Basal Tyrosine Kinase Activity Research Articles

Abstract 4248: Predictive signature of response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer cells by tyrosine kinase activity profiling

Abstract Background: There is an urgent need to develop companion diagnostics for response prediction to increase the success rate of anticancer drug development. Constitutively activated protein kinases are major targets for cancer treatment. However, blockade of target molecules may not necessarily lead to treatment response. Thus we attempted to investigate the consequences of target kinase inhibition by comprehensive tyrosine kinases activity profiling, which can be applied to establish a response prediction signature. Here we employed clinically validated EGFR-targeted inhibition as in vitro model utilizing PamChip® peptide micro-arrays. Methods: Eight lung cancer cell lines harboring either wild-type EGFR (A549, H1781 and H441) or mutant EGFR (HCC4006, PC9, HCC827, H1650 and H1975) were used. Chemosensitivity to EGFR inhibitors (gefitinib, erlotinib and afatinib) was evaluated by MTT assay and IC50 values were obtained. For assessment of protein tyrosine kinase activity, cell lysates were prepared, aliquoted and stored at -80°C until use. The tyrosine kinase activity of the lysates was measured with or without EGFR TKIs on PamChip® peptide micro-arrays, containing 144 peptides derived from known human phosphorylation sites. Data analysis was carried out on both basal and inhibition profiles. Inhibition profiles were calculated for each EGFR TKI by taking the log-ratio of peptide phosphorylation measured with or without EGFR TKIs using BioNavigator6 software. Peptides that did not show increase of signal in time (indicative of kinase activity) were excluded from the analysis. Results: Basal tyrosine kinase activity profiles showed a clear segregation between EGFR driven (HCC4006, PC9, and HCC827) and EGFR independent (H1650, H441 and A549) cells. Additionally, overall tyrosine kinase activities in EGFR mutant cells were higher than those in EGFR wild-type cells. Based on IC50 values for three EGFR TKIs, the cell lines were divided into responders (PC9, HCC4006 and HCC827 with IC50 values ≦0.1 μmol/L), non-responders (H1650, H441 and A549 with IC50 values >1 μmol/L) and intermediate responders. Basal phosphorylation signals on a number of peptides differed between responders and non-responders. The responders showed phosphorylation on sites responsible for activating RAF, ERK1/2 and MAPK7 and on sites for inactivating the phosphatases PP2AB and PTN11. The phosphorylation was absent in the non-responders, possibly as a consequence of constitutive activation of the RAS/RAF pathway. This peptide set was suggested to serve as a predictive signature of response to EGFR TKIs. Conclusions: Kinase activity profiles reflect the biochemical consequences of EGFR mutant non-small-cell lung cancer. Tyrosine kinase activity profiling may serve as companion diagnostic in drug development and this should be further explored. Citation Format: Naoki Izawa, Masakuni Serizawa, Faris Naji, Rik de Wijn, Riet Hilhorst, Rob Ruijtenbeek, Hirofumi Yasui, Takako Nakajima, Narikazu Boku, Yasuhiro Koh. Predictive signature of response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer cells by tyrosine kinase activity profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4248. doi:10.1158/1538-7445.AM2014-4248

Abstract 4655: In vitro tyrosine kinase activity profiling to identify molecular targets and predictive biomarkers in gastric cancer cell lines.

