Search

MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell–like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIκBα, decrease in nuclear p65 content, reduction of nuclear factor-κB (NF-κB) transcriptional activity, and G1 arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-κB pathway inhibition. Treatment of germinal-center B cell–like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-κB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-κB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-κB–dependent lymphomas.

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

Loss of the tumor suppressor MMAC1 has been shown to be involved in breast, prostate and brain cancer. Consistent with its identification as a tumor suppressor, expression of MMAC1 has been demonstrated to reduce cell proliferation, tumorigenicity, and motility as well as affect cell-cell and cell-matrix interactions of malignant human glioma cells. Subsequently, MMAC1 was shown to have lipid phosphatase activity towards PIP3 and protein phosphatase activity against focal adhesion kinase (FAK). The lipid phosphatase activity of MMAC1 results in decreased activation of the PIP3-dependent, anti-apoptotic kinase, AKT. It is thought that this inhibition of AKT culminates with reduced glioma cell proliferation. In contrast, MMAC1's effects on cell motility, cell - cell and cell - matrix interactions are thought to be due to its protein phosphatase activity towards FAK. However, recent studies suggest that the lipid phosphatase activity of MMAC1 correlates with its ability to be a tumor suppressor. The high rate of mutation of MMAC1 in late stage metastatic tumors suggests that effects of MMAC1 on motility, cell - cell and cell - matrix interactions are due to its tumor suppressor activity. Therefore the lipid phosphatase activity of MMAC1 may affect PIP3 dependent signaling pathways and result in reduced motility and altered cell - cell and cell - matrix interactions. We demonstrate here that expression of MMAC1 in human glioma cells reduced intracellular levels of inositol trisphosphate and inhibited extracellular Ca2+ influx, suggesting that MMAC1 affects the phospholipase C signaling pathway. In addition, we show that MMAC1 expression inhibits integrin-linked kinase activity. Furthermore, we show that these effects require the catalytic activity of MMAC1. Our data thus provide a link of MMAC1 to PIP3 dependent signaling pathways that regulate cell - matrix and cell - cell interactions as well as motility. Lastly, we demonstrate that AKT3, an isoform of AKT highly expressed in the brain, is also a target for MMAC1 repression. These data suggest an important role for AKT3 in glioblastoma multiforme. We therefore propose that repression of multiple PIP3 dependent signaling pathways may be required for MMAC1 to act as a tumor suppressor.

Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.

PTEN/MMAC1 is a recently characterized tumor suppressor. A pseudogene derived from the human PTEN/MMAC1 phosphatase, psiPTEN, has been reported. Recent analysis of the pseudogene revealed conflicting results about the expression of psiPTEN. In this study, we show that the PTEN/MMAC1 pseudogene is actively transcribed in all cells and tissues examined. In some cases, pseudogene transcripts were found to represent as much as 70% of the total PTEN/MMAC1 RNA. As psiPTEN is transcribed, there is a potential for misinterpretation of PTEN/MMAC1 mutations when RT-PCR techniques are used, as well as potential for a psiPTEN-encoded translation product. Although we were unable to detect a pseudogene protein product in the cell lines examined, a baculovirus produced GST pseudogene fusion protein exhibited phosphatase activity comparable to wild type. The results of this study, taken together, indicate the potential complication of PTEN/MMAC1 molecular analysis caused by the expression of psiPTEN.

Bruton's tyrosine kinase (BTK) is a member of the SRC-related TEC family of protein tyrosine kinases (PTKs). DT-40 lymphoma B cells, rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout, did not undergo radiation-induced apoptosis, but cells with disrupted lyn or syk genes did. Introduction of the wild-type, or a SRC homology 2 domain or a plecstrin homology domain mutant (but not a kinase domain mutant), human btk gene into BTK-deficient cells restored the apoptotic response to radiation. Thus, BTK is the PTK responsible for triggering radiation-induced apoptosis of lymphoma B cells, and its kinase domain is indispensable for the apoptotic response.

The fate of developing CD4+CD8+ thymocytes is determined by signals transduced through surface TCR complexes. Here, we report that cross-linking of TCR on CD4+CD8+ thymocytes fails to activate ZAP70 protein tyrosine kinase and fails to initiate downstream signaling events, unless the TCR are coaggregated with surface coreceptor molecules. TCR signaling in CD4+CD8+ thymocytes is impaired because the number of available p56lck molecules is diminished by intrathymic CD4–Ia interactions that initially activate p56lck molecules, which are subsequently degraded. As a consequence of intrathymic CD4–Ia interactions, TCR ζ chains are initially phosphorylated to recruit ZAP70 molecules, but the recruited ZAP70 molecules are not subsequently phosphorylated, resulting in TCR complexes that are stably associated with inactive ZAP70 molecules. Thus, intrathymic interactions that diminish p56lck regulate TCR signaling thresholds and affect TCR structure in developing CD4+CD8+ thymocytes.

