Regulation of Cbl molecular interactions by the co-receptor molecule CD43 in human T cells.

CD43, one of the most abundant glycoproteins on the T cell surface, has been implicated in selection and maturation of thymocytes and migration, adhesion, and activation of mature T cells. The adapter molecule Cbl has been shown to be a negative regulator of Ras. Furthermore, it may also regulate intracellular signaling through the formation of several multi-molecular complexes. Here we investigated the role of Cbl in the CD43-mediated signaling pathway in human T cells. Unlike T cell receptor signaling, the interaction of the adapter protein Cbl with Vav and phosphatidylinositol 3-kinase, resulting from CD43-specific signals, is independent of Cbl tyrosine phosphorylation, suggesting an alternative mechanism of interaction. CD43 signals induced a Cbl serine phosphorylation-dependent interaction with the tau-isoform of 14-3-3. protein. Protein kinase C-mediated Cbl serine phosphorylation was required for this interaction, because the PKC inhibitor RO-31-8220 prevented it, as well as 14-3-3 dimerization. Moreover, mutation of Cbl serine residues 619, 623, 639, and 642 abolished the interaction between Cbl and 14-3-3. Overexpression of Cbl in Jurkat cells inhibited the CD43-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and AP-1 transcriptional activity, confirming nevertheless a negative role for Cbl in T cell signaling. However, under normal conditions, PKC activation resulting from CD43 engagement was required to activate the MAPK pathway, suggesting that phosphorylation of Cbl on serine residues by PKC and its association with 14-3-3 molecules may play a role in preventing the Cbl inhibitory effect on the Ras-MAPK pathway. These data suggest that by inducing its phosphorylation on serine residues, CD43-mediated signals may regulate the molecular associations and functions of the Cbl adapter protein.

A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase.

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.

Inhibition of signaling through the B cell antigen receptor by the protooncogene product, c-Cbl, requires Syk tyrosine 317 and the c-Cbl phosphotyrosine-binding domain.

The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical pathways. Syk becomes phosphorylated on multiple tyrosine residues upon receptor cross-linking. Tyrosine 317 is a site of phosphorylation located within the linker region of Syk that separates the amino-terminal, tandem pair of SH2 domains from the carboxyl-terminal catalytic domain. The amino acid sequence surrounding phosphotyrosine 317 matches the consensus sequence for recognition by the phosphotyrosine-binding (PTB) domain of the protooncogene product, c-Cbl. The overexpression of c-Cbl in DT40 B cells inhibits Ag receptor-mediated activation of the NF-AT transcription factor. The ability of overexpressed c-Cbl to inhibit signaling requires both Syk tyrosine 317 and a functional c-Cbl PTB domain. Mutant forms of Syk lacking tyrosine 317 exhibit an enhanced ability to couple the BCR to pathways leading to the activation of both NF-AT and Elk-1. Coimmunoprecipitation experiments indicate that Syk phosphotyrosine 317 and the c-Cbl PTB domain enhance, but are not required for, all interactions between these two proteins. In unstimulated cells, c-Cbl and Syk can be isolated in a complex that also contains tubulin. A mutant form of Syk lacking tyrosine at position 317 exhibits an enhanced ability to interact with a diphosphopeptide modeled on the immunoreceptor tyrosine-based activation motif of the CD79a component of the Ag receptor. These studies indicate that c-Cbl may contribute to the regulation of BCR signaling by modulating the ability of Syk to associate with the BCR and couple the receptor to intracellular signaling pathways.

SLAP, a dimeric adapter protein, plays a functional role in T cell receptor signaling.