Abstract Background: Gastric cancer is among the leading causes of cancer-related deaths worldwide, notably in Asia. Though targeted therapies have been under clinical evaluation, the biological complexity of this cancer type may confer resistance to single-targeted therapies. Especially, scirrhous phenotype is known to be affected by multiple growth factors. To overcome this biological complexity, multi-targeted kinase inhibitors (MTKIs) have been clinically tested and were reported to be effective in a subset of patients. We wanted to elucidate the kinase activity signature of gastric cancer and to explore predictive biomarkers of MTKIs using gastric cancer cell lines by tyrosine kinase activity profiling. Material and methods: The growth-inhibitory effect of the MTKIs sunitinib, sorafenib, pazopanib and the Akt inhibitor MK-2206 was evaluated by the MTT assay. 11 scirrhous and 14 non-scirrhous gastric cancer cell lines were grown until semi-confluent, lysed, aliquoted and stored at -80°C until use. The basal tyrosine kinase activity of the lysates was measured on PamChip® peptide micro-arrays, containing 144 peptides derived from known human phosphorylation sites. Quantification of peptide phosphorylation and data analysis were performed using BioNavigator and SIMCA P+ software. Results: HSC-39, HSC-40A, KATO-III and HSC-43 scirrhous cells and SNU-16 non-scirrhous cells were sensitive to all three MTKIs with IC50 values < 1 μmol/L. Orthogonal partial least square-discriminant analysis (OPLS-DA) of basal tyrosine kinase activity profiles showed a clear segregation between sensitive cell lines and insensitive cell lines. Levels of putative target proteins such as PDGFR, VEGFR, Raf and Kit did not correlate with the sensitivity. All sensitive cell lines turned out to harbor an FGFR2 amplification suggesting that FGFR2 amplification may be a predictive biomarker of response to MTKI treatment. Kinases such as EGFR, EPHA, FGFR, IGF1R, INSR and Src contributed to this segregation suggesting that these are relevant to the sensitivity to MTKIs. MET overexpressing cell lines were resistant to treatment with the four inhibitors. Scirrhous cells were segregated from non-scirrhous cells by OPLS-DA among cell lines with signet-ring cell carcinoma cytology with R2, 0.994; Q2, 0.615. EGFR, ERBB2, 3, 4, EPHAs, FAK, IGF1R, INSR, MAP2Ks, RET and SRC seemed to be responsible for the segregation suggesting that they play an important role in scirrhous gastric cancer biology. Conclusions: Kinase activity profiles reflect the biological complexity of gastric cancer. FGFR2 status correlates with sensitivity to sunitinib, pazopanib and sorafenib, whereas MET status does not. These data imply that tyrosine kinase activity profiling with PamChip® peptide arrays can be utilized to identify molecular targets and predictive biomarkers in gastric cancer and this should be further explored. Citation Format: Yasuhiro Koh, Masakuni Serizawa, Rik de Wijn, Riet Hilhorst, Takako Nakajima, Hirofumi Yasui, Kazuyoshi Yanagihara, Martijn Dankers, Rob Ruijtenbeek, Narikazu Boku. In vitro tyrosine kinase activity profiling to identify molecular targets and predictive biomarkers in gastric cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4655. doi:10.1158/1538-7445.AM2013-4655

Early events of B-cell receptor signaling are not essential for the proliferation and viability of AIDS-related lymphoma