In response to invading microorganisms, neutrophils produce large amounts of superoxide and other reactive oxygen intermediates (ROI) by assembly and activation of a multicomponent enzyme complex, the NADPH oxidase. While fulfilling a microbicidal role, ROI have also been postulated to serve as signaling molecules, because activation of the NADPH oxidase was found to be associated with increased tyrosine phosphorylation (Fialkow, L., Chan, C. K., Grinstein, S., and Downey, G.P. (1993) J. Biol. Chem. 268, 17131-17137). The mechanism whereby ROI induces phosphotyrosine accumulation was investigated using electroporated neutrophils stimulated with guanosine 5'-O-3-thiotriphosphate in order to bypass membrane receptors. In vitro immune complex assays and immunoblotting were used to identify five tyrosine kinases present in human neutrophils. Of these, p56/59hck, p72syk, and p77btk were activated during production of ROI. Interestingly, the in vitro autophosphorylation activities of p53/56lyn and p59fgr were found to decline with ROI production. The mode of regulation of p56/59hck was explored in detail. Oxidizing agents were unable to activate p56/59hck in vitro and, once activated in situ, reducing agents failed to inactivate it, suggesting that the effects of ROI are indirect. Tyrosine phosphorylation of p56/59hck paralleled its activation, and dephosphorylation in vitro reversed the stimulation. We therefore conclude that tyrosine phosphorylation is central to the regulation of p56/59hck and likely also of p72syk, which is similarly phosphorylated upon activation of the oxidase. Because ROI have been shown to reduce the activity of tyrosine phosphatases, we suggest that this inhibition allows constitutively active kinases to auto/transphosphorylate on stimulatory tyrosine residues, leading to an increase in their catalytic activity. Enhanced phosphotyrosine accumulation would then result from the combined effects of increased phosphorylation with decreased dephosphorylation.

CD20, a non-glycosylated cell-surface protein expressed exclusively on B lymphocytes, is one of a family of 4-pass transmembrane molecules that also includes the β chain of the high affinity receptor for IgE. The precise function of CD20 is unknown, although in vitro effects of CD20-specific antibodies on resting B cells indicate that it is able to transduce an extracellular signal affecting the G0/G1 cell cycle transition. Previous studies have demonstrated that CD20-initiated intracellular signals involve tyrosine kinase activation and that CD20 is tightly associated with both serine and tyrosine kinases. Here, analysis of CD20-associated molecules has revealed that CD20 is associated with the Src family tyrosine kinases p56/53lyn, p56lck, and p59fyn and with 75/80-kDa proteins phosphorylated in vivo on tyrosine residues. Mutagenesis of CD20 was performed to define regions of CD20 involved in intermolecular interactions. Mutants were analyzed in the human T lymphoblastoid cell line Molt-4, in which ectopically expressed wild-type CD20 associated with p59fyn, p56lck, and 75/80-kDa phosphoproteins. Deletion of major portions of the cytoplasmic regions of CD20 did not abolish its association with either p75/80 or tyrosine kinases. The interaction between CD20 and the Src-related kinases is therefore likely to be independent of CD20 cytoplasmic domains and may occur indirectly. The interaction may be mediated by the p75/80 phosphoproteins, which were found to be tightly associated with the Src family kinases isolated from the CD20 complex.

Activation of cytoplasmic tyrosine kinases is an important aspect of signal transduction mediated by integrins. In the human monocytic cell line THP-1, either integrin-dependent cell adhesion to fibronectin or ligation of beta 1 integrins with antibodies causes a rapid and intense tyrosine phosphorylation of two sets of proteins of about 65-75 and 120-125 kDa. In addition, integrin ligation leads to nuclear translocation of the p50 and p65 subunits of the NF-kappa B transcription factor, to activation of a reporter gene driven by a promoter containing NF-kappa B sites, and to increased levels of mRNAs for immediate-early genes, including the cytokine interleukin (IL)-1 beta. The tyrosine kinase inhibitors genistein and herbimycin A block both integrin-mediated tyrosine phosphorylation and increases in IL-1 beta message levels, indicating a causal relationship between the two events. The components tyrosine phosphorylated subsequent to cell adhesion include paxillin, pp125FAK, and the SH2 domain containing tyrosine kinase Syk. In contrast, integrin ligation with antibodies induces tyrosine phosphorylation of Syk but not of FAK or paxillin. In adhering cells, pre-treatment with cytochalasin D suppresses tyrosine phosphorylation of FAK and paxillin but not of Syk, while IL-1 beta message induction is unaffected. These observations indicate that the Syk tyrosine kinase may be an important component of an integrin signaling pathway in monocytic cells, leading to activation of NF-kappa B and to increased levels of cytokine messages.

