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The Aurora kinases are essential for cell mitosis, and the dysregulation of Aurora A and B have been linked to the etiology of human cancers. Investigational agents MLN8054 (8) and alisertib (MLN8237, 10) have been identified as high affinity, selective, orally bioavailable inhibitors of Aurora A that have advanced into human clinical trials. Alisertib (10) is currently being evaluated in multiple Phase II and III clinical trials in hematological malignancies and solid tumors.

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.

Various translocations and mutations have been identified in myeloma, and certain aberrations, such as t(4;14) and del17, are linked with disease prognosis. To investigate mutational prevalence in myeloma and associations between mutations and patient outcomes, we tested a panel of 41 known oncogenes and tumor suppressor genes in tumor samples from 133 relapsed myeloma patients participating in phase 2 or 3 clinical trials of bortezomib. DNA mutations were identified in 14 genes. BRAF as well as RAS genes were mutated in a large proportion of cases (45.9%) and these mutations were mutually exclusive. New recurrent mutations were also identified, including in the PDGFRA and JAK3 genes. NRAS mutations were associated with a significantly lower response rate to single-agent bortezomib (7% vs 53% in patients with mutant vs wild-type NRAS, P = .00116, Bonferroni-corrected P = .016), as well as shorter time to progression in bortezomib-treated patients (P = .0058, Bonferroni-corrected P = .012). However, NRAS mutation did not impact outcome in patients treated with high-dose dexamethasone. KRAS mutation did not reduce sensitivity to bortezomib or dexamethasone. These findings identify a significant clinical impact of NRAS mutation in myeloma and demonstrate a clear example of functional differences between the KRAS and NRAS oncogenes.

We report the molecular cloning and initial characterization of a novel fatty acid acylated serine/threonine protein kinase. The putative open reading frame is predicted to encode a 305 amino acid protein possessing a carboxy-terminal protein kinase domain and amino-terminal myristylation and palmitylation sites. The protein kinase has been accordingly denoted as the myristylated and palmitylated serine/threonine protein kinase (MPSK). Human and mouse MPSKs share approximately 93% identity at the amino acid level with complete retention of acylation sites. Radiation hybridization localized the human MPSK gene to chromosome 2q34-37. Northern analysis demonstrated that the human MPSK 1.7 kilobase mRNA is widely distributed. Epitope tagged human MPSK was found to be acylated by myristic acid at glycine residue 2 and by palmitic acid at cysteines 6 and/or 8. Palmitylation of MPSK in these experiments was found to require an intact myristylation site. While epitope tagged MPSK in immune complexes or purified human glutathione S transferase-MPSK was found to autophosphorylate at one or more threonine residues, the enzyme was not found to phosphorylate several other common exogenous substrates. Indeed, only PHAS-I was identified as an exogenous substrate which was found to be phosphorylated on threonine and serine residues.

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.

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2+ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC50 2 nM) and MCF7 (IC50 20 nM), while OVCAR3 was only moderately sensitive (IC50 2 microM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced.

Exposure of human B-cell precursors (BCP) to ionizing radiation results in cell cycle arrest at the G2-M checkpoint as a result of inhibitory tyrosine phosphorylation of p34cdc2 . Here, we show that ionizing radiation promotes physical interactions between p34cdc2 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of human BCP leading to tyrosine phosphorylation of p34cdc2. Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as a full-length glutathione S-transferase (GST)-Lyn fusion protein-phosphorylated recombinant human p34cdc2 on tyrosine 15. Furthermore, Lyn kinase physically associated with and tyrosine-phosphorylated p34cdc2 kinase in vivo when co-expressed in COS-7 cells. Binding experiments with truncated GST-Lyn fusion proteins suggested a functional role for the SH3 rather than the SH2 domain of Lyn in Lyn-p34cdc2 interactions in BCP. The first 27 residues of the unique amino-terminal domain of Lyn were also essential for the ability of GST-Lyn fusion proteins to bind to p34cdc2 from BCP lysates. Ionizing radiation failed to cause tyrosine phosphorylation of p34cdc2 or G2 arrest in Lyn kinase-deficient BCP, supporting an important role of Lyn kinase in radiation-induced G2 phase-specific cell cycle arrest. Our findings implicate Lyn as an important cytoplasmic suppressor of p34cdc2 function.

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.

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.

