases. Mutation of the MEK1 Thr-386 residue, which is phosphorylated by mitogen-activated protein kinase in vitro, reduces binding to Grb10 in a two-hybrid assay. Interaction of Grb10 with Raf1 is constitutive, while interaction between Grb10 and MEK1 needs insulin treatment of the cells and follows mitogen-activated protein kinase activation. Random mutagenesis of the SH2 domain demonstrated that the Arg-betaB5 and Asp-EF2 residues are necessary for binding to the epidermal growth factor and insulin receptors as well as to the two kinases. In addition, we show that a mutation in Ser-betaB7 affects binding only to the receptors, while a mutation in Thr-betaC5 abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domains induces apoptosis in HTC-IR and COS-7 cells. These effects can be competed by co-expression of the wild type protein, suggesting that these mutants act by sequestering necessary signaling components.     

The T cell receptor associated CD3-epsilon protein is phosphorylated upon T cell activation in the two tyrosine residues of a conserved signal transduction motif

Signal transduction through the T cell receptor for antigen, the TcR/CD3 complex, involves phosphorylation of tyrosine residues in the CD3-zeta chain. Since both CD3-epsilon and the zeta chain contain a tyrosine-based signaling motif, we examine phosphorylation of CD3-epsilon in human T cells. Engagement of the TcR/CD3 complex induced tyrosine phosphorylation of CD3-epsilon in vivo. Induction of CD3-epsilon phosphorylation followed similar kinetics to that of the zeta chain phosphorylation. In contrast to zeta, CD3-epsilon phosphorylation was strictly dependent upon cell surface expression of this member of the TcR/CD3 complex. Chemical and proteolytic cleavage combined with peptide-specific Western blotting established that CD3-epsilon phosphorylation occurred in the two tyrosine residues located in the signal transduction motif in the C-terminal portion of the molecule. Taken together, these data indicated that phosphorylation of CD3-epsilon by tyrosine protein kinases may serve to couple the TcR/CD3 complex to other effector molecules in the signaling cascade.     

In vivo association of Grb2 with pp116, a substrate of the T cell antigen receptor-activated protein tyrosine kinase

Numerous recent studies have implicated the src homology 2 and 3 domain-containing protein, Grb2, in coupling protein tyrosine kinase signaling pathways with the Ras signaling pathway. Ligation of the T cell antigen receptor results in the activation of both a PTK, and Ras; therefore, we investigated whether Grb2 may serve a similar function in T cells. Here we report that a GST/Grb2 fusion protein associates with several tyrosine phosphoproteins from lysates of T cell antigen receptor-stimulated Jurkat T cells. Two of these proteins, pp36 and pp116, bind to the Grb2 fusion protein with high affinity. Through the use of mutated Grb2 fusion proteins, we demonstrate that pp116 binds the amino-terminal src homology 3 domain of Grb2, the same domain of Grb2 thought to be primarily responsible for its interaction with SOS. We demonstrate further that pp116 associates with Grb2 in vivo, and we provide evidence that in the Jurkat T cell line Grb2 may exist complexed with either pp116 or with SOS.     

The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling

We have characterized an SH3-SH2-SH3 linker protein that is prominently expressed in lymphoid tissues. This protein has 58% sequence identity to Grb2. An identical protein called Grap has been found in hematopoietic cells. In Jurkat cells, T cell receptor activation leads to the association of Grap with phosphoproteins p36/38 and, to a lesser degree, Shc. This interaction is mediated by the Grap SH2 domain, which has similar binding specificity to the Grb2 SH2 domain. Grap also associates via its SH3 domains with Sos, the Ras guanine nucleotide exchange factor; with dynamin, a GTPase involved in membrane protein trafficking; and with Sam68, a nuclear RNA-binding protein that serves as a substrate of Src kinases during mitosis. T cell activation effects an increase in Grap association with p36/38, Shc, Sos, and dynamin. Sam68 binding is constitutive. Phospholipase C-gamma1 and Fyn are also found in activated Grap signaling complexes, although these interactions may not be direct. We conclude that Grap is a prominent component of lymphocyte receptor signaling. Based on the known functions of bound effector molecules, Grap-mediated responses to antigen challenge may include endocytosis of the T cell receptor, cellular proliferation, and regulated entry into the cell cycle.     