Intracellular responses to antigen receptor engagement involve the activation of protein tyrosine kinases and the tyrosine phosphorylation of cellular proteins, including components of the antigen receptor. Phosphorylation of two tyrosine residues within an 18 amino acid segment of the cytoplasmic domain of antigen receptor subunits, and the subsequent association of either the Syk or Zap protein tyrosine kinase, has recently been shown to be required for successful antigen receptor signal propagation. The recent finding that distinct primary human immunodeficiencies result from mutations in genes encoding two non-transmembrane protein tyrosine kinases underscores the importance of this class of enzyme in antigen receptor signal transduction.

We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction.

Several mammalian responses to UV irradiation, including the activation of NF-kappa B, are believed to involve tyrosine phosphorylation. UV irradiation and H2O2 treatment of T lymphocytes induce protein tyrosine phosphorylation and Ca2+ signals similar to those observed following biological stimulation. We have examined the role of cell surface molecules in these responses. Normal T lymphocytes whose surface expression of CD3 was depleted showed impaired UV-induced tyrosine phosphorylation and Ca2+ signals. Similarly, Jurkat T cell lines deficient in CD3 or CD45 expression also gave impaired UV responses. However, all these cell types still gave strong Ca2+ and tyrosine phosphorylation responses to H2O2. The T cell tyrosine kinase ZAP-70 was found to be highly responsive to UV and H2O2 treatment. ZAP-70 responsiveness to UV required expression of both CD3 and CD45, whereas only CD3 was required for the response to H2O2. UV-induced activation of NF-kappa B was blocked by CD3 depletion, indicating the importance of such cell surface molecules in biological responses to UV. In nonlymphoid cells, the epidermal growth factor receptor displayed increased tyrosine phosphorylation within seconds of UV irradiation. These results suggest that UV-induced signal transduction is mediated via cell surface receptors that normally respond to biological stimulation, whereas H2O2 is able to partially bypass this requirement.

Signal transduction via the B cell antigen receptor complex is regulated by changes in tyrosine phosphorylation of several proteins. The equilibrium between tyrosine phosphorylation and dephosphorylation is regulated by the combined action of protein tyrosine kinase and protein tyrosine phosphatase enzymes. In particular, the protein tyrosine phosphatase, CD45, has been shown to play an essential role in signal transduction via the B cell antigen receptor. Therefore, experiments were performed to examine the intermolecular associations between CD45 and phosphotyrosine-containing proteins in the B cell to identify potential substrates for CD45. Based on coprecipitation experiments, CD45 was found to be physically associated with multiple components of the B cell antigen receptor complex including the MB-1/B29 heterodimer. Additionally, CD45 was selectively associated with the src family protein tyrosine kinase, lyn. Neither blk nor fyn were observed to interact with CD45 even though they have been implicated in antigen receptor signal transduction. This finding suggests that CD45 may preferentially regulate the phosphorylation of lyn and thus, its activity. In summary, these studies provide evidence to support the hypothesis that CD45 regulates antigen receptor-mediated signal transduction by controlling the tyrosine phosphorylation of multiple components of the antigen receptor complex.

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.

Interleukin-2 (IL-2) is a growth factor involved in the clonal expansion of antigen-activated T lymphocytes. Interaction of IL-2 with its receptor triggers tyrosine phosphorylation of a series of proteins and results in the activation of p21ras. We report here that Shc, an SH2-containing adaptor protein, is tyrosine-phosphorylated following IL-2 stimulation. IL-2-induced tyrosine phosphorylation of Shc was detectable within seconds following IL-2 addition, reaching its highest level by 15 min. Tyrosine phosphorylation of Shc was induced in multiple IL-2-dependent T cell lines and was found to correlate with IL-2-dependent cell proliferation. Tyrosine-phosphorylated Shc was found to be capable of associating with the SH2 domain of Grb2 following IL-2 stimulation. These results indicate that tyrosine phosphorylation of Shc and its association with Grb2 may be important events in IL-2-initiated signal transduction events in T cells.