We have evaluated whether targeting the Src family kinase cascade, an early component of B-cell receptor (BCR) signaling, is an effective strategy to treat AIDS-related lymphoma (ARL). Src kinases are activated after ligation of the BCR and they phosphorylate downstream signaling proteins in tyrosine residues relaying B-cell signaling cascades that lead to B cell activation and proliferation. These kinases play an important role in lymphoma pathogenesis. We have shown that Src kinases are constitutively active in diffuse large cell lymphoma (DLBCL) cells occurring in immunocompetent individuals and inhibition of these kinases using dasatinib inhibits proliferation of the lymphoma cells. Therefore, we postulated that targeting Src tyrosine kinases pharmacologically would also inhibit the survival and growth of the ARL cells. We tested the effect of dasatinib in 11 ARL cell lines, including six primary effusion lymphomas, three EBV(+) DLBCL (immunoblastic) lymphomas, one EBV(-) DLBCL, and one EBV(+) Burkitt lymphoma. Cell proliferation and viability in the presence of increasing doses of dasatinib were determined by the MTT assay. In contrast to DLBCL in HIV-negative individuals, most of which show sensitivity to dasatinib in the nanomolar range, none of the ARL cell lines tested showed sensitivity at doses as high as 1 uM. One EBV(+) Hodgkin lymphoma cell line was also evaluated, and found to be resistant to dasatinib treatment. Non-AIDS DLBCLs have activation of SRC family kinases (SFK). The activity of SFK including Src, Lyn, Hck and Blk are invariably inhibited in both sensitive and resistant DLBCL cell lines, whereas the activity of two downstream signaling molecules, Syk and PLCγ2, are inhibited by dasatinib only in sensitive cell lines. To determine the molecular events that occur in ARL after dasatinib treatment in comparison to non-AIDS HIV DLBCL, we performed immunoblot analyses and phosphospecific flow cytometry. Both primary effusion lymphoma (KSHV+) cell lines evaluated (BC2 and VG-1) revealed very low basal levels of total phosphotyrosine, which did not change upon treatment with dasatinib. In contrast, the EBV-associated (LCL-JP06 and BCKN-1) and the virus-negative ARL cell line (BCHN-1) had significantly higher basal tyrosine kinase activity. The HL cell line, L591 has low but detectable level of phosphotyrosines. In these non-PEL cell lines, dasatinib was able to inhibit tyrosine phosphorylation at doses between 10 to 50 nM. In cell lines that had total tyrosine phosphorylation, basal phosphorylation of Src and Lyn was detected, and their activities were efficiently inhibited by treatment with dasatinib. When we examined ARL cell lines for phosphorylation of Syk and PLCγ2, in those that have little or no basal phosphotyrosines including the PEL cell lines (BC2 and VG-1) and the HL cell line L591, cells did not respond to dasatinib treatment. In contrast, the DLBCL cell line BCHN-1 that had basal tyrosine phosphorylation, pSyk and p-PLCγ2 responded to BCR stimulation and dasatinib treatment in a similar fashion to the control DLBCL cell lines. However, in spite of this molecular response, this cell line did not respond to the inhibitor at cellular level as no inhibition in proliferation or viability was detected. Our results indicate that in ARLs, inhibition of BCR signaling is not detrimental to cellular survival or proliferation. These results suggest that viral proteins may provide survival and proliferation signals through interaction with cellular proteins that are further downstream from these early events of BCR signaling. Lack of inhibition by dasatinib was unexpected and in marked contrast to observations made in DLBCL occurring in immunocompetent individuals. These findings have therapeutic as well as pathobiological implications. From the therapeutic standpoint, while dasatinib is likely to be beneficial in lymphoma patients without HIV infection, this approach is unlikely to be successful in patients with AIDS.

Chronic-alcohol exposure alters IGF1 signaling in H9c2 cells via changes in PKC delta

Previously, we have demonstrated that chronic-alcohol exposure alters insulin-like growth factor 1 (IGF1) signaling in adult rat heart cells. This report examines the effects of alcohol in vitro on the expression of protein kinase C (PKC) alpha, delta, and epsilon using the embryonic heart cell line, H9c2, and how this may be linked to changes in IGF1 signal transduction. Western blot analyses of H9c2 protein preparations demonstrate that there are significant increases in the total protein levels of PKC delta and epsilon after 4 days exposure to alcohol, and similar increases were found after 2 and 6 days exposure. In addition, there was a significant increase in PKC delta and epsilon in the membranal fractions and a decrease in the cytosolic fractions. No change was found in the expression or activity levels for PKC alpha. Chronic-alcohol exposure (100 mM, 4 days) increased the basal tyrosine kinase activity of the IGF1 receptor (IGF1R), and altered its rate of activation. Chronic-alcohol exposure also reduced the rate of Erk1/Erk2 activation by IGF1. Chronic alcohol blocked the proliferative effects of IGF1 on cell growth and reduced cell viability both in the presence and absence of IGF1, and this alcohol-induced reduction in cell viability was blocked using siRNA to inhibit PKC delta. In addition, a reduction in the amount of myosin light chain 2 was found in the alcohol-exposed cells. In conclusion, chronic alcohol alters PKC delta and epsilon expression and activity, and suppresses the IGF1 signaling pathway in embryonic heart cell culture. Blockage of PKC delta expression using siRNA inhibits the suppressive effects of alcohol on cell viability.

