Biomedical subjects

A Y Tsygankov

Publications and source records attributed to A Y Tsygankov.

At least 19 recordsLinked to original sources

PubMed2004

Src: regulation, role in human carcinogenesis and pharmacological inhibitors.

The cellular signaling machinery is a complex network of cross-talking proteins that enables dynamic communication between upstream causal factors and downstream effectors. Non-receptor tyrosine kinases, including Src, are the intermediates of signal transfer, controlling pathways as diverse as cell growth, death, differentiation, migration, and genome maintenance. When expressed as viral genes these proteins are potent carcinogens. Furthermore, analogous genetic alterations are observed, albeit not frequently, in human tumors. In a variety of tumors including those derived from the colon and breast, Src is either over expressed or constitutively active in a large percentage of patients. Increased expression or activity of Src correlates with the stage and metastatic potential of some neoplasia. The detailed knowledge of Src activation facilitates rational design of drugs that potentially interfere with either binding of ATP or substrate peptides. Several existing inhibitors are available as lead compounds for further development of Src inhibitors.

Animals↗

PubMed2001

Beyond the RING: CBL proteins as multivalent adapters.

Following discovery of c-Cbl, a cellular form of the transforming retroviral protein v-Cbl, multiple Cbl-related proteins have been identified in vertebrate and invertebrate organisms. c-Cbl and its homologues are capable of interacting with numerous proteins involved in cell signaling, including various molecular adapters and protein tyrosine kinases. It appears that Cbl proteins play several functional roles, acting both as multivalent adapters and inhibitors of various protein tyrosine kinases. The latter function is linked, to a substantial extent, to the E3 ubiquitin-ligase activity of Cbl proteins. Experimental evidence for these functions, interrelations between them, and their biological significance are addressed in this review, with the main accent placed on the adapter functions of Cbl proteins.

Animals↗

PubMed2001

Transient tyrosine phosphorylation of human ryanodine receptor upon T cell stimulation.

The ryanodine receptor of Jurkat T lymphocytes was phosphorylated on tyrosine residues upon stimulation of the cells via the T cell receptor/CD3 complex. The tyrosine phosphorylation was transient, reaching a maximum at 2 min, and rapidly declined thereafter. In co-immunoprecipitates of the ryanodine receptor, the tyrosine kinases p56(lck) and p59(fyn) were detected. However, only p59(fyn) associated with the ryanodine receptor in a stimulation-dependent fashion. Both tyrosine kinases, recombinantly expressed as glutathione S-transferase (GST) fusion proteins, phosphorylated the immunoprecipitated ryanodine receptor in vitro. In permeabilized Jurkat T cells, GST-p59(fyn), but not GST-p56(lck), GST-Grb2, or GST alone, significantly and concentration-dependently enhanced Ca(2+) release by cyclic ADP-ribose. The tyrosine kinase inhibitor PP2 specifically blocked the effect of GST-p59(fyn). This indicates that intracellular Ca(2+) release via ryanodine receptors may be modulated by tyrosine phosphorylation during T cell activation.

Calcium↗

PubMed2001

c-Cbl facilitates fibronectin matrix production by v-Abl-transformed NIH3T3 cells via activation of small GTPases.

The protooncogenic protein c-Cbl has been shown to act as a multivalent adaptor and a negative regulator of protein tyrosine kinase-mediated signaling. Recent studies have implicated it in the regulation of cell adhesion-related events. We have previously shown that c-Cbl facilitates adhesion and spreading of v-Abl-transformed fibroblasts, and that these effects are dependent on its tyrosine phosphorylation. However, the mechanisms mediating effects of c-Cbl on fibroblast adhesion remain poorly understood. In this study we demonstrate that the tyrosine phosphorylation-dependent effect of c-Cbl on adhesion of v-Abl-transformed fibroblasts is primarily mediated by an increase in fibronectin matrix deposition by these cells. This increase in fibronectin matrix deposition and, hence, in cell adhesion is dependent on cytoskeletal rearrangements induced by RhoA, Rac1 and, possibly, Rap1 activation caused by c-Cbl. The observed activation of these GTPases is mediated by the recruitment of phosphatidylinositol-3' kinase, CrkL and Vav2 to the C-terminal tyrosine residues of c-Cbl. The results of this study also demonstrate that ubiquitination is essential for the observed effects of c-Cbl on fibronectin matrix production and cell adhesion.

3T3 Cells↗

PubMed2001

Herpesvirus saimiri oncoproteins Tip and StpC synergistically stimulate NF-kappaB activity and interleukin-2 gene expression.