We present evidence that the CD19 receptor is functionally operative and transmits pleiotropic signals throughout the pro-B, pre-pre-B, pre-B, early B, and mature B cell stages of human B-cell ontogeny. The signaling ability of CD19 does not depend on the existence of a functional B-cell antigen receptor complex (ARC). In B-cell precursors (BCP) lacking a functional ARC, CD19 is physically and functionally associated with Src family protein tyrosine kinases (PTK). The engagement of the CD19 receptor on BCP with a high affinity anti-CD19 monoclonal antibody (mAb) or its homoconjugate rapidly activates the associated PTK and results in tyrosine phosphorylation of CD19. Moreover, this proximal PTK activation step triggers downstream stimulation of several different intracellular messenger systems. Remarkably, CD19 becomes rapidly phosphorylated on tyrosine residues upon engagement of several other surface receptors as well, suggesting that it may function as a common response element linked via tyrosine phosphorylation to multiple BCP/B-cell receptors and signaling pathways. Furthermore, in all B-lineage lymphoid cell populations, co-approximation of the receptors CD19 and CD72 (ligand for the CD5 T-cell receptor) generates a stronger signal than the engagement of either individual receptor. These convergent observations constitute a strong argument for an important regulatory function of CD19 in human BCP and prompt the hypothesis that the CD19 receptor may play an important role in cognate interactions between B- and T-lineage lymphoid compartments as well as the coordinate production of BCP at multiple stages of human B-cell ontogeny.

We have generated a novel "receptor tyrosine kinase" by fusing the extracellular and transmembrane domain of the mouse platelet-derived growth factor receptor (PDGFR) to the cytoplasmic domain of CD4 and coexpressing the construct with the murine cytoplasmic tyrosine protein kinase p56lck. NMuMG cells, which are mouse mammary gland epithelial cells that lack endogenous platelet-derived growth factor (PDGF) receptor expression, were stably transfected with both PDGFR-CD4 and p56lck. The PDGFR-CD4 chimeric protein was expressed at the cell surface and formed a complex with p56lck. Addition of PDGF to these cells led to increased tyrosine phosphorylation of a 56-kDa protein likely to be p56lck and several unidentified cellular proteins. The enzymatic activity of p56lck was increased after treatment with PDGF, indicating that dimerization (or oligomerization) mediated by ligand binding at the cell surface is capable of inducing the activation not only of receptor tyrosine kinases but nonreceptor tyrosine kinases as well. However, the PDGFR-CD4.p56lck complex was, in contrast to the wild type PDGF receptor, not able to induce a PDGF-dependent mitogenic response or DNA synthesis in NMuMG cells. Analysis of several known substrates of the PDGFR-signaling pathway indicates an early block in the transduction of the signal generated by p56lck.

IgE molecules bind mast cells via a heterotetrameric receptor termed Fc epsilon RI. Cross-linking of bound IgE by specific allergen (Ag) initiates a signal transduction cascade resulting in a degranulation response. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in isoprenoid and sterol biosynthesis, by the cholesterol-lowering drug, lovastatin, blocks Fc epsilon RI-dependent [3H] serotonin ([3H]5HT) release from the mast cell line, RBL-2H3. We studied the mode and locus of action of lovastatin in these cells. Lovastatin inhibited Ag-stimulated degranulation, as well as that evoked by ionomycin or by phorbol 12-myristate 13-acetate and ionomycin, stimuli which bypass early receptor events. Inhibition was concentration-dependent, occurred at levels which reduce lipid synthesis, and was reversible by addition of mevalonic acid, the product of the reductase reaction. The effects of lovastatin were not mimicked by treatment with the sterol demethylase inhibitor, econazole, suggesting that nonsterol isoprenoid synthesis is required for the degranulation response. Conversely, tyrosine kinase inhibitors from three disparate chemical classes reduced stimulus-evoked [3H]5HT release in a manner similar to lovastatin, suggesting that these agents share similar loci of action. Accordingly, lovastatin altered the phosphorylation pattern in unstimulated RBL-2H3, and reduced the phosphorylation response to IgE cross-linking. By analogy to 5HT release, this effect was concentration-dependent and mevalonic acid-reversible. The tyrosine kinase inhibitor, geldanamycin, also reduced the phosphorylation response to Ag. Lyn, a Src-related tyrosine kinase activated upon IgE cross-linking, was little influenced by either lovastatin or geldanamycin. Thus, lipid synthesis inhibition by lovastatin results in impaired tyrosine phosphorylation in RBL-2H3. This impairment is reflected in the subsequent exocytotic response. While lovastatin may inhibit tyrosine phosphorylation via an indirect mechanism, our results with tyrosine kinase inhibitors support the concept that multiple tyrosine kinases participate in the Fc epsilon RI-dependent signal transduction process.

