RESUMO
Antigen receptor-triggered T-cell activation is mediated by the sequential action of the Src and Syk/Zap-70 families of protein tyrosine kinases (PTKs). Previously, we reported that another PTK termed p50(csk) was a potent negative regulator of T-cell receptor (TCR) signaling because of its ability to inactivate Src-related kinases. This inhibitory effect required the catalytic activity of Csk, as well as its Src homology (SH)3 and SH2 domains. Subsequent studies uncovered that, via its SH3 domain, p50(csk) was associated with PEP, a proline-enriched protein tyrosine phosphatase (PTP) of unknown function expressed in hemopoietic cells. Herein, we have attempted to identify the role of the Csk-PEP complex in T lymphocytes. The results of our experiments showed that, like Csk, PEP was a strong repressor of TCR signaling. This property was dependent on the phosphatase activity of PEP, as well as on the sequence mediating its binding to p50(csk). Through reconstitution experiments in Cos-1 cells, evidence was obtained that Csk and PEP act synergistically to inhibit protein tyrosine phosphorylation by Src-related kinases, and that this effect requires their association. Finally, experiments with a substrate-trapping mutant of PEP suggested that PEP functions by dephosphorylating and inactivating the PTKs responsible for T-cell activation. In addition to giving novel insights into the mechanisms involved in the negative regulation of T-cell activation, these findings indicate that the association of an inhibitory PTK with a PTP constitutes a more efficient means of inhibiting signal transduction by Src family kinases in vivo.
Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Quinases da Família src/metabolismo , Animais , Sítios de Ligação , Células COS , Proteína Tirosina Quinase CSK , Hibridomas/metabolismo , Camundongos , Mapeamento de Peptídeos , Fosfoproteínas/análise , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases , Transdução de Sinais , Transfecção/genética , Domínios de Homologia de srcRESUMO
Accumulating data show that the tyrosine protein kinase Zap-70 plays an essential role in T cell receptor-mediated signal transduction. However, the model of action, as well as the physiologically relevant substrates of Zap-70, have not been determined. We have attempted to identify a 120-kD tyrosine-phosphorylated protein (p120) that associates with Zap-70 in activated T lymphocytes. The results of our analyses showed that p120 is largely encoded by the c-cbl protooncogene. Furthermore, the association of Zap-70 with c-Cbl was shown to be induced by T cell receptor stimulation, implying that it required posttranslational modification of one or both of these products. FynT, but not Lck, also associated with c-Cbl in activated T cells. Finally, using a heterologous system, it was demonstrated that the ability of Zap-70 to cause tyrosine phosphorylation of p120c-cbl was dependent on Lck- or FynT-mediated signals. As c-Cbl can associate with several other signaling molecules, it may couple Zap-70 to downstream effectors during T cell activation.
Assuntos
Ativação Linfocitária , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Ubiquitina-Proteína Ligases , Animais , Camundongos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-cbl , Proteínas Proto-Oncogênicas c-fyn , Proteína-Tirosina Quinase ZAP-70RESUMO
Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59fyn in T cell activation and to test the hypothesis that p59fynT serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59fyn on the physiology of an antigen-specific mouse T cell hybridoma. Our results demonstrated that the two forms of fyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59fynT increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59fyn isoform expressed in T lymphocytes regulates the coupling of TCR stimulation by antigen/major histocompatibility complex to lymphokine production.
Assuntos
Antígenos/imunologia , Ativação Linfocitária , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/imunologia , Animais , Sequência de Bases , Encéfalo/fisiologia , Vetores Genéticos , Interleucina-2/biossíntese , Cinética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fyn , Receptores de Antígenos de Linfócitos T/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
We have identified a novel Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76)-related molecule which we have termed Clnk (for cytokine-dependent hemopoietic cell linker). Unlike its relatives SLP-76 and B cell linker protein (Blnk), Clnk is not expressed uniformly within a given hemopoietic cell lineage. Even though it can be detected in several cell types, including T cells, natural killer cells, and mast cells, its expression seems to be strictly dependent on sustained exposure to cytokines such as interleukin (IL)-2 and IL-3. Strong support for the notion that Clnk is involved in immunoreceptor signaling was provided by the observation that it inducibly associated with at least one tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. Moreover, transient expression of Clnk caused an increase in immunoreceptor-mediated signaling events in a T cell line. Taken together, these results show that Clnk is a novel member of the SLP-76 family selectively expressed in cytokine-stimulated hemopoietic cells. Furthermore, they suggest that Clnk may be involved in a cross-talk mechanism between cytokine receptor and immunoreceptor signaling.