Engagement of the T cell antigen receptor (TCR) leads to rapid activation of protein tyrosine kinases, which in turn phosphorylate downstream enzymes and adapter proteins. Some adapter proteins, such as SLP-76, Vav, and LAT, positively regulate TCR-mediated signal transduction, whereas others, such as Cbl, play an inhibitory role. SLAP (Src-like adapter protein), an adapter protein containing a Src homology 3 and a Src homology 2 domain, was isolated from a yeast interacting screen by using N-terminal Cbl as bait. N-terminal Cbl interacts with SLAP in vivo and in vitro in a tyrosine phosphorylation-independent manner. We observed that SLAP is expressed in T cells, and upon TCR activation, SLAP interacts with ZAP-70, Syk, LAT, and TCRzeta chain in Jurkat T cells. In transiently transfected COS-7 cells, SLAP forms separate complexes with ZAP-70, Syk, and LAT through its Src homology 2 domain. Overexpression of a C-terminal-truncated SLAP mutant down-regulates nuclear factor of activated T cells-AP1 activity. We have evidence that SLAP forms homodimers through its C-terminal region. Serial truncations and mutations in the C terminus of SLAP demonstrate that there is a correlation between the loss of dimerization and the inhibition of nuclear factor of activated T cells-AP1 activity. The in vivo association of SLAP with key signaling molecules and its inhibition of T cell activation suggests that SLAP plays an important role in TCR-mediated signal transduction.

Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase.

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.

The small GTP-binding protein Rho potentiates AP-1 transcription in T cells.

The Rho family of small GTP-binding proteins is involved in the regulation of cytoskeletal structure, gene transcription, specific cell fate development, and transformation. We demonstrate in this report that overexpression of an activated form of Rho enhances AP-1 activity in Jurkat T cells in the presence of phorbol myristate acetate (PMA), but activated Rho (V14Rho) has little or no effect on NFAT, Oct-1, and NF-kappaB enhancer element activities under similar conditions. Overexpression of a V14Rho construct incapable of membrane localization (CAAX deleted) abolishes PMA-induced AP-1 transcriptional activation. The effect of Rho on AP-1 is independent of the mitogen-activated protein kinase pathway, as a dominant-negative MEK and a MEK inhibitor (PD98059) did not affect Rho-induced AP-1 activity. V14Rho binds strongly to protein kinase Calpha (PKCalpha) in vivo; however, deletion of the CAAX site on V14Rho severely diminished this association. Evidence for a role for PKCalpha as an effector of Rho was obtained by the observation that coexpression of the N-terminal domain of PKCalpha blocked the effects of activated Rho plus PMA on AP-1 transcriptional activity. These data suggest that Rho potentiates AP-1 transcription during T-cell activation.

Tyrosine-phosphorylated Cbl binds to Crk after T cell activation.

Crk is a Src homology 2 (SH2)/Src homology 3 (SH3)-containing adapter protein that has been implicated in intracellular signaling in fibroblasts and PC12 pheochromocytoma cells. Crk has been shown to bind to a tyrosine-phosphorylated protein of 116 kDa after TCR-mediated T cell activation. Here we demonstrate that the Crk-associated p116 phosphoprotein is not the Crk-associated substrate (Cas) but, rather, is a protein product of the c-cbl proto-oncogene. Whereas Cas was not tyrosine-phosphorylated after T cell activation, Cbl became highly phosphorylated. Crk immunoprecipitates from activated T cell lysates contain tyrosine-phosphorylated Cbl. This association is mediated by the SH2 domain of Crk, as evidenced by the interaction between Cbl and the fusion protein product of a glutathione S-transferase (GST) expression construct encoding the Crk-SH2 domain in vitro. Furthermore, phosphopeptide-binding studies revealed that the GST-Crk SH2 domain binds to a tyrosine-phosphorylated peptide corresponding to amino acids 770-781 of Cbl with high affinity. Cbl is a protein tyrosine kinase (PTK) substrate that becomes phosphorylated after engagement of numerous cell surface receptors including the TCR. Data revealed by genetic studies in the nematode, Caenorhabditis elegans, implicates a Cbl-like molecule, Sli-1, as a negative regulator of the Let-23-signaling pathway. Because the signal from the Let-23 pathway affects the activation status of the Let-60 (Ras homologue in C. elegans) pathway, the activation-dependent association between Crk and Cbl may represent another TCR-generated signal leading to Ras-related pathways.