The Src family of tyrosine protein kinases (TPKs) represents a class of closely related intracellular enzymes that participate in the signal transduction pathways in a variety of hemopoietic cells. The Src TPKs associate with multiple cell surface molecules rendering these receptors capable of activating tyrosine phosphorylation of cellular protein targets. Despite phenotypic differences between various hemopoietic cells, the signal transduction pathways that involve Src TPKs demonstrate clear similarities. Accumulating data on the antigen-induced activation in T cells, B cells, and mast cells indicate that the Src TPKs participate in early antigen receptor responses in these cells.

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.

In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

Several styryl-based compounds were evaluated for their capacity to act as inhibitors of the non-receptor tyrosine protein kinase p56lck. Our results demonstrate that α-cyanocinnamamide compounds can inhibit both the in vitro tyrosine autophosphorylation of p561ck as well as p56lck phosphorylation of exogenous substrates. Compound 67B-83-A was found to inhibit p561ck protein kinase activity with a calculated IC50 of 7 to 10 μM. This compound did not significantly inhibit the tyrosine protein kinase activity of the epidermal growth factor receptor and was found to be a less effective tyrosine protein kinase inhibitor for other members of the src family of protein kinases.

A structure activity study was conducted on two important members of the styryl class of tyrosine-specific protein kinase inhibitors to examine relative roles which the aryls rings and vinyl side chains play in their inhibitory activity. The ability of four analogs 1a–d to inhibit autophosphorylation of epidermal growth factor receptor (EGFR) and p56 lck tyrosine kinases was examined, with results showing that both the pattern of aromatic hydroxylation and the type of side chain functionality can greatly influence both selectivity and potency.

B cell express paired immunoglobulin-like receptor B (PIR-B) contains four potential ITIMs (immunoreceptor tyrosine-based inhibitory motifs) in the cytoplasmic domain. Coligation of PIR-B with B cell antigen receptor (BCR) blocks antigen-induced B cell activation. This inhibition is mediated in part by recruitment of SH2-containing tyrosine phosphatases SHP-1 and SHP-2 to the phosphorylated ITIMs in the cytoplasmic domain of PIR-B. PIR-B ligation inhibits the BCR-induced tyrosine phosphorylation of Igα/Igβ, Syk, Btk, and phospholipase C (PLQ-γ2. Overexpression of a catalytically inactive form of SHP-1 prevents the PIR-B-mediated inhibition of tyrosine phosphorylation of Syk, Btk, and PLC-γ2. Dephosphorylation of Syk and Btk mediated by SHP-1 leads to a decrease of their kinase activity, which in turn inhibits tyrosine phosphorylation of PLC-γ2. In addition, Lyn is required for tyrosine phosphorylation of PIR-B.

MMAC1, also known as PTEN or TEP-1, was recently identified as a gene commonly mutated in a variety of human neoplasias. Sequence analysis revealed that MMAC1 harbored sequences similar to those found in several protein phosphatases. Subsequent studies demonstrated that MMAC1 possessed in vitro enzymatic activity similar to that exhibited by dual specificity phosphatases. To characterize the potential cellular functions of MMAC1, we expressed wild-type and several mutant variants of MMAC1 in the human glioma cell line, U373, that lacks endogenous expression. While expression of wild-type MMAC1 in these cells significantly reduced their growth rate and saturation density, expression of enzymatically inactive MMAC1 significantly enhanced growth in soft agar. Our observations indicate that while wild-type MMAC1 exhibits activities compatible with its proposed role as a tumor suppressor, cellular expression of MMAC1 containing mutations in the catalytic domain may yield protein products that enhance transformation characteristics.

Palmitylation of Src family tyrosine kinases has been shown to play a role in directing their membrane localization. Here we demonstrate that palmitylation can also regulate recognition and tyrosine phosphorylation of the B cell Src kinase substrate Ig alpha. Blk and Src, which are not palmitylated, phosphorylate co-expressed Ig alpha in Cos cells, whereas palmitylated Src kinases do not. Addition of a palmitylation site to Blk abrogates its phosphorylation of the substrate, while mutation of Fyn's palmitylation sites results in recognition and phosphorylation of Ig alpha. These results indicate that palmitylation, a reversible protein modification, aids in regulating recognition of physiologic substrates by Src family tyrosine kinases.