Assuntos
Citocinas/farmacologia , Sistema Hematopoético/química , Proteínas Nucleares , Fosfoproteínas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas de Transporte/fisiologia , Linhagem Celular , DNA Complementar/análise , Proteínas de Ligação a DNA/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Receptores Imunológicos/fisiologia , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Domínios de Homologia de srcRESUMO
Accumulating data suggest that the target cells for selection events leading to establishment of the mature T cell repertoire are the functionally immature double-positive (CD4+/CD8+) thymocytes, and that the CD4 and CD8 antigens expressed on these cells play important roles in these processes. In an attempt to define the biochemical pathways implicated in these events, we have studied the effects of engagement of accessory molecules on tyrosine protein phosphorylation. The results of our experiments demonstrate that engagement of CD4 and CD8 expressed on double-positive thymocytes is coupled with a rapid tyrosine protein phosphorylation signal. Further analyses have revealed that these two surface molecules are physically associated with the internal membrane tyrosine protein kinase p56lck in immature thymocytes, and that the catalytic function of lck expressed in double-positive thymocytes is significantly enhanced upon engagement of CD4. These data provide evidence that tyrosine-specific protein phosphorylation pathways coupled to the CD4 and CD8 T cell surface antigens are functional in immature thymocytes, and therefore, formally prove that signaling functions of CD4 and CD8 molecules are operative in immature thymocytes.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Animais , Complexo CD3 , Linfócitos T CD4-Positivos/citologia , Antígenos CD8 , Diferenciação Celular , Reagentes de Ligações Cruzadas , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Fosforilação , Fosfotirosina , Timo/citologia , Tirosina/metabolismoRESUMO
T cell activation and clonal expansion is the result of the coordinated functions of the receptors for antigen and interleukin (IL)-2. The protein tyrosine kinase p56(lck) is critical for the generation of signals emanating from the T cell antigen receptor (TCR) and has also been demonstrated to play a role in IL-2 receptor signaling. We demonstrate that an IL-2-dependent, antigen-specific CD4(+) T cell clone is not responsive to anti-TCR induced growth when propagated in IL-2, but remains responsive to both antigen and CD3epsilon-specific monoclonal antibody. Survival of this IL-2-dependent clone in the absence of IL-2 was supported by overexpression of exogenous Bcl-xL. Culture of this clonal variant in the absence of IL-2 rendered it susceptible to anti-TCR-induced signaling, and correlated with the presence of kinase-active Lck associated with the plasma membrane. The same phenotype is observed in primary, resting CD4(+) T cells. Furthermore, the presence of kinase active Lck associated with the plasma membrane correlates with the presence of ZAP 70-pp21zeta complexes in both primary T cells and T cell clones in circumstances of responsive anti-TCR signaling. The results presented demonstrate that IL-2 signal transduction results in the functional uncoupling of the TCR complex through altering the subcellular distribution of kinase-active Lck.
Assuntos
Complexo CD3/metabolismo , Interleucina-2/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cricetinae , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70 , Proteína bcl-XRESUMO
Proto-oncogenes encode products that comprise a select group of cellular regulatory proteins whose mutation or aberrant expression can result in oncogenic transformation. With the exception of certain growth factors and their receptors, the definition of normal functions for most proto-oncogene products has been elusive. The discovery that a member of the src-family of tyrosine protein kinases (p56lck) is associated with both the CD4 and CD8 T-lymphocyte surface glycoproteins provides the first clue to understanding the potential physiological functions of this family of proto-oncogenes.