Tyrosine phosphatase inhibitors selectively antagonize beta-adrenergic receptor-dependent regulation of cardiac ion channels.

beta-Adrenergic receptor stimulation regulates the activity of several different cardiac ion channels through an adenylate cyclase/cAMP/protein kinase A-dependent mechanism. Previous work has suggested that basal tyrosine kinase activity attenuates the beta-adrenergic responsiveness of these cardiac ion channels, supporting the idea that tyrosine phosphorylation exerts an inhibitory effect at some point in the common signaling pathway. To determine which element in the beta-adrenergic pathway is regulated by tyrosine kinase activity, we studied the effects of various protein tyrosine phosphatase (PTP) inhibitors on the cAMP-dependent regulation of the L-type Ca(2+) current in guinea pig ventricular myocytes. Three such compounds, sodium orthovanadate, peroxovanadate, and bpV(phen), had no effect on the basal Ca(2+) current, yet each caused a pronounced inhibition of the Ca(2+) current stimulated by the beta-adrenergic receptor agonist isoproterenol. These observations are consistent with the idea that basal tyrosine kinase activity is capable of inhibiting beta-adrenergic responses. However, these PTP inhibitors had no effect on cAMP-dependent stimulation of the Ca(2+) current via activation of adenylate cyclase with forskolin or activation of H(2)-histaminergic receptors with histamine. These results are consistent with the idea that inhibition of PTP activity produces an inhibitory effect involving a tyrosine kinase-dependent mechanism acting selectively at the level of the beta-adrenergic receptor. This signaling mechanism does not seem to be linked to tyrosine kinase activity associated with insulin and insulin-like growth factor receptors, because acute exposure to agonists of these receptors did not inhibit isoproterenol regulation of the Ca(2+) current.

Mechanisms of allergen-specific immunotherapy.

Mechanisms of allergen-specific immunotherapy.

Polyethylene glycol-mediated infection of non-permissive mammalian cells with semliki forest virus: application to signal transduction studies

Semliki Forest Virus (SFV) vectors allow the subcloning of a gene of interest directly in the expression vector, thus avoiding the need to select and purify viral recombinants, making this viral expression system attractive over many others for mammalian protein expression. We now describe a novel and generally applicable method for infection of non-permissive mammalian cells with SFV, that greatly enhances the utility of this expression system. We demonstrate that the hygroscopic polymer poly (ethylene glycol), PEG, promotes the infectivity of cells by SFV under conditions that did not promote cell–cell fusion. We also found that the PEG-induced infection and expression of an exogenous protein (green fluorescent protein, GFP) did not elevate the basal tyrosine kinase activity, induce a stress-activated responses, or result in aberrant cell responses. Expression of GFP tagged-Vav, an activator of stress-activated protein kinase (SAPK/JNK), resulted in the expected induction of JNK activity and in the normal redistribution of Vav in response to engagement of the high affinity receptor for IgE (FcεRI). Thus, our findings that PEG allows the infection of non-permissive cells by SFV makes this system extremely attractive for expression of proteins in mammalian cells, and studies on signal transduction and cellular localization in immune and non-immune cells.

Differential expression of EGFR during early reparative phase of the gastric mucosa between young and aged rats.

Aging is associated with decreased reparative ability of the gastric mucosa. Our recent data suggest a role for epidermal growth factor receptor (EGFR) in the mucosal reparative processes. Thus we examined changes in EGFR tyrosine kinase activity as well as expression and subcellular localization of EGFR and its ligand transforming growth factor-alpha (TGF-alpha) in the gastric mucosa of young (4-mo-old) and aged (24-mo-old) Fischer 344 male rats during the early reparative phase after acute injury induced by 2 M NaCl. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. Expansion of the neck region and initiation of foveolar cell migration could be seen within 45 min of injury in young rats but not until 90 min in aged rats. In young rats mucosal EGFR tyrosine kinase activity increased at 45 min after injury and subsequently fell to basal levels. Mucosal EGFR mRNA increased throughout the reparative phase as did content of the EGFR ligand TGF-alpha. In contrast, although the basal tyrosine kinase activity and levels of EGFR mRNA and TGF-alpha were elevated in the gastric mucosa of aged rats, injury did not cause increases in these parameters. Immunofluorescent localization suggests that internalization and/or degradation of EGFR may be higher in aged than in young rats. We suggest that diminished induction of EGFR tyrosine kinase activity and increased EGFR internalization after injury may in part be responsible for the age-related decrease in the reparative capacity of the gastric mucosa.