Saimiriine herpesvirus 2 (Herpesvirus saimiri) is capable of inducing lethal T-cell lymphoproliferative diseases in primates and of immortalizing human T lymphocytes in vitro. Two viral oncoproteins, Tip and StpC, are essential for T-cell transformation by Herpesvirus saimiri strains of the subgroup C, which exhibits a higher transformation potential than other subgroups of this virus. Despite the importance of these proteins, the molecular basis of their effects on T cells is poorly understood. It remains unclear how Tip and StpC affect gene expression and what is the molecular basis of their cooperation. To address these issues, we expressed Tip and StpC in T lymphoblastoid cells and assessed both their effects on and transcription factors involved in IL-2 gene expression. Our study shows that Tip and StpC cooperate to upregulate IL-2 gene expression, that their effect is mediated primarily by NF-kappaB and NF-AT, which is partially dependent on tyrosine phosphorylation.

Cell Line, Transformed↗

PubMed1999

Altered replication of human immunodeficiency virus type 1 (HIV-1) in T cell lines retrovirally transduced to express Herpesvirus saimiri proteins StpC and/or Tip.

Human peripheral blood T lymphocytes are transformed in vitro to continuous proliferation by Herpesvirus saimiri subgroup C strains. It has been previously shown that H. saimiri-transformed human T cell lines are a permissive system for HIV-1 and 2 replication and are highly susceptible to infection by HIV-1 and 2. Two open reading frames of H. saimiri, StpC and Tip, are required for T cell transformation and are unique to this herpesvirus. The successful transduction of human T cells with retroviral vectors expressing H. saimiri proteins StpC and Tip has allowed us to extend the previously mentioned observations and investigate the role of StpC and Tip in replication of HIV-1 T-tropic strains (IIIB, MN, and RF) in human T cell lines. StpC expression in Molt4 dramatically enhanced HIV-1 replication as measured by Tat protein expression, syncytia formation, and accumulation of reverse transcriptase activity. In contrast, Tip expression in Molt4 cells inhibited HIV-1 replication and cytopathic effects relative to Molt4 cells transduced with the empty vector alone. The StpC-induced phenotype dominated in Molt4 cells transduced to express both StpC and Tip, suggesting that StpC is responsible for facilitating HIV-1 replication in H. saimiri-transformed T cells. Colony-forming ability of Tip-expressing Molt4 cells following HIV-1 infection was greatly enhanced over Molt4 cells expressing either StpC or no H. saimiri proteins at all. HIV-1 proviral DNA could be detected by PCR in surviving Molt4 cells expressing StpC or Tip, indicating that a persistent infection was established. A better understanding of the effects of Tip and StpC proteins on the biology of human hemopoietic stem cells may lead to novel therapeutic interventions for the treatment of AIDS.

Base Sequence↗

PubMed1999

Tyrosine phosphorylation of C-Cbl facilitates adhesion and spreading while suppressing anchorage-independent growth of V-Abl-transformed NIH3T3 fibroblasts.

The protooncogenic protein c-Cbl becomes tyrosine phosphorylated in normal cells in response to a variety of external stimuli, as well as in cells transformed by oncogenic protein tyrosine kinases. Tyrosine phosphorylation of c-Cbl upregulates its binding to multiple crucial signaling molecules. However, the biological consequences of c-Cbl-mediated signaling are insufficiently understood. To analyse the biological functions of c-Cbl, we overexpressed wild-type c-Cbl and its tyrosine phosphorylation-defective mutant form in v-Abl-transformed NIH3T3 fibroblasts. In this system, wild-type c-Cbl facilitated adhesion and spreading of v-Abl-transformed fibroblasts on the extracellular matrix, while reducing anchorage independence of these cells, as measured by their colony-forming efficiency in soft agar. Therefore, overexpression of wild-type c-Cbl exhibits an overall transformation-suppressing effect. By contrast, overexpression of a tyrosine phosphorylation-defective form of c-Cbl increases neither adhesion nor anchorage dependence of v-Abl-transformed fibroblasts. Analysis of the role of individual tyrosine phosphorylation sites of c-Cbl in these phenomena indicates that both phosphatidylinositol-3' kinase and the CrkL adaptor protein may be involved in the observed effects of c-Cbl. To summarize, the results presented in this report indicate that c-Cbl is involved in regulation of cell adhesion and cytoskeletal rearrangements, and that these effects of c-Cbl are dependent on its tyrosine phosphorylation.

3T3 Cells↗

PubMed1999

Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1.