Assuntos
Proto-Oncogenes/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Humanos , Ativação Linfocitária , Dados de Sequência Molecular , Proteínas Tirosina Quinases/genética , Proto-Oncogene Mas , Linfócitos T/imunologiaRESUMO
Upon antigen stimulation, the T-cell receptor for antigen transduces an intracellular protein tyrosine phosphorylation signal that is critical for subsequent T-lymphocyte activation. As the antigen receptor does not possess an intrinsic protein tyrosine kinase activity, the mechanism by which it regulates protein tyrosine phosphorylation is unconventional. Evidence is increasing that the Src-related protein tyrosine kinases P56lck and p59fyn, as well as the protein tyrosine phosphatase CD45, are involved in this process.
Assuntos
Genes src/genética , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/fisiologia , Proteínas Tirosina Quinases/metabolismoRESUMO
Immunoreceptor engagement results in the sequential activation of several classes of protein tyrosine kinases, including the Src and Syk/Zap-70 families. Recent progress has been made in our understanding of the regulation and function of these molecules. First, it was revealed that membrane compartmentation of protein tyrosine kinases may be essential for their proper biological function. Second, Src family kinases were found to act not only as positive regulators, but also as inhibitors of cell activation. Third, it was appreciated that Csk, a potent inhibitor of Src kinases, is regulated by an assortment of protein-protein interactions. Fourth, differences in the regulation of Syk and Zap-70 were observed, suggesting significant distinctions in the purpose of these two kinases in immunoreceptor signaling. And fifth, it was suggested that proximal kinases implicated in immunoreceptor-mediated signal transduction may be regulated by protein degradation via binding to c-Cbl, a ubiquitin ligase.
Assuntos
Proteínas Tirosina Quinases/fisiologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Animais , Humanos , Linfócitos/enzimologia , Linfócitos/imunologia , Microdomínios da Membrana/enzimologia , Receptores Proteína Tirosina Quinases/fisiologiaRESUMO
Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Animais , Sequência de Bases , Compartimento Celular , Linhagem Celular , Membrana Celular/enzimologia , DNA/genética , Análise Mutacional de DNA , Ativação Enzimática , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Linfócitos/enzimologia , Camundongos , Dados de Sequência Molecular , Mutação , Ácido Mirístico , Ácidos Mirísticos , Oligonucleotídeos , Fosforilação , Fosfotirosina , Processamento de Proteína Pós-Traducional , Ratos , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
As a result of alternative splicing, the fyn gene encodes two different tyrosine protein kinase isoforms. While one protein (p59fynB) is abundantly expressed in the brain, the alternative product (p59fynT) is contained only in cells of hemopoietic lineages, especially T lymphocytes. Sequence analyses have revealed that these two isoforms differ exclusively within a stretch of 52 amino acids which overlaps the end of the Src homology 2 (SH2) motif and the beginning of the catalytic domain. Consistent with the idea that FynT provides a specialized function in hemopoietic cells, we have previously shown that expression of activated FynT molecules, but not that of activated FynB polypeptides, enhanced the antigen responsiveness of a mouse T-cell line (BI-141) (D. Davidson, L. M. L. Chow, M. Fournel, and A. Veillette, J. Exp. Med. 175:1483-1492, 1992). In this study, we examined the basis for the distinct signalling capabilities of the two Fyn isoforms in T lymphocytes. Our biochemical analyses revealed that FynT is more adept than FynB at promoting antigen receptor-triggered calcium fluxes. This phenomenon likely contributes to the improved biological function of FynT during antigen stimulation, as the calcium ionophore ionomycin partially rescued the inability of FynB to enhance antigen-induced lymphokine secretion. To establish the structural basis for these observations, we also created and analyzed a series of chimeras of FynT and FynB. These studies demonstrated that the distinct catalytic domain of FynT, and not its altered SH2 motif, is responsible for the improved ability to augment antigen responsiveness. Similarly, this sequence enhances the ability to mobilize cytosolic calcium in response to antigen receptor stimulation. Taken together, these data show that the distinct biological impacts of FynT and FynB in T cells are related to limited structural differences in the amino-terminal portion of their catalytic domains and that they reflect, at least in part, the greater ability of FynT to mobilize cytoplasmic calcium.