Defective TCR stimulation in anergized type 2 T helper cells correlates with abrogated p56(lck) and ZAP-70 tyrosine kinase activities.

Development of IgE-mediated allergic conditions is dependent on the secretion of a Th2 cytokine pattern, including IL-4, IL-5, and IL-13. The induction of anergy would be one mechanism to abrogate cytokine secretion by Th2 cells, which may be pivotal to the allergic response. We demonstrate here that incubation of cloned human CD4+ phospholipase A2 (PLA)-specific Th2 cells with antigenic peptide, in the absence of professional APC, results in a state of nonresponsiveness. The anergic T cells failed to proliferate or secrete IL-4 in response to optimal stimulation with PLA and autologous, professional APC. Secretion of IL-5 and IL-13, however, was only partially inhibited. The anergic state of the Th2 cells was not associated with CD3 or CD28 down-regulation. However, anergy did appear to be closely related to alterations in signaling pathways, mediated through the TCR, of the cells. In contrast to untreated Th2 cells, anergized Th2 cells failed to respond to anti-CD3 mAb with either increased tyrosine kinase activity or increased levels of tyrosine phosphorylation of p56(lck) or ZAP70. A strong and sustained intracellular calcium flux, observed in untreated Th2 cells in response to anti-CD3 mAb, was absent in anergic Th2 cells. Furthermore, the induction of anergy seems to represent an active process, associated with increased levels of basal tyrosine kinase activity, cytokine production, and CD25 up-regulation in anergic Th2 cells. Together, our results indicate that anergy in Th2 cells is associated with defective transmembrane signaling through the TCR.

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.

Effects of metformin on tyrosine kinase activity, glucose transport, and intracellular calcium in rat vascular smooth muscle.

Metformin enhances peripheral insulin action and reduces blood pressure in hypertensive rats. Our group has previously reported that insulin and insulin-like growth factor I (IGF-1) attenuate both agonist-induced vascular smooth muscle cell (VSMC) contraction and associated increases in cytosolic free calcium ([Ca]i). Thus, changes in insulin actions may explain in part metformin's vascular effects. However, metformin's mechanism of action at the vasculature had not been elucidated. Therefore, the purpose of this study was to determine whether metformin evokes alterations in VSMC insulin and IGF-I receptors, glucose transport, and/or [Ca]i. We quantitated hormone binding and tyrosine kinase (TK) activity in partially purified insulin and IGF-I receptors prepared from metformin-treated (100 microM) and control rat aortic VSMC in culture. Glucose transport was assessed by 2-deoxyglucose uptake. Metformin exposure for 24 h 1) increased basal TK activity (metformin, 3.49 +/- 0.39; control, 1.77 +/- 0.39 pmol 32P incorporated/mg protein; P < 0.01) without changes in insulin-or IGF-I stimulated TK activity, 2) increased 2-deoxyglucose transport in a dose-dependent manner, 3) decreased thrombin-induced elevation in [Ca]i (metformin, 10.3%; control, 35.3% over basal; P < 0.05), These insulin/IGF-I-like effects of metformin may help explain some of its vascular actions.

Investigation of intracellular signals mediating the anti-apoptotic action of prolactin in Nb2 lymphoma cells.