Experimental acute adult T cell leukemia-lymphoma is associated with thymic atrophy in human T cell leukemia virus type I infection.

Human T cell leukemia virus type I (HTLV-1) infection may lead to an acutely fatal adult T cell leukemia-lymphoma (ATLL), but HTLV-1-infected people usually remain asymptomatic. Why only certain HTLV-I infections lead to acute ATLL, which is characterized by leukemic infiltration of multiple organs and immune suppression, remains unknown. A readily accessible animal model in which the spectrum of consequences resulting from HTLV-I infection can be observed would greatly aid studies of this retrovirus. New Zealand White rabbits inoculated with either HTLV-1-infected CD25+ T cells or cell-free virus, were serially necropsied at different intervals after death or humane sacrifice. Tissues were preserved at necropsy or cultured in vitro and subsequently prepared for morphologic or molecular examination. Rabbits inoculated with RH/K34, a productively infected rabbit T cell line that contains a monoclonally integrated full-length HTLV-I provirus, developed acute ATLL-like biologically malignant lymphoproliferative disease with lymphocyte infiltration of viscera; lymphomas consisting primarily of monoclonal expansions of RH/K34 manifested a variety of diffuse pleomorphic histologic types. Concurrently, lymphoproliferative disease was associated with onset of thymic atrophy in the presence of rapidly increasing thymic proviral load. In contrast, rabbits given two other HTLV-1 inocula, originally derived (as was RH/K34) using the human T cell line MT-2 as virus source, also became infected but did not develop thymic atrophy or the ATLL-like disease. HTLV-1 infection, thymic atrophy, and leukemic infiltration similar to acute ATLL occurred reproducibly in a New Zealand White rabbit model independent of RH/K34 inoculum and host histocompatibility. Thymic atrophy in RH/K34-inoculated rabbits, but not in rabbits given other similar HTLV-1, was consistent with immunosuppression sufficient to prevent rejection of the inoculum. Although the short, 8-day course of the experimental ATLL precludes its having a molecular pathogenesis identical to the human condition, the systemic consequences of acute ATLL, including its association with thymic atrophy, are closely modeled.

Replication of HIV type 1 in rabbit cell lines is not limited by deficiencies in tat, rev, or long terminal repeat function.

HIV-1 infection has been documented in rabbits, but infection proceeds slowly in this species. Human and rabbit cell lines were compared in order to identify barriers to efficient HIV-1 infection of rabbit cells. A direct comparison of human and rabbit CD4 as receptor for HIV-1 indicated that the rabbit CD4 homolog did not function well even when expressed by human cells. Examination of viral RNA production indicated that the major HIV transcripts were produced in HIV-infected rabbit cells, but were present at levels significantly lower than those found for human cells. Ability of HIV-1 LTRs to direct protein expression in human and rabbit cells was compared using gene constructs with the chloramphenicol acetyltransferase (cat) gene flanked by HIV-1 LTRs. Chloramphenicol acetyltransferase protein expression was equivalent in rabbit and human cell lines transfected with the HIV-1/CAT constructs and cotransfections with the HIV-1 tat gene led to similar increases in CAT expression. Subsequent transfections with an infectious molecular HIV clone yielded approximately equal levels of HIV protein expression in rabbit and human cell lines, suggesting that major barriers to virus production in rabbit lines exist at steps prior to transcription of the viral genome. Because HTLV-I replicates with high efficiency in rabbit cells, a chimeric virus clone was constructed consisting of the 5' portion of HIV-1 through the nef coding sequence followed by the 3' HTLV-I LTR. Transfection of most rabbit cell lines with the chimera produced levels of p24gag protein higher than those transfected with the parent HIV-1 clone. By contrast, the unmodified HIV clone replicated more efficiently in all human cell lines tested.

Crk interacts with tyrosine-phosphorylated p116 upon T cell activation.