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

Caenorhabditis elegans Proteins↗

PubMed1998

Fyn, Yes, and Syk phosphorylation sites in c-Cbl map to the same tyrosine residues that become phosphorylated in activated T cells.

Protooncogenic protein c-Cbl undergoes tyrosine phosphorylation in response to stimulation through the receptors for antigens, immunoglobulins, cytokines, and growth factors as well as through the integrins. Tyrosine phosphorylation of c-Cbl may play a functional role in signal transduction, since c-Cbl interacts with many crucial signaling molecules including protein-tyrosine kinases, adaptor proteins, and phosphatidylinositol 3'-kinase. Therefore, it is essential for our understanding of the functions of c-Cbl in signal transduction to identify its tyrosine phosphorylation sites, to determine the protein-tyrosine kinases that phosphorylate these sites, and to elucidate the role of these sites in the interactions of c-Cbl with other signaling proteins. In this report, we demonstrate that tyrosines 700, 731, and 774 are the major tyrosine phosphorylation sites of c-Cbl in T cells in response to pervanadate treatment, as well as in response to TcR/CD3 ligation. Coexpression experiments in COS cells demonstrate that among T cell-expressed Src- and Syk-related protein-tyrosine kinases, Fyn, Yes, and Syk appear to play a major role in phosphorylation of c-Cbl, whereas Lck and Zap phosphorylate c-Cbl ineffectively. Fyn, Yes, and Syk phosphorylate the same sites of c-Cbl that become phosphorylated in stimulated T cells. Among these kinases, Fyn and Yes demonstrate strong binding to c-Cbl, which involves both phosphotyrosine-dependent and phosphotyrosine-independent mechanisms.

Amino Acid Sequence↗

PubMed1996

Specific association of tyrosine-phosphorylated c-Cbl with Fyn tyrosine kinase in T cells.

Fyn is a Src family protein-tyrosine kinase functionally associated with the T-cell antigen receptor (TcR)/CD3 receptor complex. We have demonstrated earlier that the TcR/CD3-induced activation of Fyn results in tyrosine phosphorylation of several Fyn-associated proteins, including a protein of 116 kDa. In this report, we identify the Fyn-associated 116-kDa phosphoprotein (p116) as c-Cbl. The identity of p116 has been demonstrated by its specific reactivity with anti-Cbl and similarity of phosphopeptides generated by V8 proteolysis of phospho-Cbl and p116. We demonstrate here that the association of Fyn and c-Cbl is direct and does not require the presence of other proteins. We also demonstrate that Fyn is the Src family kinase that preferentially interacts with c-Cbl in T cells. The fraction of c-Cbl capable of coprecipitating with Fyn is increased by TcR/CD3 ligation. This increase is likely due to the involvement of Fyn SH2 in the interactions between Fyn and tyrosine-phosphorylated c-Cbl.

Clone Cells↗

PubMed1996

Selective activation of T cell kinase p56lck by Herpesvirus saimiri protein tip.

Infection with Herpesvirus saimiri, a T lymphotropic virus of non-human primates, immortalizes human T cells in vitro. The cells show a mature activated phenotype and retain their antigen specificity. We have previously shown that in H. saimiri transformed cells a viral gene product termed tyrosine kinase interacting protein (Tip) associates with the T cell-specific tyrosine kinase p56lck and becomes phosphorylated by the enzyme on tyrosine residues. Here we show that p56lck is activated by recombinant and native Tip in cell-free systems. A dramatic increase of Lck activity was also observed in T cell lines transfected with Tip. p60fyn and p53/56lyn, the other Src-related kinases expressed in H. saimiri transformed T cells, did not phosphorylate Tip, and they were not activated by the protein. The selective activation of p56lck by Tip could contribute to the transformed phenotype of H. saimiri infected cells, and it might explain the T cell selectivity of the transformation event.

Base Sequence↗

PubMed1995

The product of the Herpesvirus saimiri open reading frame 1 (tip) interacts with T cell-specific kinase p56lck in transformed cells.