Studies were undertaken to identify intracellular mediators of prolactin inhibition of glucocorticoid-induced apoptosis in Nb2 lymphoma cells. A short-term assay was implemented that quantitates fragmented DNA released from the genome by reaction with diphenylamine. Induction and inhibition of internucleosomal DNA cleavage (indicative of apoptosis) was verified by agarose gel electrophoresis of extracted cellular DNA. Synchronized Nb2 cells (G0/G1) exhibited increased DNA fragmentation after 4-hr incubation with dexamethasone (DEX) (25-100 nM) which was inhibited by ovine prolactin (oPRL) (0.1-1 ng/ml), the glucocorticoid receptor antagonist, RU486 (500 nM), and the nuclease inhibitor, aurintricarboxylic acid (100 microM). Signals previously implicated in prolactin induction of mitogenesis in Nb2 cells were investigated for their role in prolactin inhibition of apoptosis including: protein kinase C activation, arachidonic acid metabolism, polyamine production, tyrosine phosphorylation, and extracellular calcium. Protein kinase C agonists, phorbol-12-myristate-13-acetate, and 1,2-dioctanoyl-sn-glycerol, +/- the calcium ionophore, A23187 (200 nM), did not mimic oPRL inhibition of DEX-induced DNA fragmentation. Protein kinase C inhibitors, gossypol and quercetin, did not block prolactin action. Arachidonic acid did not mimic prolactin protection against DEX-induced DNA fragmentation. Inhibitors of arachidonic acid metabolism, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and indomethacin did not block prolactin action. The polyamine, spermine, inhibited DEX-induced DNA fragmentation at 1.5 to 2.5 mM. However, inhibition of polyamine synthesis with alpha-difluoromethyl ornithine or methylglyoxal bis(guanylhydrazone) did not inhibit prolactin action. Prolactin action was not blocked by inhibitors of tyrosine kinase activation, genistein and tyrphostin-47. On the other hand, pervanadate, a potent tyrosine phosphatase inhibitor, consistently inhibited DEX-induced DNA fragmentation. Prolactin action and DEX-induced apoptosis both occurred in calcium-free PBS. In summary, protein kinase C activation and eicosanoid production do not appear to mediate this prolactin action. Although spermine could block DNA fragmentation, blockade of the polyamine cascade did not inhibit prolactin action, suggesting that polyamines do not mediate this prolactin effect. While inhibitors of tyrosine kinase activation did not block prolactin action, tyrosine phosphatase inhibition in the presence of basal tyrosine kinase activity mimicked prolactin action, suggesting tyrosine phosphorylation participation in the anti-apoptotic effect. Extra-cellular calcium was not required for prolactin or DEX action.

Insulin receptor autophosphorylation and signaling is altered by modulation of membrane physical properties.

Many membrane functions are modulated by the bulk biophysical properties of the membrane. Various compounds which alter membrane physical properties were investigated for their ability to modulate insulin receptor autophosphorylation and signaling. Compounds which raise the bilayer to hexagonal phase transition temperature in model membranes, including carbobenzoxydipeptides, apolipoprotein A-I, acyl carnitines, and lysophosphatidylcholine, inhibited insulin stimulation of insulin receptor tyrosine phosphorylation of isolated receptors as well as in cells overexpressing human insulin receptor. For compounds of similar structure, the inhibition of insulin receptor tyrosine phosphorylation correlates well with their bilayer-stabilizing potency. Most of the compounds which inhibit tyrosine phosphorylation of the insulin receptor also inhibited glucose uptake in the same cells. Compounds which lower the bilayer to hexagonal phase transition temperature in model membranes enhanced insulin stimulation of autophosphorylation in isolated receptors, with no effect on insulin receptor activity in NIH 3T3 HIR 3.5 cells. The effects of cationic amphiphiles were not readily predictable from their membrane modulating activity. All of the compounds tested exert their effects independent of changes in insulin binding to the receptor or changes in the basal tyrosine kinase activity of the receptor. This provides evidence that mechanism of modulation of insulin signaling by these additives lies in their ability to alter the bulk physical properties of the membrane. The results suggest that membrane monolayer curvature strain is a factor contributing to the efficiency of insulin signal transduction.

Hyperinsulinemia induces a reversible impairment in insulin receptor function leading to diabetes in the sand rat model of non-insulin-dependent diabetes mellitus.