Here, we present evidence that exposure of B-lineage lymphoid cells to low energy electromagnetic fields (EMF) stimulates the protein tyrosine kinases Lyn and Syk, results in tyrosine phosphorylation of multiple electrophoretically distinct substrates, and leads to downstream activation of protein kinase C (PKC). EMF exposure enhances protein tyrosine phosphorylation in Syk deficient but not in Lyn-deficient B-lineage lymphoid cells and stimulates Lyn kinase activity in wild-type as well as Syk-deficient B-lineage lymphoid cells. These results indicate that activation of Lyn kinase is sufficient and mandatory for EMF-induced tyrosine phosphorylation in B-lineage lymphoid cells. The PKC activity increases later than the Lyn activity and pretreatment with the PTK inhibitors genistein or herbimycin A abrogates the EMF-induced PKC signal. Thus, stimulation of Lyn is a proximal and mandatory step in EMF-induced activation of PKC in B-lineage lymphoid cells. Our observations prompt the hypothesis that a delicate growth regulatory balance might be altered in B-lineage lymphoid cells by EMF-induced activation of Lyn.

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.

The nonreceptor 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 nonreceptor 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 nonreceptor PTK loss-of-function mutations in different immunodeficiency diseases has revealed the important contribution of this group of enzymes to lymphocyte development.

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.

Syk is a cytoplasmic protein-tyrosine kinase containing two amino-terminal Src homology 2 domains that is activated following ligation of the B cell antigen receptor. Syk activation in B cells correlates with Syk tyrosine phosphorylation as well as with Syk SH2-mediated association with the tyrosine-phosphorylated Igα and Igβ B cell antigen receptor subunits. Tyrosine-phosphorylated peptide 20-mers representing Igα and Igβ immunoreceptor tyrosine activation motifs were synthesized and found to stimulate the specific activity of Syk by as much as 10-fold in vitro. Maximal phosphopeptide-induced Syk activation required both Syk SH2 domains and phosphorylation of both tyrosine residues present in the immunoreceptor tyrosine activation motif. The biochemical mechanism responsible for the phosphopeptide-induced Syk enzyme activation appears to be a function of Syk autophosphorylation. Our observations suggest the association of Syk tandem SH2 domains with the tyrosine-phosphorylated Igα and/or Igβ immunoreceptor tyrosine activation motifs in B cells stimulates Syk autophosphorylation leading to Syk enzyme activation.

Northern blot analysis of polyadenylated RNA prepared from RBL-2H3 cells revealed the presence of three distinct mRNAs encoding p72Syk, a protein-tyrosine kinase previously shown to be associated with the high affinity IgE receptor present on the surface of these cells (Hutchcroft, J. E., Geahlen, R. L., Deanin, G. G., and Oliver, J. M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9107-9111). Here we report the full-length nucleotide sequence of two of these messages, as well as the complete predicted amino acid sequence of the rodent p72Syk protein-tyrosine kinase. In addition, we report evidence indicating alternative splicing of p72Syk mRNAs within RBL-2H3 cells. This splicing event results in the expression of two distinct protein isoforms that differ with respect to the presence of a 23-amino acid insert located within the region of the protein that separates the two SH2 domains from the catalytic domain. Both mRNAs arising from this splicing event appear to encode functional protein-tyrosine kinases, as expression of the corresponding cDNAs in COS cells results in the production of proteins of the expected sizes that possess intrinsic tyrosine specific kinase activity.

Although the cytoplasmic portion of the low-affinity receptor for immunoglobulin G, Fc gamma RII, does not contain a kinase domain, rapid tyrosine phosphorylation of intracellular substrates occurs in response to aggregation of the receptor. The use of specific tyrosine kinase inhibitors has suggested that these phosphorylations are required for subsequent cellular responses. We previously demonstrated the coprecipitation of a tyrosine kinase activity with Fc gamma RII, suggesting that non-receptor tyrosine kinases might associate with the cytoplasmic domain of Fc gamma RII. Anti-receptor immune complex kinase assays revealed the coprecipitation of several phosphoproteins, most notably p56/53lyn, an Src-family protein tyrosine kinase (PTK), and a 72 kDa phosphoprotein. Here we identify the 72 kDa Fc gamma RII-associated protein as p72syk (Syk), a member of a newly described family of non-receptor PTKs. A rapid and transient tyrosine phosphorylation of Syk was observed following Fc gamma RII activation. Syk was also tyrosyl-phosphorylated following aggregation of the high-affinity Fc gamma receptor, Fc gamma RI. The Fc gamma RI activation did not result in association of Syk with Fc gamma RII, implying that distinct pools of Syk are activated upon aggregation of each receptor in a localized manner. These results demonstrate a physical association between Syk and Fc gamma RII and suggest that the molecules involved in Fc gamma RII signalling are very similar to the ones utilized by multichain immune recognition receptors such as the B-cell antigen receptor and the high-affinity IgE receptor.

