Search

PDF file - 34K

Purpose: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays.Experimental Design: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3′-fluoro-3′-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation.Results: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response.Conclusions: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors. Clin Cancer Res; 17(24); 7614–24. ©2011 AACR.

Purpose: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM).Experimental Design: Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease.Results: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model.Conclusions: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. Clin Cancer Res; 17(23); 7313–23. ©2011 AACR.

PDF file - 108K

PDF file - 27K

PDF file - 65K

PDF file - 36K

BackgroundThe effectiveness of many targeted therapies is limited by toxicity and the rise of drug resistance. A growing appreciation of the inherent redundancies of cancer signaling has led to a rise in the number of combination therapies under development, but a better understanding of the overall cancer network topology would provide a conceptual framework for choosing effective combination partners. In this work, we explore the scale-free nature of cancer protein-protein interaction networks in 14 indications. Scale-free networks, characterized by a power-law degree distribution, are known to be resilient to random attack on their nodes, yet vulnerable to directed attacks on their hubs (their most highly connected nodes).ResultsConsistent with the properties of scale-free networks, we find that lethal genes are associated with ∼5-fold higher protein connectivity partners than non-lethal genes. This provides a biological rationale for a hub-centered combination attack. Our simulations show that combinations targeting hubs can efficiently disrupt 50% of network integrity by inhibiting less than 1% of the connected proteins, whereas a random attack can require inhibition of more than 30% of the connected proteins.ConclusionsWe find that the scale-free nature of cancer networks makes them vulnerable to focused attack on their highly connected protein hubs. Thus, we propose a new strategy for designing combination therapies by targeting hubs in cancer networks that are not associated with relevant toxicity networks.

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.

Purpose: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. Experimental Design: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3′-fluoro-3′-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. Results: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. Conclusions: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors. Clin Cancer Res; 17(24); 7614–24. ©2011 AACR.

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.

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.

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.

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.

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.

Trophoblast giant cell differentiation is characterized by endoreduplication and expression of members of the prolactin (PRL) gene family and can be simulated in vitro via manipulations of the Rcho-1 trophoblast cell line. The regulation of trophoblast cell proliferation and differentiation involves tyrosine protein kinase signaling pathways. Treatment of Rcho-1 trophoblast cells with tyrosine kinase inhibitors disrupted differentiation-dependent expression of members of the PRL gene family and cytoskeletal organization. Activated p60 c-src , p62 c-yes , and p53/56 lyn were present in the Rcho-1 rat trophoblast cell line and in differentiated trophoblast cells isolated from the developing rat placenta. p60 c-src and p62 c-yes were active in proliferating and differentiating trophoblast cells. During proliferation, p62 c-yes exhibited distinct associations with other phosphoproteins (34, 66, 76, and 150 kDa). p53/56 lyn was activated only in differentiating trophoblast cells. p53/56 lyn showed a differentiation-dependent accumulation in cytoskeletal and membrane fractions, whereas p60 c-src levels were virtually invariant in both fractions. Expression patterns of csk, a negative regulator of Src family kinase activities, were not consistent with its involvement in the differentiation-dependent activation of p53/56 lyn ; however, there was some indication of the participation of a tyrosine phosphatase in the regulation of p53/56 lyn . In conclusion, p60 c- src , p62 c-yes , and p53/56 lyn patterns of activation in trophoblast cells are consistent with their involvement in the control of trophoblast cell proliferation and differentiation.

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.

Takeda's Oncology Discovery Strategy is tightly integrated and focused on first and fast-best- in-class products and product combinations. Core areas of expertise include hormones, protein homeostasis, biotherapeutics and signal transduction. Strategic imperatives for research success are understanding of unmet needs, focus on biological expertise in founda- tional areas of leadership and flexibility to adapt to new information.

In attempt to elucidate the mechanism of the HIV infection induced T cell unresponsiveness, we studied signal-transducing molecules proximal to the T cell receptor (TCR) in T lymphocytes of HIV-infected individuals. Total amounts of protein tyrosine kinases (PTKs) Lck, Fyn, and ZAP-70 and the zeta chain of the TCR were found significantly decreased in T cells of symptomatic/AIDS patients as well as in T cells of individuals in acute and early asymptomatic stages of HIV infection. Unexpectedly, the detection of Lck, Fyn, and ZAP-70 was reversed after the treatment of cell lysates with dithiothreitol. This suggests that PTKs Lck, Fyn, and ZAP-70 were modified by a mechanism altering the status of sulfhydryl groups. Moreover, this mechanism seems to affect selectively T cells of HIV infected patients since B cell PTKs Syk and Lyn were detected structurally and functionally intact. Interestingly, similar alterations of signaling molecules were not detected in T cells of HIV-infected long-term asymptomatic individuals. Modification of T cell PTKs may thus underlie the HIV-induced impairment of lymphocyte function and may potentially predict disease progression.

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 WEHI-231 B lymphoma line is representative of immature B cells, which undergo growth arrest/apoptosis following cross-linking of surface immunoglobulin M (sIgM). In B cells, sIgM engagement has been shown to induce immediate (within seconds) activation of src family protein tyrosine kinases (PTKs) such as p53 lyn 56 lyn , p55blk, p56lck and p59fyn which are associated with B cell antigen receptor (BCR) complex. However, p59fyn expression is very low in both normal immature B cells and apoptosis-prone B cell lines, including WEHI-231. Such a finding prompted us to investigate the effects of ectopic expression of p59fyn in growth regulation of WEHI-231 cells. We have obtained WEHI-231 transfectants expressing the exogenous p59fyn by retroviral mediated gene transfer method. The transfectants demonstrated increased [Ca2+]i level in both the non-stimulated condition and sIgM cross-linking. The expression of ectopic p59fyn also increased the sensitivity of the transfectants to growth arrest signal by sIgM cross-linking. The results suggest that p59fyn can modulate signal transduction and growth regulation when expressed in the immature B cell line.

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.

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.

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.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon tyrosine activation motifs in RBL-2H3 cells.

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.

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.

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.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that critically regulates the viability, proliferation, and differentiation of granulocytic precursors and the function of neutrophils by signaling through its receptor. Cloning of the human G-CSF receptor (G-CSFR) cDNA has demonstrated sequence homology with other members of the hematopoietic/cytokine receptor superfamily. G-CSF stimulates the appearance of phosphotyrosine proteins in several types of human and murine myeloid cells. Since the receptor does not possess intrinsic tyrosine kinase activity, we hypothesized that G-CSFR interacts with and activates cytosolic protein-tyrosine kinases (PTKs). In vitro protein kinase assay of human G-CSFR immunoprecipitates demonstrated at least two tyrosine phosphoproteins, pp55 and pp70. We observed that G-CSF activated p53/p56lyn, a Src-related PTK, and p72syk, a non-Src-related PTK. Lyn and Syk were recovered in anti-G-CSFR immunoprecipitates; Lyn was detected in the absence of ligand. In addition, upon G-CSF stimulation, Lyn coimmunoprecipitated with Syk. Analysis of the G-CSFR amino acid sequence revealed a potential receptor activation motif for Syk. On the basis of immunoprecipitation and sequence analysis data, we propose that the human G-CSFR forms a three-component signaling complex with Lyn and Syk. Their sequential recruitment into the G-CSFR signaling complex demonstrates the coordinated involvement of two PTKs with a member of the hematopoietic/cytokine receptor superfamily.

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

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.

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.

Purpose: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Experimental Design: Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease. Results: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Conclusions: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. Clin Cancer Res; 17(23); 7313–23. ©2011 AACR.

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.