The germinal center kinase (GCK)-related protein kinases HPK1 and KHS are candidates for highly selective signal transducers of Crk family adapter proteins

Adapter proteins function by mediating the rapid and specific assembly of multi-protein complexes during the signal transduction which guards proliferation, differentiation and many functions of higher eukaryotic cells. To understand their functional roles in different cells it is important to identify the selectively interacting proteins in these cells. Two novel candidates for signalling partners of Crk family adapter proteins, the hematopoietic progenitor kinase 1 (HPK1) and the kinase homologous to SPS1/STE20 (KHS), were found to bind with great selectivity to the first SH3 domains of c-Crk and CRKL. While KHS bound exclusively to Crk family proteins, HPK1 also interacted with both SH3 domains of Grb2 and weakly with Nck, but not with more than 25 other SH3 domains tested. The interaction of HPK1 with c-Crk and CRKL was studied in more detail. HPK1-binding to the first SH3 domain of CRKL is direct and occurs via proline-rich motifs in the C-terminal, non-catalytic portion of HPK1. In vitro complexes were highly stable and in vivo complexes of c-Crk and CRKL with HPK1 were detectable by co-immunoprecipitation with transiently transfected cells but also with endogenous proteins. Furthermore, c-Crk II and, to a lesser extent, CRKL were substrates for HPK1. These results make it likely that HPK1 and KHS participate in the signal transduction of Crk family adapter proteins in certain cell types.     

T cell receptor-mediated tyrosine phosphorylation of Cas-L, a 105-kDa Crk-associated substrate-related protein, and its association of Crk and C3G

Cas-L (pp105), a Crk-associated substrate (p130(Cas))-related protein, was first identified as a 105-kDa protein that is tyrosine-phosphorylated following beta1 integrin cross-linking in T cells. Cas-L contains possible multiple binding sites for the Src homology (SH) 2 domains of various signaling molecules, and appears to be involved in signal transduction through phosphorylated tyrosine-mediated protein-protein interaction. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocyte-specific signals. Here, we show the involvement of Cas-L in the T cell receptor (TCR)/CD3 signaling pathway. Cas-L is transiently phosphorylated following CD3 cross-linking, and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant that lacks the SH3 domain, the binding site for focal adhesion kinase (FAK), is also tyrosine-phosphorylated upon CD3 cross-linking, but not upon beta1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Taken together, the present study indicates a novel signaling pathway mediated by tyrosine-phosphorylated Cas-L upon the TCR/CD3 stimulation.     

Interactions of indomethacin with lysosomes and proteins

The stabilizing action of indomethacin on lysosomes and protein in vivtro was studied. The magnitude of the stabilizing action on lysosomes was greater than that of aspirin or oxyphenbutazone, similar to that of phenylbutazone, but less than that of prednisolone. The stabilizing action of indomethacin on lysosomes possibly may contribute to its anti-inflammatory properties.     

RANTES-induced T cell activation correlates with CD3 expression

The chemokine RANTES induces a unique biphasic cytoplasmic Ca2+ signal in T cells. The first phase of this signal, similar to that of other chemokines, is G-protein mediated and chemotaxis associated. The second phase of this signal, unique to RANTES and evident at concentrations greater than 100 nM, is tyrosine kinase linked and results in a spectrum of responses similar to those seen with antigenic stimulation of T cells. We show here that certain jurkat T cells responded to RANTES solely through this latter pathway. A direct correlation between the RANTES-induced second phase response and CD3 expression was demonstrated in these cells. Sorting the Jurkat cells into CD3(high) and CD3(low) populations revealed that only the CD3(high) cells were responsive to RANTES. Furthermore, stimulation of these Jurkat cells with anti-CD3 mAb significantly depresses their subsequent response to RANTES. While a RANTES-specific chemokine receptor is expressed at a low level on these Jurkat cells, the RANTES-induced activation is dependent on the presence of the TCR. Thus, stimulation through TCR may partially account for RANTES' unique pattern of signaling in T cells.     