Products of the crk oncogene are expressed in all tissues. Crk proteins are composed exclusively of Src homology 2 (SH2) and Src homology 3 (SH3) domains, and they have been implicated in intracellular signaling. For example, they participate as mediators of Ras activation during nerve growth factor stimulation of PC12 pheochromocytoma cells. We examined the role of Crk proteins during T cell receptor-mediated signaling and observed that Crk proteins specifically interact, via their SH2 domains, with a tyrosine-phosphorylated 116-kDa protein upon T cell activation. p116 may be related to the recently cloned fibroblast p130cas and/or p120-Cbl. In addition, we observed that GST-Crk fusion proteins and Crk-L bind, most likely via their SH3 domain, to C3G, a Ras guanine nucleotide exchange factor. Thus, the interaction of Crk with p116 and C3G strongly implicates Crk as a mediator of T cell receptor signaling, possibly involved in Ras activation.

Heat-shock proteins expressed on the surface of human T cell leukemia virus type I-infected cell lines induce autoantibodies in rabbits.

Eight human T cell leukemia virus type I (HTLV-I)-infected cell lines were derived in vitro from peripheral blood mononuclear cells of 8 rabbits. Each rabbit was then inoculated with its own HTLV-I-transformed cells, after which all but 1 rabbit had anti-heat-shock protein (hsp) antibodies in sera. Cell line RH/K34, which failed to raise a response to hsp70, caused lethal leukemia when > 2 x 10(8) live cells were injected into unrelated outbred rabbits. Rabbits injected with cell-free virus isolated from RH/K34 cells produced anti-hsp70 antibodies and became infected but developed no fatal disease. ELISA inhibition and flow cytometry analyses indicated that hsp molecules are expressed on the surface of RH/K34 and RH/K30, a nonlethal HTLV-I cell line used for comparison; surface hsp expression does not occur normally. Two proteins of approximately 72 and 93 kDa were detected by Western blot in extracts of RH/K30 cells. Presence of anti-hsp70 antibodies correlated with resistance to lethal doses of live RH/K34 cells, suggesting that hsp immunity may influence the outcome of RH/K34 pathogenicity.

Selection of rabbit CD4- CD8- T cell receptor-gamma/delta cells by in vitro transformation with human T lymphotropic virus-I.

In vitro transformation of rabbit peripheral blood mononuclear cells (PBMC) with human T lymphotropic virus-I (HTLV)-infected human or rabbit cells resulted in CD4- CD8- cell lines, some of which caused acute leukemia when injected into rabbits. Structural analyses of the proviruses from cell lines with diverse pathogenic effects provided no clear correlation with lethality. The rabbit lines were provisionally designated T cells because they express interleukin 2R (IL-2R) and CD5 and lack surface immunoglobulin, but none express functional T cell receptor (TCR) alpha or beta transcripts. A more detailed characterization of the HTLV-I-infected cells was required to determine cell lineage and its potential influence on pathogenic consequences. Probes for rabbit TCR gamma and delta genes were derived and used to detect gamma and delta TCR RNA transcripts, identifying the in vitro transformed lines as gamma/delta T cells. CD4+ and CD8+ lines were derived from PBMC of HTLV-I-infected rabbits and CD4+ TCR-alpha/beta HTLV-I lines were derived from rabbit thymus, eliminating the possibility that the HTLV-I isolates used here transform only CD4- CD8- TCR-gamma/delta cells. The percentage of gamma/delta cells in rabbit PBMC is relatively high (23% in adult rabbits); this with diminution of CD4+ and CD8+ cells in IL-2-supplemented PBMC or thymocyte cultures may account for selection of rabbit HTLV-I-infected gamma/delta T cell lines in vitro. The availability of well-characterized T cell lines with diverse in vivo effects in the rabbit HTLV-I disease model allows evaluation of roles played by cell type in HTLV-I-mediated disease.

Variation in HTLV-I sequences from rabbit cell lines with diverse in vivo effects.