Subgroup C strains of Herpesvirus saimiri, a leukemogenic virus of non-human primates, transform human T cells to permanent growth in culture. These cell retain their antigen specificity, and they are becoming widely used as a model for activated human T cells. Though a variety of human cell types can be infected by H. saimiri, transformation appears to be specific for CD4+ and CD8+ T cells. Our investigation of early signaling events in H. saimiri-transformed T cells revealed a novel 40-kDa phosphoprotein complexed with the T cell-specific tyrosine protein kinase p56lck. This protein, termed Tip (tyrosine kinase interacting protein), is identified as a viral protein encoded by the open reading frame 1 (ORF1). In the transformed cells Tip is expressed together with the gene product of ORF2, the viral oncoprotein StpC, which acts on epithelial cells. The H. saimiri genome has 75 ORFs, but only ORF1 and ORF2 are transcribed in transformed human cells. Tip is phosphorylated on tyrosine in cell-free systems containing Lck, indicating that the viral protein is a substrate for this T cell-specific kinase. Alteration of T cell signaling pathways by Tip may be the second event complementing the action of StpC in a new mechanism of T cell transformation.

Amino Acid Sequence↗

PubMed1994

Activation-dependent tyrosine phosphorylation of Fyn-associated proteins in T lymphocytes.

While previous studies have implicated the tyrosine protein kinase p60fyn in antigenic activation of T lymphocytes, it is clear that signal transduction initiated through the antigen receptor requires the concerted actions of several proteins. Here, we report our finding that the activation of p60fyn following TcR cross-linking results in the tyrosine phosphorylation of two Fyn-associated proteins of 82 and 116 kDa. In the cells analyzed, p116 appears to represent one of the major substrates of T-cell antigen receptor-mediated tyrosine phosphorylation. In activated T-cells, the interaction of these proteins is specific for p60fyn since neither p56lck nor p62c-yes were found to detectably associate with p82 or p116. Furthermore, the p60fyn-p82/p116 complex could be dissociated and then reconstituted in vitro using purified recombinant Fyn. Using this technique we demonstrated that both p82 and p116 were capable of binding to the Fyn SH2 domain while p82 was to some extent capable of independent binding to the Fyn SH3 domain. An association between p60fyn and phosphoproteins possibly related to the T-cell p82 and p116 was also observed in other hematopoietic cells. Thus, the activation-induced phosphorylation of the p60fyn-associated proteins p116 and p82 and the wide distribution of potentially similar p60fyn-associated proteins in hematopoietic cells suggest that p116 and p82 may play a role as physiological substrates and/or regulators of p60fyn.

Blotting, Western↗

PubMed1994

Engagement of the CD4 receptor inhibits the interleukin-2-dependent proliferation of human T cells transformed by Herpesvirus saimiri.

Infection with Herpesvirus saimiri, a tumor virus of non-human primates, transformed human CD4+ T cell clones to permanent interleukin (IL)-2-dependent growth without need for restimulation with antigen and accessory cells. The IL-2-dependent proliferation of these cells was dramatically inhibited by soluble anti-CD4 whole antibodies, F(ab')2 and Fab fragments, and also by gp 120 of human immunodeficiency virus. The inhibition was not due to cell death and could be overcome by high concentrations of exogenous IL-2. Cell surface expression of CD4, and to a lesser degree the density of the IL-2 receptor alpha chain, were reduced upon anti-CD4 treatment. After long lasting (> 12 h) incubation with anti-CD4, abundance and activity of CD4-bound p56lck were diminished while the free fraction of p56lck remained unchanged. Since IL-2 binding to its receptor activated only the CD4-bound fraction of p56lck, the IL-2-induced p56lck activity was diminished after long-term CD4 ligation. Taken together, our results suggest a cross talk between CD4- and IL-2 receptor-mediated signaling via p56lck.

Antibodies, Monoclonal↗

PubMed1994

Diminished tyrosine protein kinase activity in T cells unresponsive to TCR stimulation.

Tyrosine phosphorylation is thought to be one of the earliest steps in antigenic activation of T cells. Three nonreceptor tyrosine kinases, p56lck, p60fyn, and ZAP-70, are known to be involved in T cell receptor (TCR) signaling, albeit their functional roles appear to be different. Whereas p60fyn and ZAP-70 are functionally associated with the T cell antigen receptor, p56lck is essential for TCR signaling without being directly coupled to the TCR. We have studied a mutant variant of the Jurkat T cell line (J32-3.2), in which basal activities of p56lck and p60fyn are 2- to 2.5-fold reduced relative to those in its parental line (J32) while basal activity of ZAP-70 remains unchanged, and compared responses of J32-3.2 and J32 to TCR stimulation. We have demonstrated that tyrosine phosphorylation following CD3 cross-linking in J32-3.2 cells was extremely short-lived and thus insufficient for the induction of subsequent physiological responses. This was at least partially due to the diminished tyrosine kinase activity in these cells. A decrease in the activity of src-related kinases was caused primarily by their lower expression, whereas expression of ZAP-70 was unchanged but its response to CD3 cross-linking was diminished, correlating with the deficient tyrosine phosphorylation of the CD3 zeta-chain, recently observed in J32-3.2. These data are consistent with the idea that src-related kinases phosphorylate the zeta-chain, which in turn recruits ZAP-70 required to sustain the signal.