The insulin receptor was evaluated at different disease stages in the sand rat (Psammomys obesus), a model for nutrition-induced diabetes. Nondiabetic sand rats showed markedly low receptor number in liver compared with albino rats. Their receptor had an intact tyrosine kinase activity but a higher Km for ATP in the phosphorylation reaction of exogenous substrates. The initial effects of overeating (i.e., development of hyperinsulinemia without hyperglycemia) were associated in the sand rat with a dramatic decrease in in vitro and in vivo insulin-induced receptor tyrosine kinase activity in both liver and muscle. In muscle, this coincided with a decrease in receptor number and an increase in basal tyrosine kinase activity. Similar changes were observed upon development of hyperinsulinemia with hyperglycemia. Upon recovery from the diabetic state by diet restriction, the impaired receptor kinase activation was corrected. Complete restoration occurred only in animals that fully recovered from the diabetic state and became normoinsulinemic. These observations indicate that loss and gain of receptor tyrosine kinase activity were dependent on insulin levels. Thus, overeating may lead to the development of hyperinsulinemia through ineffective extraction of excess insulin by the scarce liver receptors. Hyperinsulinemia, in turn, causes a reversible reduction in receptor kinase activity, leading to insulin resistance. This sequence of events may be relevant to diet-related changes in human non-insulin-dependent diabetes mellitus.

Differential heat stress stability of epidermal growth factor receptor and erbB‐2 receptor tyrosine kinase activities

The epidermal growth factor (EGF) and erbB-2 receptors are structurally related membrane-bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH-3T3 or NR6 cells transfected with wild-type EGF-R and incubated at 37 degrees C or heat shocked at 46 degrees C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46 degrees C, however, the basal tyrosine kinase activity of the wild-type erbB-2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF-R extracellular domain and intracellular domain of erbB-2 were heat stressed, 125I-EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF-stimulated chimeric erbB-2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46 degrees C. The loss of chimeric receptor erbB-2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF or erbB-2 receptors. Sensitivity of erbB-2 to heat stress is also not a result of denaturation of this receptor's carboxy-terminal domain. Insertion of the erbB-2 tyrosine kinase domain into the EGF-R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF-R and erbB-2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized in distinct manners.

Genistein Induces Apoptosis in Immature Human Thymocytes by Inhibiting Topoisomerase-II

The toxicity of genistein, an inhibitor of tyrosine kinases and topoisomerase-II, on human thymocytes was investigated. Genistein induced marked chromatin fragmentation indicative of apoptosis in human thymocyte cultures. Genistein-induced thymocyte apoptosis is unlikely due to an inhibition of basal tyrosine kinase activity, since another tyrosine kinase inhibitor, herbimycin A, does not induce thymocyte apoptosis, whereas other topoisomerase-II inhibitors do. The thymocyte subpopulation most sensitive to genistein-induced apoptosis exhibited a CD3−CD4+CD8+ phenotype. This subpopulation of thymocytes is also sensitive to glucocorticoid-induced apoptosis; however, differences between genistein- and glucocorticoid-induced apoptosis were noted. In particular, unlike glucocorticoid-induced apoptosis, genistein-induced apoptosis does not involve changes in [Ca2+]i and cannot be blocked by activation of protein kinase C.

Ethanol modulates epidermal growth factor-stimulated tyrosine kinase and phosphorylation of PLC- γ1

A431 cells have an abundance of Epidermal Growth Factor (EGF) receptors which possess intrinsic tyrosine kinase activity. Treatment of membranes isolated from A431 cells with EGF caused a 2–3 fold increase in phosphorylation of a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) which is a substrate for tyrosine kinase. Treatment of these membranes with 0.1 to 100 mM ethanol altered basal tyrosine kinase activity in a biphasic manner: increase at 10 mM and decrease at 100 mM ethanol. The treatment of the membranes with the same concentrations of ethanol also altered EGF's ability to stimulate tyrosine kinase activity: increase at 0.1 mM ethanol and decrease at 10 mM. Strikingly, EGF-stimulated tyrosine kinase was more sensitive to ethanol than the basal activity. Experiments with other alcohols showed a relationship between chain length and the inhibitory ability of the alcohol. These data demonstrate a biochemical effect of low concentrations of ethanol on tyrosine kinase. Interestingly, ethanol treatment of A431 cells inhibited EGF-stimulated phosphorylation of PLC- γ 1 which is a substrate for EGF receptor tyrosine kinase. It is concluded that ethanol at low concentrations has significant modulatory effect on basal and EGF-stimulated tyrosine kinase, as well as PLC- γ 1 phosphorylation.