Comparison of nucleotide sequences determined for HTLV-I integrated provirus from two rabbit cell lines, RH/K30 and RH/K34, revealed greater than 99% identity to one another. Substitutions encoding amino acid interchanges were observed in the gag, pol, and rex regions whereas the env and tax proteins were identical in the two lines. Comparison with the human prototypic HTLV-I sequence revealed considerably more variation, especially in the viral envelope region where the rabbit sequences are identical. The HTLV-I lines differed in their potential to cause disease in rabbits: injection of the RH/K34 cell line caused human adult T-cell leukemia/lymphoma-like (ATLL) disease which was fatal within 10 days, whereas all rabbits injected with the same or higher doses of RH/K30 survived with a low-grade leukemia that showed evidence of acute rejection. Correlation of lethality with viral sequence was tested by injection of rabbits with two other rabbit cell lines with HTLV-I provirus identical to RH/K34 in LTR, gag, and env regions. The fact that only one of these lines produced fatal disease suggests that pathogenic determinants lie outside of these regions or, alternatively, that the structure of the integrated virus is not the sole factor in the cell lines' ability to cause ATLL-like disease.

Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.

To examine the intrinsic properties of postnatal mesolimbic dopamine (DA) neurons, we dissociated the ventral tegmental area (VTA) from postnatal rats, enriched for DA neurons by microdissection or gradient purification, and grew the cells in culture. In these cultures, up to 50% of neurons were dopaminergic. DA neurons resembled their in vivo counterparts in soma shapes, and in showing two levels of tyrosine hydroxylase (TH) expression, axodendritic differentiation, two sizes of synaptic vesicles, nest-like synaptic arrangements with non-DA cells, and synaptic specializations. Electrophysiologically, however, they could not be distinguished from non-DA cells, which could be consistent with heterogeneity in cell properties. To examine a functional subset of VTA DA neurons, we retrogradely labeled VTA neurons projecting to the nucleus accumbens. These mesoaccumbens neurons were 86% TH positive, 56% cholecystokinin positive, and 0% neurotensin positive; they also displayed the soma shapes characteristic of DA neurons more generally and two levels of TH expression. Like their in vivo counterparts, mesoaccumbens cells generally fired single broad spikes that were triggered by slow depolarizations and had robust spike afterhyperpolarizations, low- and high-threshold Ca2+ spikes, rapid accommodation of firing, time-dependent anomalous rectification, and hyperpolarizing autoreceptor responses. Strikingly, the expression of these active properties did not change with time in culture. Mesoaccumbens DA cells could be identified by a distinctive subset of properties that made up an electrophysiological signature; however, unlike their in vivo counterparts, they were less often spontaneously active and never fired in bursts. These results suggest that most DA cell properties are intrinsic to the cells, including a significant heterogeneity that is maintained in postnatal culture; their level and mode of activity, however, appear to require afferent input. Culturing identified postnatal VTA DA neurons now makes possible examination of the impact of their individual properties on synaptic function.

CD4 and its role in infection of rabbit cell lines by human immunodeficiency virus type 1.

Human CD4 (HuCD4) is the principal receptor for human immunodeficiency virus type 1 (HIV-1) in human cell infection. Susceptibility of rabbit cell lines to infection with HIV-1 raised questions concerning whether a CD4 homolog serves as HIV-1 receptor on rabbit cells. Sequence comparisons of rabbit CD4 (RbCD4) cloned from a rabbit thymus cDNA library showed that 6 of the 18 residues implicated in HIV-1 binding by CD4 differ between the human and rabbit proteins. No correlation between RbCD4 expression by rabbit cell lines and their ability to support HIV-1 infection was seen. Transfection of RbCD4-negative, HTLV-I-transformed cell lines with HuCD4 significantly enhanced HIV-1 infectivity, suggesting that these lines lack a receptor present on other RbCD4-negative lines that produce high levels of p24 in their native state. Inhibition of HIV-1 infection with soluble HuCD4 was demonstrated for all rabbit lines tested, but complete inhibition was obtained only with a rabbit T-cell line expressing RbCD4 and with HuCD4 transfectants. The results suggest that HIV-1 infection of the RbCD4-positive line proceeds through a receptor similar to HuCD4 but that an additional receptor or receptors may serve this purpose in RbCD4-negative lines.