Assuntos
Processamento Alternativo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Sanguíneas/enzimologia , Encéfalo/enzimologia , Cálcio/metabolismo , Bovinos , Citosol/metabolismo , Primers do DNA , Éxons , Insulina/farmacologia , Ionomicina/farmacologia , Isoenzimas/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-fyn , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
T-cell antigen receptor (TCR) signalling has been shown to involve two classes of tyrosine protein kinases: the Src-related kinases p56(lck) and p59(fyr), and the Zap-70/Syk family kinases. Lck and FynT are postulated to initiate TCR-triggered signal transduction by phosphorylating the CD3 and zeta subunits of the TCR complex. This modification permits the recruitment of Zap-70 and Syk, which are presumed to amplify the TCR-triggered signal, by phosphorylating additional intracellular proteins. While Zap-70 is expressed in all T cells, Syk is present in thymocytes and mature T-cell populations such as intraepithelial gammadelta T cells and naive alphabeta T cells. To better understand the role of Syk in these cells, its impact on the physiology of an antigen-specific T-cell line was tested. Our results showed that compared to Zap-70 alone, Syk was a strong positive regulator of antigen receptor-induced signals in BI-141 cells. Surprisingly, they indicated that, like Src family kinases, Syk augmented TCR-triggered tyrosine phosphorylation of CD3/zeta. Syk, but not Zap-70 alone, could also stimulate tyrosine phosphorylation of a zeta-bearing chimera in transiently transfected Cos-1 cells. Finally, evidence was provided that Syk has the capacity to directly phosphorylate a zeta-derived peptide in vitro. These findings suggested that Syk may have a unique role in T cells, as a consequence of its ability to efficiently phosphorylate multiple components of the TCR signalling cascade. Furthermore, they raised the possibility that Syk can regulate the initiation of TCR signalling, by promoting phosphorylation of the immunoreceptor tyrosine-based activation motifs of the TCR complex.
Assuntos
Precursores Enzimáticos/imunologia , Proteínas de Membrana/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Animais , Antígenos/imunologia , Complexo CD3/metabolismo , Células COS , Linhagem Celular , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Insulina/imunologia , Interleucina-2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ativação Linfocitária , Proteínas de Membrana/metabolismo , Camundongos , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Interleucina-2/genética , Proteínas Recombinantes de Fusão , Quinase Syk , Linfócitos T/imunologia , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70RESUMO
The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.
Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Linfócitos T/enzimologia , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Primers do DNA/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Camundongos , Dados de Sequência Molecular , Mutação , Fosforilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We have tested the possibility that tkl, a partially characterized avian tyrosine protein kinase gene, is the chicken homolog of lck, a lymphocyte-specific mammalian gene. Using polymerase chain reactions, we have cloned sequences encoding the previously unidentified amino terminus of the tkl gene product. The newly defined unique domain of Tkl displayed significant identity (68%) to the equivalent region of the mammalian lck gene product, p56lck. This identity included a glycine residue at position 2 (present in all Scr-related tyrosine protein kinases) and a cysteine motif at positions 20 and 23, which allows binding of p56lck to CD4 and CD8 in mammalian T lymphocytes. A specific RNase protection assay revealed that, in contrast to a previous report (K. Strebhardt, J. I. Mullins, C. Bruck, and H. Rübsamen-Waigmann, Proc. Natl. Acad. Sci. USA 84:8778-8782, 1987), tkl expression is restricted to the lymphoid tissues thymus and spleen. Moreover, the absence of tkl transcripts in the bursa of Fabricius suggested that this gene is expressed in avian T lymphocytes but not in B lymphocytes. A polyclonal rabbit antiserum raised against the unique domain of Tkl recognized a 56-kDa polypeptide with associated protein kinase activity from avian thymus-derived