▪ Abstract Intracellular signal transduction following the extracellular ligation of a wide variety of different types of surface molecules on leukocytes involves the activation of protein tyrosine kinases. The dependence of successful intracellular signaling on the functions of the nontransmembrane class of protein tyrosine kinases coupled with the cell type–specific expression patterns for several of these enzymes makes them appealing targets for therapeutic intervention. Development of drugs that can interfere with the catalytic functions of the nontransmembrane protein tyrosine kinases or that can disrupt critical interactions with regulatory molecules and/or substrates should find clinical applications in the treatment of allergic diseases, autoimmunity, transplantation rejection, and cancer.

We demonstrate that the CD19 receptor associates with the beta1 family integrin receptors on human B-cell precursors as well as mature B-lymphocytes, and engagement of the beta1 family integrin receptors with monoclonal antibody homoconjugates leads to rapid activation of the CD19-associated protein-tyrosine kinases (PTK) and results in hyperphosphorylation of CD19 on tyrosine residues. Our findings prompt the hypothesis that homoconjugate-induced integrin clustering may effect the approximation and, by intermolecular cross-phosphorylation, activation of the CD19-associated PTK and subsequent tyrosine phosphorylation of the CD19 receptor. The ability of the beta1 family integrin receptors to transmit a biochemical signal triggering the CD19-linked multifunctional PTK pathway provides a possible explanation for the pleiotropic biologic responses generated though adhesive VLA-4- and VLA-5-mediated contacts.

Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34cdc2 kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34cdc2 in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of p34cdc2 in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G2-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34cdc2. We detected no increase of human WEE1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34cdc2 amino-terminal region or (c) recombinant human p34cdc2-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34cdc2 and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49 WEE1 HU and enzymes which augment the tyrosine kinase activity of p49 WEE 1HU.

The non-receptor protein tyrosine kinases (PTKs) have been grouped into 10 different enzyme families based on predicted amino acid sequences. As the number of enzymes belonging to the nonreceptor class of PTK is increasing, one challenge is to determine how these various classes of PTKs interact within the cell to promote signal transduction. Herein, the activation of four classes of non-receptor PTKs is discussed in relation to their interactions with each other as well as with other signaling molecules during the process of lymphocyte surface antigen receptor mediated activation. Recent findings of non-receptor PTK loss of function mutations in different immunodeficiency diseases has revealed the important contribution of this group of enzymes to lymphocyte development.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.

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 crosslinking was diminished, correlating with the deficient tyrosine phosphorylation of the CD3 -chain, recently observed in J32–3.2. These data are consistent with the idea that sir-related kinases phosphorylate the -chain, which in turn recruits ZAP-70 required to sustain the signal. J. Leukoc. Biol. 55: 289–298; 1994.

The nonreceptor protein tyrosine kinases (PTKs) represent cellular enzymes grouped together based upon their lack of defined extracellular sequences. The currently identified nonreceptor PTKs can be divided into nine different enzyme groups based upon predicted structural features. With the exception of the Focal Adhesion Kinase (Fak) and the Activated Cdc42Hs-associated Kinase (Ack) which are the only known members of these two individual PTK groups, the remaining PTKs appear to be members of distinct PTK families. They range in size from about 50 kDa for the C-src Kinase (Csk) family to approximately 150 kDa for the Abl kinase family. All of these PTKs are likely to be involved in one or more signaling pathways that modulate growth, differentiation, and mature cell function.

A baculovirus vector system that expresses cloned DNA sequences as glutathione S-transferase fusion proteins was developed. This system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. This recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione resin. By SDS gel analysis recombinant p56lck was found to migrate as two species with molecular masses of approximately 56,000 Da. p56lck purified in this manner retained a high level of activity, phosphorylated an exogenous substrate on tyrosine residues, and underwent autophosphorylation on tyrosine residues. The Km (approximately 0.33 mmol) and Vmax (approximately 83 pmol min-1 mg-1) values were also determined by using enolase as a substrate.

Immunoglobulin (Ig) antigen receptors are composed of a noncovalently-associated complex of Ig and two other proteins, Ig alpha and Ig beta. The cytoplasmic domain of both of these Ig associated proteins contains a consensus sequence that is shared with the signaling proteins of the T cell and Fc receptor. To test the idea that Ig alpha-Ig beta heterodimers are the signaling components of the Ig receptor, we have studied Ig mutations that interfere with signal transduction. We find that specific mutations in the transmembrane domain of Ig that inactivate Ca2+ and phosphorylation responses also uncouple IgM from Ig alpha-Ig beta. These results define amino acid residues that are essential for the assembly of the Ig receptor. Further, receptor activity can be fully reconstituted in Ca2+ flux and phosphorylation assays by fusing the cytoplasmic domain of Ig alpha with the mutant Igs. In contrast, fusion of the cytoplasmic domain of Ig beta to the inactive Ig reconstitutes only Ca2+ responses. Thus, Ig alpha and Ig beta are both necessary and sufficient to mediate signal transduction by the Ig receptor in B cells. In addition, our results suggest that Ig alpha and Ig beta can activate different signaling pathways.