Effects of exercise training on the relationship between insulin binding and insulin-stimulated tyrosine kinase activity in rat skeletal muscle

The effect of exercise training on insulin binding and insulin receptor tyrosine kinase activity was studied using detergent solubilized wheat germ agglutinin (WGA)-agarose purified receptor preparations from rat biceps femoris (BF) and tensor fascia lata (TFL) muscles. Insulin receptor activity, as assessed by A14 [ 125I] insulin binding, was significantly elevated in BF of exercise-trained rats when compared with similar preparations from a sedentary control group. This increase in binding activity was due to change in B max not K D. In contrast, neither the B max nor the K D of insulin binding to TFL changed with exercise training. The structure of insulin receptors isolated from BF or TFL was unaltered by exercise training as determined by affinity labeling (α-subunit, molecular weight (mol wt) ∼131 kilodaltons [kDa]) and electrophoretic mobility of the α- and β-subunit. Furthermore, basal tyrosine kinase activity was not affected by exercise training in extracts from either BF or TFL. However, the insulin dependent increase in maximal tyrosine kinase activity (V max) of the BF, but not TFL, was enhanced by exercise training. Specifically, insulin stimulated phosphorylation of both the β-subunit of the insulin receptor (auto phosphorylation) and of a synthetic peptide (exogenous phosphorylation) were increased over control values in BF from exercise-trained rats, whereas both measurements of tyrosine kinase activity of TFL from the two experimental groups were similar. In contrast, both insulin-stimulated autophosphorylation and tyrosine kinase activity were significantly decreased in BF of exercise-trained rats when normalized to insulin binding activity. This disassociation was only seen in BF from exercise-trained rats, and was not true of TFL. These data indicate that excercise training can lead to increases in insulin receptor number and tyrosine kinase activity, as well as modifying the relationship between these two variables. The changes noted are not observed in all exercising muscles, and their development seems to depend upon the fiber composition. These results emphasize the complex relationship that exists in the regulation of insulin action at the level of its receptor.

Alterations in the tyrosine kinase activity of the insulin receptor produced by in vitro hyperinsulinemia.

The tyrosine kinase activity of the insulin receptor was examined in fat cells made insulin resistant by hyperinsulinemia. Adipocytes previously treated for 16 h in vitro with 0.1 microM insulin were markedly insensitive to insulin as shown by the requirement of 2.3-fold higher concentration of insulin to stimulate half-maximal activation of glucose metabolism (glucose oxidation and glucose conversion to lipids). A similar rightward shift in the insulin dose-response curve was also evident for an insulin action not dependent on glucose metabolism, i.e. the inhibition of hormone-stimulated lipolysis. The almost 60% loss of insulin sensitivity occurred in conjunction with a 37% loss in insulin-binding activity, an alteration caused by a reduction in the number of insulin receptors. Studies of tyrosine kinase activity demonstrated a preferential alteration in this insulin receptor property, in addition to the receptor loss. The insulin-stimulated Vmax for the phosphorylation of the artificial substrate, Glu80-Tyr20, was significantly reduced by almost 40%, when the activity was expressed per insulin binding and measured in a receptor preparation purified by wheat germ affinity chromatography and immunoprecipitation. An elevation in basal tyrosine kinase activity was also present, which correlated with a lower apparent Km for ATP (0.025 mM) in comparison to the Km of 0.070 mM for the receptors from control cells. These findings indicate the presence of two types of alterations that involve the insulin receptor and hyperinsulinemia: one constituted by a modest reduction in receptor number and a second by modifications in the tyrosine kinase activity of the remaining receptors. Both alterations are required to explain the magnitude of the insulin resistance that develops after 16 h of insulin treatment.