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 Jurkat T cells the time-dependent responses of Fyn, Lck, Syk, and Zap following antibody-mediated cross-linking of the T cell antigen receptor. Our results show that the protein kinase activities of Fyn and Lck were activated within seconds of receptor cross-linking. Fyn activity, as measured by autophosphorylation and tyrosine phosphorylation of an exogenous substrate, was maximal 5 s to 1 min following receptor cross-linking. Lck was also found to be activated within 5 s of antigen receptor cross-linking but differed from Fyn in that Lck activity was elevated for at least 30 min. Syk and Zap protein kinase activities were found to peak between 5 and 10 min following receptor cross-linking, returning to approximately basal activity levels by 60 min. The protein kinase activities of both Syk and Zap were found to parallel their reactivity in immunoblotting experiments with anti-phosphotyrosine antibodies. Both Syk and Zap were found to associate with the tyrosine-phosphorylated zeta subunit of the T cell antigen receptor. These observations imply that T cell antigen receptor signal transduction involves the activation of multiple members of at least two different families of non-transmembrane protein tyrosine kinases.

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.

The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59fyn and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of p59fyn and ZAP-70. Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fyn association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59fyn binding was not disrupted. Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activating motif occurs in four discrete steps: binding of p59fyn, phosphorylation of the motif, binding of ZAP-70, and activation of ZAP-70 kinase activity.The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59fyn and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of p59fyn and ZAP-70. Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fyn association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59fyn binding was not disrupted. Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activating motif occurs in four discrete steps: binding of p59fyn, phosphorylation of the motif, binding of ZAP-70, and activation of ZAP-70 kinase activity.

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.

Aggregation of Fc gamma RII (CD 32), a low affinity receptor for immunoglobulin G (IgG), on the monocytic cell line THP-1 induces protein tyrosine kinase (PTK) activity. Several distinct cellular proteins, including Fc gamma RII itself, are phosphorylated on tyrosine following cross-linking of the receptor. Fc gamma RII lacks intrinsic PTK activity. In this report we demonstrate that a kinase activity was coprecipitated with Fc gamma RII in THP-1 cells. The kinetics of the receptor-associated kinase activity paralleled the appearance of tyrosine phosphorylation events observed following Fc gamma RII activation of THP-1 cells. Several proteins were associated with the receptor. Reimmunoprecipitation analysis demonstrated that lyn gene products were among the proteins coprecipitated with Fc gamma RII. p59hck (Hck) and p56lyn (Lyn) were the most abundant Src-related PTKs (Src-PTKs) in THP-1 cells. Enzymatic activity of both kinases, as measured by an in vitro kinase assay, was increased following specific cross-linking of Fc gamma RII. Furthermore, Fc gamma RII was specifically associated with both enzymes following its engagement and served as a substrate for both of these kinases. The association of Fc gamma RII with Src-PTK was specific for Fc gamma RII activation of THP-1 cells, since activation of cells via the high affinity Fc gamma receptor, Fc gamma RI (CD 64), did not result in association of Fc gamma RII with Hck or Lyn. Our data demonstrate a functional and physical association of Fc gamma RII with Hck and Lyn consistent with the involvement of Src-PTK in Fc gamma RII-mediated signal transduction.

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.

SummaryMLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations.

Stimulation of the human monocytic cell line THP-1 by cross-linking either Fc gamma receptor I (Fc gamma RI) or Fc gamma receptor II (Fc gamma RII) gave rise to the rapid phosphorylation of multiple intracellular proteins. The pattern of proteins that were phosphorylated appeared to be identical. Analysis of these proteins by specific immunoprecipitation indicated that stimulation through either receptor did indeed give rise to phosphorylation of the same set of proteins. These included: Fc gamma RII, phospholipase C (PLC) gamma 1, PLC gamma 2, Vav, GAP, and a protein that co-precipitated with the Fc gamma receptors and migrated with a molecular weight of about 70,000. Co-cross-linking an F(ab')2 anti-CD45 monoclonal antibody together with monoclonal antibodies to either of the Fc gamma receptors inhibited phosphorylation of all these proteins. Analysis of the tyrosine kinases in the cells revealed that both receptors stimulated the phosphorylation and activation of a kinase recognized by antibodies to Syk. Furthermore, the Syk kinase became associated with the Fc gamma RII following receptor cross-linking. These data indicate that although the two Fc gamma receptors have different cytoplasmic tails, they are coupled to the same signal transduction cascade that is regulated by CD45 and involves the activation of Syk.