cells. Additional studies showed that p56tkl is structurally similar to mammalian p56lck and that it is physically associated with the avian CD4 and CD8 T-cell surface antigens. It was also determined that tkl transcripts have one major type of 5' untranslated region (UTR), which differs greatly from the two known 5' UTRs of mammalian lck mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas Tirosina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Antígenos CD4/metabolismo , Galinhas , DNA , Éxons , Expressão Gênica , Íntrons , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Ribonucleases , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The protein tyrosine kinase (PTK) Csk is a potent negative regulator of several signal transduction processes, as a consequence of its exquisite ability to inactivate Src-related PTKs. This function requires not only the kinase domain of Csk, but also its Src homology 3 (SH3) and SH2 regions. We showed previously that the Csk SH3 domain mediates highly specific associations with two members of the PEP family of nonreceptor protein tyrosine phosphatases (PTPs), PEP and PTP-PEST. In comparison, the Csk SH2 domain interacts with several tyrosine phosphorylated molecules, presumed to allow targetting of Csk to sites of Src family kinase activation. Herein, we attempted to understand better the regulation of Csk by identifying ligands for its SH2 domain. Using a modified yeast two-hybrid screen, we uncovered the fact that Csk associates with PTP-HSCF, the third member of the PEP family of PTPs. This association was documented not only in yeast cells but also in a heterologous mammalian cell system and in cytokine-dependent hemopoietic cells. Surprisingly, the Csk-PTP-HSCF interaction was found to be mediated by the Csk SH2 domain and two putative sites of tyrosine phosphorylation in the noncatalytic portion of PTP-HSCF. Transfection experiments indicated that Csk and PTP-HSCF synergized to inhibit signal transduction by Src family kinases and that this cooperativity was dependent on the domains mediating their association. Finally, we obtained evidence that PTP-HSCF inactivated Src-related PTKs by selectively dephosphorylating the positive regulatory tyrosine in their kinase domain. Taken together, these results demonstrate that part of the function of the Csk SH2 domain is to mediate an inducible association with a PTP, thereby engineering a more efficient inhibitory mechanism for Src-related PTKs. Coupled with previously published observations, these data also establish that Csk forms complexes with all three known members of the PEP family.
Assuntos
Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Proteína Tirosina Quinase CSK , Sequência Conservada , Células-Tronco Hematopoéticas/enzimologia , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases não Receptoras , Proteínas Tirosina Quinases/genética , Ratos , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Tirosina/química , Domínios de Homologia de src , Quinases da Família srcRESUMO
Accumulating data suggest that the CD4 T-cell surface antigen transduces an independent intracellular signal during antigen-mediated T-cell activation. CD4 is physically associated with the internal membrane tyrosine protein kinase p56lck and can mediate, after antibody-mediated cross-linking, the rapid enzymatic activation of Lck, implying that CD4 signalling may involve changes in tyrosine protein phosphorylation. In this report, we describe that cross-linking of CD4 results in a series of rapid changes in intracellular tyrosine protein phosphorylation. The most prominent CD4-induced tyrosine phosphorylation change involved p56lck, which became extensively phosphorylated on the carboxy-terminal tyrosine residue 505 and, to a lesser extent, lymphocytes can transduce an intracellular signal resulting in tyrosine protein phosphorylation and strongly suggest that this property of CD4 is mediated through p56lck.
Assuntos
Antígenos Ly/metabolismo , Antígenos CD4/imunologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Animais , Antígenos Ly/imunologia , Brometo de Cianogênio , Camundongos , Peso Molecular , Fosfopeptídeos/análise , Fosforilação , Fosfotirosina , Agregação de Receptores/imunologia , Agregação de Receptores/fisiologia , Tirosina/análiseRESUMO
To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.