CD4, CD8 and CD45 regulate the coupling of the T-cell receptor complex (CD3-TCR) to tyrosine kinase activation and phosphorylation of key substrates such as phospholipase C gamma 1. CD4 and CD8 contribute to activation signals through their cytoplasmic association with p56lck. Expression of the zeta-chain is required for functional synergy of the T-cell receptor with CD4 in the activation of phospholipase C gamma 1, which probably reflects an interaction between p56lck and zeta-associated kinase ZAP-70. CD45 expression is required for CD3-TCR signaling. CD45 may positively regulate signaling by dephosphorylating the carboxyl-terminal tyrosine of p56lck and p59fyn, and negatively regulate signaling by dephosphorylation of other TCR-associated substrates directly. One ligand for CD45 receptor has been identified as the B cell CD22 molecule. The positive and negative effects of CD45 are sensitive to the composition of CD45 in receptor complexes, and may be regulated by specific associations of CD45 isoforms with other receptors such as CD3-TCR, CD2 and CD4.

The Src family of tyrosine protein kinases represent an expanding class of closely related intracellular enzymes that participate in the signal transduction pathways of a variety of surface receptors. One of the more surprising aspects of the information relating Src protein kinases to receptor signaling is the apparent diversity of receptor types with which the Src-related enzymes are reported to interact physically or functionally. Traditional biochemical and genetic approaches have yielded much information regarding the interactions between the Src tyrosine protein kinases and other cellular proteins in defined cell types, and emerging technologies, most notably homologous recombination in embryonal stem cells to achieve gene "knockouts," are providing new insights into the participation of the Src-related gene products in signal transduction and development.

The high-affinity IgE receptor (Fc epsilon RI), which is expressed on the surface of mast cells and basophils, has a central role in immediate allergic responses. In the rat basophilic leukaemia cell line RBL-2H3, which is a model system for the analysis of Fc epsilon RI-mediated signal transduction, surface engagement of Fc epsilon RI induces histamine release and the tyrosine phosphorylation of several distinct proteins. Although the alpha, beta, and gamma subunits of Fc epsilon RI lack intrinsic tyrosine protein kinase (TPK) activity, a kinase that copurifies with Fc epsilon RI phosphorylates the beta and gamma subunits of the receptor on tyrosine residues. We report here that in RBL-2H3 cells, p56lyn and pp60c-src are activated after Fc epsilon RI crosslinking, and p56lyn coimmunoprecipitates with Fc epsilon RI. In the mouse mast-cell line PT-18, another cell type used to study FC epsilon RI-mediated signalling, tyrosine phosphorylation of proteins is also an immediate consequence of receptor crosslinking. Notably, the only detectable src protein-related TPK in PT-18 cells is p62c-yes, and it is this TPK that is activated on Fc epsilon RI engagement and coimmunoprecipitates with the receptor. Therefore, it seems that different src protein-related TPKs can associate with the same receptor and become activated after receptor engagement.

Early events in both T-cell receptor (CD3)- and CD4-induced signal transduction pathways include tyrosine phosphorylation of protein substrates, the generation of phosphatidylinositol-phosphate breakdown products, and the mobilization of intracellular Ca2+. Oxidative stress in T cells mediated by sulfhydryl-reactive nonpolar maleimides was shown previously to down-regulate both receptor-mediated Ca2+ mobilization and interleukin 2 production. Here we show that N-ethylmaleimide suppresses both CD3- and CD4-induced Ca2+ responses in human T cells correlating with a reduction in the level of phospholipase C gamma 1 (PLC gamma 1) tyrosine phosphorylation. The inhibition of tyrosine phosphorylation of PLC gamma 1 and additional protein substrates was observed at concentrations of N-ethylmaleimide above 20 microM, whereas lower concentrations of oxidant appeared to increase tyrosine kinase activity following cell stimulation. Sulfhydryl oxidation did not directly affect the catalytic activity of PLC gamma 1, since immunopurified enzyme from N-ethylmaleimide-treated T cells was fully active. Although N-ethylmaleimide treatment of T cells did not cause a direct effect on total pp56lck kinase activity measured in vitro, the interaction between CD4 and pp56lck was oxidation-sensitive in vivo. However, CD3-induced signaling was inhibited at N-ethylmaleimide concentrations lower than that required for CD4/pp56lck dissociation, suggesting that CD3-associated tyrosine kinase activity involves acutely sensitive regulatory thiols. In addition to chemically induced sulfhydryl oxidation, naturally regulated cellular redox states appear to dictate the potential for T-cell responsiveness, since degranulating human peripheral blood neutrophils inhibited CD3-induced Ca2+ mobilization in T lymphocytes. These data indicate that signal transduction in T cells involves the activation of PLC gamma 1 by tyrosine phosphorylation through an oxidation-sensitive intermediate between surface receptors and tyrosine kinases, perhaps including the interaction between CD4 and pp56lck.

Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate protein-tyrosine kinases (PTKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr 54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRC was detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation.

Tyrosine-specific protein kinases (TPK) are important signal transducing enzymes involved in normal cellular growth and differentiation and have been implicated in the etiology of a number of human neoplastic processes. Efforts to develop agents which inhibit the function of these enzymes by interfering with the binding of substrate have been limited by the lack of detailed three-dimensional structural data. Many inhibitors of substrate binding share a common styrene nucleus 1 which has been postulated to function as a conformationally constrained analogue of tyrosine. In an effort to develop high-affinity compounds based on this hypothesis, a number of derivatives were synthesized in which either methylphosphonate (4a-c) or (hydroxymethyl)phosphonate (3a-c) were appended to the aromatic 4-position of styrene-containing moieties. The intent of this approach was to prepare hydrolytically stable analogues which expressed additional enzyme recognition features present during the phosphorylation of tyrosine itself. None of the analogues showed inhibitory activity up to the maximum concentration tested (1000 microM) when assayed against autophosphorylation of A-431-derived epidermal growth factor receptor (EGFR) or p56lck (autophosphorylation and transphosphorylation of rabbit muscle enolase). Additionally, a series of naphthalene-based inhibitors including (1-naphthalenylhydroxymethyl)phosphonic acid (14), its known 2-positional isomer 16, and sulfonate (19, 20) and phosphate derivatives (17, 18) were also tested under similar conditions. Only (2-naphthalenyl-hydroxymethyl)phosphonic acid (16) showed activity (IC50 = 250 microM in EGFR, in agreement with the reported literature value). These results suggest that the interaction of styrene-based inhibitors with the substrate binding domain of TPKS may not occur in a manner analogous to the interaction of tyrosine with this domain.

THE T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck (reviewed in ref. 1). In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphory lation of various cellular proteins at tyrosine residues2–4. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity5. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV)6,7, binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways.We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.

Rabbit antisera raised against human FYN-specific peptides were used to evaluate the expression of the fyn gene product in normal and murine polyomavirus middle tumor antigen (MTAg)-transformed rat cells. The antisera were capable of detecting p60fyn in both normal and MTAg-transformed cells. Two different antisera directed against unique p60fyn sequences were found to detect p60fyn-MTAg complexes in cell lysates from the MTAg-transformed cells. The MTAg molecules immunoprecipitated by FYN antisera were phosphorylated on tyrosine during immune-complex kinase reactions at sites similar to those found on MTAg in complexes with pp60c-src. Whereas the abundance of p60fyn was estimated to be less in the MTAg-transformed cells than in their normal counterparts, the specific activities of p60fyn molecules in the normal and transformed cells were similar.

An Epstein-Barr virus-encoded protein, LMP2, blocks the effects of surface immunoglobulin (slg) cross-linking on calcium mobilization and on lytic reactivation of EBV in latently infected and growth-transformed primary human B lymphocytes. In wild-type EBV-transformed cells, LMP2 is constitutively tyrosine phosphorylated and is associated with Lyn and Syk protein-tyrosine kinases (PTKs). Baseline Lyn PTK activity is substantially reduced, and slg cross-linking fails to activate Lyn, Syk, Pl3-K, PLC gamma 2, Vav, Shc, and MAPK. Syk, Pl3-K, PLC gamma 2, and Vav are constitutively tyrosine phosphorylated, and their tyrosine phosphorylation does not change following slg cross-linking. In contrast, cross-linking slg on cells transformed by LMP2 null mutant EBV recombinants triggers the same protein tyrosine kinase cascade as in noninfected B lymphocytes. These data are consistent with a model in which LMP2 is a constitutive dominant negative modulator of slg receptor signaling through its effects on Lyn, Syk, or regulators of these kinases.

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.