Bacterial lipopolysaccharide (LPS) induces a pleiotropic activation of the immune system which might subsequently result in septic shock. One of the cell surface receptors for LPS is the glycophosphatidylinositol-anchored protein CD14. Binding of LPS to CD14 induces production of lymphokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-8, and CD14 is subsequently released from the cell surface. However, the mechanism of signaling via CD14 is still not known. We report here that protein tyrosine kinase (PTK) p56lyn is coupled to the LPS receptor CD14 in human monocytes. LPS rapidly activates CD14-associated p56lyn simultaneously with PTKs p58hck and p59c-fgr. Inhibition of PTKs by herbimycin A completely blocks LPS-induced down-modulation of CD14 and production of TNF-alpha and IL-1. These data suggest a critical role of PTKs in the LPS/CD14-mediated signal transduction pathway in human monocytes.

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.

CD45 is a tyrosine phosphatase expressed in all hematopoietic cells which is important for signal transduction through the T cell antigen receptor (TCR). Studies using CD45-deficient cells have revealed that Lck, a tyrosine kinase thought to be essential for TCR signaling, is hyperphosphorylated on Y505 in the absence of CD45. This site of tyrosine phosphorylation negatively regulates the function of the Src family of kinases. Here we provide evidence that CD45 can modulate the binding of the Lck to an 11 amino acid tyrosine phosphorylated peptide containing the carboxy-terminus of Lck (lckP). Significantly, CD45 did not influence the binding of Fyn, PLC gamma 1, GAP and Vav to the same phosphopeptide. Lck protein which bound the peptide was dephosphorylated on Y505 and consisted of only 5-10% of the total cellular Lck. Interestingly, there was a marked increase in binding 15-30 min after CD4 or TCR cross-linking. Taken together, our data suggest that CD45 specifically modulates the conformation of Lck in a manner consistent with the intramolecular model of regulation of Src-like kinases.

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.

The gamma subunit of the high affinity IgE receptor, Fc epsilon RI, is a member of a family of proteins which form disulfide-linked dimers. This family also includes the zeta- and eta-chains of the T cell receptor. Engagement of Fc epsilon RI activates src-related protein tyrosine kinases in basophils and mast cells. However, the role of individual subunits of Fc epsilon RI in this activation is still not known. In an effort to determine the function of Fc epsilon RI-gamma, we used chimeric proteins containing the extracellular and transmembrane domains of the alpha chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domains of either T cell receptor-zeta or Fc epsilon RI-gamma. We show that while cross-linking of the Tac chimeras in the rat basophilic leukemia cell line RBL-2H3 resulted in the tyrosine phosphorylation of a subset of proteins and a portion of the degranulation normally observed after Fc epsilon RI-mediated stimulation, no detectable activation of p56lyn or pp60c-src was observed. In contrast, an apparent transient deactivation of these two kinases was observed after Tac chimera cross-linking. These observations suggest that Fc epsilon RI-gamma is responsible for some, but not all, of the signaling that occurs after engagement of its receptor, and that other receptor subunits may also play important roles in this signaling process.

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.

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.

Tyrosine protein kinases (TPKs) represent a diverse group of enzymes that contribute to cellular signal transduction. The generally low abundance of TPKs, coupled with their rapid activation and deactivation, usually precludes their purification through conventional biochemical means. Using immune-complex protein kinase assays, the presence or absence of a given TPK can be established and an estimation of its functional state obtained. In the Basic Protocol of this unit, TPKs are immunoprecipitated, allowed to autophosphorylate in the presence of labeled ATP, run out on an SDS-PAGE gel, and detected by autoradiography. Alternate protocols are provided for the assessment of the functional state of TPKs by providing a potential substrate along with the labeled ATP in the reaction mixture. In the first alternate protocol, the exogenous substrate is a protein, permitting simultaneous assessment of autophosphorylation and exogenous substrate phosphorylation. The second alternate protocol utilizes a peptide substrate, resulting in a rapid, high-throughput assay that evaluates only exogenous substrate phosphorylation.

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.

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.