Processing, subcellular localization, and function of 519 (granulysin), a human late T cell activation molecule with homology to small, lytic, granule proteins

CTL and NK cells share a common cytolytic mechanism that involves regulated exocytosis of lytic molecules stored within cytoplasmic granules. Here we describe the processing, subcellular localization, and function of a T and NK cell-specific granule protein that shares homology with small, lytic granule-associated molecules. The gene coding for this protein, 519, is expressed late after T cell activation. Antisera raised against a 519/glutathione-S-transferase fusion protein and a series of peptides derived from the 519 protein sequence permitted the identification of two small CTL protein products of 15 and 9 kDa that are exocytosed after stimulation through the TCR. The 9-kDa product is a processed form of 519 and differs from the 15-kDa product in both its amino and carboxyl terminus. While both 519 proteins are found in cytoplasmic granules, the 9-kDa form is also present in dense, highly cytolytic granules. Functional studies indicate that this protein is lytic against tumor cell targets. The cell type- and stage-specific expression pattern of 519 along with its subcellular localization are reminiscent of molecules that play a vital role in granule-mediated cytolysis by CTL and NK cells. Its lytic activity suggests the involvement of 519 in CTL effector function.     

Fidelity of T cell activation through multistep T cell receptor zeta phosphorylation

The T cell receptor (TCR) alphabeta heterodimer interacts with its ligands with high specificity, but surprisingly low affinity. The role of the zeta component of the murine TCR in contributing to the fidelity of antigen recognition was examined. With sequence-specific phosphotyrosine antibodies, it was found that zeta undergoes a series of ordered phosphorylation events upon TCR engagement. Completion of phosphorylation steps is dependent on the nature of the TCR ligand. Thus, the phosphorylation steps establish thresholds for T cell activation. This study documents the sophisticated molecular events that follow the engagement of a low-affinity receptor.     

Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors

B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation. The BCR activates Ras, presumably by the formation of a Shc-Grb2 adaptor complex, which recruits the Grb2-associated guanine nucleotide exchange factor Sos to the plasma membrane. In order to reveal additional BCR-induced signaling events involving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins. Using the yeast two-hybrid system and bacterial fusion proteins, Vav and C3G were identified as Grb2 binders. Vav is a putative nucleotide exchange factor and a target for BCR-induced tyrosine phosphorylation. C3G exerts nucleotide exchange activity on the Ras-related Rap1 protein. While Sos binds to both Grb2 Src homology-3 (SH3) domains, Vav was found to associate selectively with the carboxyl-terminal SH3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2. Despite the association of Vav with Grb2 in vitro, we could not demonstrate an interaction between endogenous Vav and Grb2 molecules in primary B cells. Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BCR stimulation. C3G did not bind to either Grb2, Shc, or Crk in vivo. Instead, C3G was found in association with the Crk-L adaptor, both before and after BCR stimulation. We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylated Cbl. We conclude that, in addition to Sos, Vav and C3G play a role in BCR-mediated signal transduction. These guanine nucleotide exchange factors selectively associate with Grb2, Crk, and Crk-L, respectively, which may serve to direct them to different target molecules. Since Cbl binds to Grb2, Crk, as well as Crk-L, we hypothesize that Cbl may affect the function of all three exchangers.     

The BCR/ABL oncogene alters interaction of the adapter proteins CRKL and CRK with cellular proteins

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary leukemic neutrophils from patients with CML, the major tyrosine phosphorylated protein is CRKL, an SH2-SH3-SH3 adapter protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene. In cell lines transformed by BCR/ABL, CRKL was tyrosine phosphorylated, while CRK was not. We looked for changes in CRK- and CRKL-binding proteins in Ba/F3 hematopoietic cell lines which were transformed by BCR/ABL. Anti-CRK II or anti-CRKL immunoprecipitates were probed by far Western blotting with CRK II- or CRKL-GST fusion proteins to display CRK- and CRKL-coprecipitating proteins. There was a striking qualitative difference in the proteins coprecipitating with CRKL and CRK II. In untransformed cells, three major proteins coprecipitated with CRKL, identified as C3G, SOS and c-ABL. Each of these proteins was found to interact with the CRKL-SH3 domains, but not the SH2 domain. After BCR/ABL transformation, the CRKL SH3-domain binding proteins did not change, with the exception that BCR/ABL now coprecipitated with CRKL. Compared to CRKL, very few proteins coprecipitated with CRK II in untransformed, quiescent cells. After BCR/ABL transformation, both the CRKL- and CRK-SH2 domains bound to a new complex of proteins of approximate molecular weight 105-120 kDa. The major protein in this complex was identified as p120CBL. Thus, in these hematopoietic cell lines, CRKL is involved to a greater extent than CRK II in normal signaling pathways that involve c-ABL, C3G and SOS. In BCR/ABL-transformed cells, CRKL but not CRK II, appears to form complexes which potentially link BCR/ABL, c-ABL, C3G, and SOS to the protooncoprotein, p120CBL.