CD3 Complex↗

PubMed1993

Immortalization of human T cell clones by Herpesvirus saimiri. Signal transduction analysis reveals functional CD3, CD4, and IL-2 receptors.

Investigation of human activated T cells has been complicated by the need for periodic restimulation with Ag/mitogen and accessory cells and by the limited life span of most human T cell clones. To overcome these problems, we have transformed established human T cell clones to permanent growth with Herpesvirus saimiri, a lymphoma-inducing virus of nonhuman primates. Three human CD4+ T cell clones were investigated in detail. They have been growing in the presence of exogenous IL-2 but without restimulation with mitogen or feeder cells for more than 11 mo with doubling times between 2 and 4 days. In contrast, their nontransformed parent clones needed to be restimulated with PHA and feeder cells every 14 to 21 days. To compare responses of H. saimiri-transformed clones with those of their parent clones, we stimulated the cells with IL-2 or with anti-CD3 and/or anti-CD4 mAb with and without cross-linking on the cell surface. Transformed and nontransformed T cell clones were strikingly similar in parameters of early signal transduction, namely, tyrosine phosphorylation and mobilization of calcium. Ligation of their TcR/CD3 complexes by mAb or by Ag in the presence of autologous accessory cells increased the proliferation and the secretion of IFN-gamma. Taken together, we have shown that human T cell clones immortalized with H. saimiri express functional CD3, CD4, and IL-2R. They constitute a simple, stable, reproducible and accessory cell-free model system for the investigation of signal transduction events in activated human T cells.

Antigens, Bacterial↗

PubMed1993

Preincubation with anti-CD4 influences activation of human T cells by subsequent co-cross-linking of CD4 with CD3.

Under physiological conditions, T cell activation by major histocompatibility complex (MHC)-antigen complexes requires engagement of both the T cell receptor (TcR) and the CD4 (or CD8) accessory molecules. It has been shown, however, that ligation of CD4 and CD8 can also inhibit T cell activation in an MHC-independent way. Therefore, the role of CD4 in T cell activation and the mechanism of the suppression of T cell functions by anti-CD4 are as yet unclear. We activated T cells by CD4/CD3 co-cross-linking and studied the effect of preincubation with anti-CD4 on this activation. We show here that anti-CD4 effects T cell activation in a complex, time-dependent manner. Whereas short preincubations with anti-CD4 usually enhanced T cell proliferation in response to subsequent co-cross-linking of CD3 with CD4, longer preincubations led to its decrease. The observed suppression of proliferation after a long preincubation with anti-CD4 was apparently due to impairment of TcR signaling, as assessed by measurement of Ca2+ mobilization and tyrosine phosphorylation in T cells. These results add a temporal element to the previously observed synergism between the TcR and CD4 in T cell activation.

Antibodies, Monoclonal↗

PubMed1992

Activation of tyrosine kinase p60fyn following T cell antigen receptor cross-linking.

Activation of T cells by specific antigens in the context of major histocompatibility complex encoded proteins is mediated by the T cell antigen receptor (TcR), consisting of a variable (Ti) and an invariant (CD3) subunits. Tyrosine phosphorylation is considered to be one of the earliest steps in TcR-mediated signal transduction. There are indications that the p60fyn protein tyrosine kinase is involved in signaling via TcR. However, enzymatic activation of the Src-related tyrosine kinases upon TcR triggering has not been shown yet, therefore the identity of TcR-activated tyrosine kinase(s) remains unclear. We demonstrate that cross-linking of CD3 activates p60fyn and induces tyrosine phosphorylation of cellular proteins in human T cells (resting peripheral T cells, a helper T cell clone, a helper T cell clone immortalized with Herpesvirus saimiri, and a leukemic T cell line). Activation of p60fyn was fast, and its maximum (2-4-fold activation as compared with the basal activity) was followed by a decline. The amount of p60fyn in the cells remained unchanged. None of the other T cell Src-related tyrosine kinases was activated after cross-linking of CD3. Activation of p60fyn was induced by anti-CD3, but not by anti-CD4, anti-CD2, or anti-CD28. The activation was correlated with an increase of the phosphotyrosine content of p60fyn. These studies provide direct proof for the functional association between p60fyn and the TcR.

Cells, Cultured↗