Here we show that exposure of human B-cell precursors to gamma-rays stimulates the enzymatic activity of the Src protooncogene family protein tyrosine kinase LYN. LYN activation in irradiated cells is not triggered by DNA damage or a nuclear signal since gamma-rays effectively stimulated LYN kinase in enucleated B-cell precursors as well. LYN activation in irradiated cells was abrogated by presence of the OH* radical scavenger dimethylsulfoxide and exposure of intact or enucleated B-cell precursors to chemically generated OH* radicals instead of gamma-rays also triggered LYN kinase activation and enhanced tyrosine phosphorylation of multiple electrophoretically distinct protein substrates. Thus, OH* radicals appear to be both mandatory and sufficient for radiation-induced LYN kinase activation in irradiated B-cell precursors. We further present evidence which indicates that OH* radicals activate LYN by a novel mechanism which involves disruption of inactive LYN-LYN homodimers and monomerization of the LYN kinase after proteolytic degradation of a putative LYN-associated adapter protein through a cytoplasmic TPCK-sensitive chymotrypsin-like protease following its oxidation. LYN kinase plays a pivotal role in initiation of signal cascades that affect the proliferation, differentiation, and survival of B-cell precursors. Our results prompt the hypothesis that a growth regulatory balance might be altered in human B-cell precursors by radiation-induced stimulation of LYN kinase.

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.

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.

The human leukemic Jurkat cell line is commonly used as a model cellular system to study T lymphocyte signal transduction. Various clonal derivatives of Jurkat T cells exist which display different characteristics with regard to responses to external stimuli. Among these, the E6-1 clone of Jurkat T cells has been used as a parental line from which numerous important somatic mutant clones have been generated. During the course of experiments examining signals initiated by the T cell antigen receptor in an E6-1-derived Jurkat cell clone J.CaM1, we observed that the 72-kilodalton Syk protein tyrosine kinase previously found in other Jurkat cells was not detected. Upon further analysis it was determined that Syk transcripts from the J.CaM1 cells as well as the parental E6-1 cells contain a single guanine nucleotide insertion at position 92. This nucleotide insertion results in a shift in the Syk open reading frame leading to alternate codon usage as well as the generation of a termination codon at position 109. Thus, Syk transcripts in E6-1 cells and E6-1-derived clones are predicted to be capable of encoding only the first 33 amino acids of the 630-amino acid wild type Syk. These findings are incompatible with a recently proposed model of T cell antigen receptor signal transduction based, in part, on experiments conducted using E6-1-derived cells, suggesting that Syk might play a role upstream of Lck and Zap70.

One of the primary responses observed following antigen-induced cross-linking in mast cells is an increase in the phosphorylation of certain cellular proteins on tyrosine residues. Stimulation of protein-tyrosine kinase activity appears to be necessary for induction of downstream responses such as degranulation. The role of nonreceptor protein-tyrosine kinases in the signal transduction pathway initiated by Fc epsilon RI engagement in an interleukin-3-dependent mast cell line has been examined. The results presented here show that the enzymatic activity of Lyn is increased within seconds of receptor engagement. Syk activity also undergoes a rapid and transient increase, reaching a peak at approximately 30 s. Similarly, the activity of Fer, representing a third class of nontransmembrane protein-tyrosine kinase increases as well, with its activity peak reached at 1 min poststimulation. The enzymatic activities of Syk and Fer were found to correspond to anti-phosphotyrosine antibody reactivity. Phosphorylation of tyrosine residues of the beta and gamma chains of Fc epsilon RI increased concomitant with increased protein-tyrosine kinase activity. These results indicate that at least three classes of nontransmembrane protein-tyrosine kinases are involved in mast cell FceRI signaling and that the activation of these classes of enzymes is temporally regulated.

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.

Subgroup C strains of Herpesvirus saimiri, a leukemogenic virus of non-human primates, transform human T cells to permanent growth in culture. These cells 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.

We used the polymerase chain reaction with degenerate oligonucleotide primers to search for Csk-related kinases. A cDNA coding for a Csk-like protein-tyrosine kinase was cloned from mouse brain and was designated ctk, for csk-type protein-tyrosine kinase. The 1.9-kb ctk mRNA was found to be expressed predominantly in brain and capable of encoding a 52-kDa protein-tyrosine kinase. The amino acid sequence of Ctk was found to possess 53% identity with mouse Csk, shared all the predicted structural features of Csk, and was capable of phosphorylating the carboxyl-terminal conserved tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of structurally and functionally related Csk-like protein-tyrosine kinases.