Assuntos
Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/fisiologia , Animais , Células Cultivadas , Análise Mutacional de DNA , Técnicas In Vitro , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Camundongos , Mutagênese Sítio-Dirigida , Miristatos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina , Proteínas Tirosina Quinases/química , Transdução de Sinais , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismo , Tirosina/análogos & derivados , Tirosina/fisiologiaRESUMO
Adapters are typically viewed as molecules coordinating the recruitment of positive effectors of cell signaling. Herein, we report the identification of Dok-3, a novel adapter molecule belonging to the Dok family. Our studies show that Dok-3 is highly expressed in several hemopoietic cell types, including B cells and macrophages. It undergoes rapid tyrosine phosphorylation in response to immunoreceptor-mediated cellular activation, seemingly as a result of the action of Src family kinases. This phosphorylation induces the binding of Dok-3 to at least two inhibitory molecules, the 5' inositol phosphatase SHIP and the protein tyrosine kinase Csk. We also demonstrate that augmented expression of wild-type Dok-3 in a B-cell line results in an inhibition of immunoreceptor-mediated nuclear factor of activated T-cells (NFAT) activation and cytokine release, while introduction of a Dok-3 mutant with impaired ability to associate with SHIP and Csk enhances B-cell responsiveness. Taken together, these results indicate that Dok-3 is an adapter involved in the recruitment of inhibitory molecules and that it may play a significant role in the negative regulation of immunoreceptor signaling in hemopoietic cells such as B cells and macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/imunologia , Fosfoproteínas/imunologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Proteínas de Transporte/genética , Clonagem Molecular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fosfoproteínas/genética , Linfócitos T/imunologiaRESUMO
Previous studies from our laboratory have shown that the cytosolic tyrosine protein kinase p50csk is involved in the negative regulation of T-cell activation (L.M. L. Chow, M. Fournel, D. Davidson, and A. Veillette, Nature [London] 365:156-160, 1993). This function most probably reflects the ability of Csk to phosphorylate the inhibitory carboxy-terminal tyrosine of p56lck and p59fynT, two Src-related enzymes abundantly expressed in T lymphocytes. Herein, we have attempted to better understand the mechanisms by which Csk participates in the inhibitory phase of T-cell receptor signalling. Our results demonstrated that the Src homology 3 (SH3) and SH2 domains of p50csk are crucial for its negative impact on T-cell receptor-mediated signals. As these two sequences were not essential for phosphorylation of the carboxy-terminal tyrosine of a Src-like product in yeast cells, we postulated that they mediate protein-protein interactions allowing the recruitment of p50csk in the vicinity of activated Lck and/or FynT in T cells. In complementary studies, it was observed that linkage of a constitutive membrane targeting signal to the amino terminus of Csk rescued the deleterious impact of a point mutation in the SH2 domain of p50csk. This observation suggested that the SH2 sequence is in part necessary to translocate p50csk from the cytoplasm to the plasma membrane, where Src-related enzymes are located. Nevertheless, constitutive membrane localization was unable to correct the effect of complete deletion of the SH3 or SH2 sequence, implying that these domains provide additional functions necessary for the biological activity of p50csk.
Assuntos
Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/enzimologia , Domínios de Homologia de src , Quinases da Família src/fisiologia , Animais , Sequência de Bases , Proteína Tirosina Quinase CSK , Compartimento Celular , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/enzimologia , Primers do DNA/química , Ativação Linfocitária , Linfocinas/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The lymphocyte-specific tyrosine protein kinase p56lck is abundantly expressed in L3T4+ (CD4+) and Lyt-2+ (CD8+) T-lymphocytes, where it is predominantly phosphorylated in vivo on the carboxy-terminal tyrosine residue 505 (Y-505). Upon exposure to activating signals (mitogenic lectins, antibodies to the T-cell receptor), the p56lck expressed in normal cloned murine T-cells is modified into a product which migrates at approximately 59 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels and which possesses several amino-terminal serine phosphorylations. The changes in both mobility and amino-terminal phosphorylation can be reproduced by known activators of protein kinase C (4 alpha-phorbol 12 beta-myristate, dioctanoylglycerol), suggesting that this signal transduction pathway (or related pathways) mediates at least part of these events. Interestingly, agents raising intracellular calcium (such as A23187) cause the appearance of several of these amino-terminal phosphorylation changes but do not cause the pronounced shift in electrophoretic mobility. These data suggest that at least two serine kinase systems are implicated in the alterations of p56lck associated with T-cell activation and that the lck gene product plays a critical role in normal T-cell physiology.