Publications

Galectin-8 (Gal-8) belongs to a family of animal lectins that modulate cell adhesion, cell proliferation, apoptosis, and immune responses. Recent studies have shown that mammalian Gal-8 induces in an autocrine and paracrine manner, the expression and secretion of cytokines and chemokines such as RANKL, IL-6, IL-1β, SDF-1, and MCP-1. This involves Gal-8 binding to receptor complexes that include MRC2/uPAR/LRP1, integrins, and CD44. Receptors ligation triggers FAK, ERK, Akt, and the JNK signaling pathways, leading to induction of NF-κB that promotes cytokine expression. Indeed, immune-competent Gal-8 knockout (KO) mice express systemic lower levels of cytokines and chemokines while the opposite is true for Gal-8 transgenic animals. Cytokine and chemokine secretion, induced by Gal-8, promotes the migration of cancer cells toward cells expressing this lectin. Accordingly, Gal-8 KO mice experience reduced tumor size and smaller and fewer metastatic lesions when injected with cancer cells. These observations suggest the existence of a 'vicious cycle' whereby Gal-8 expression and secretion promotes the secretion of cytokines and chemokines that further promote Gal-8 expression. This 'vicious cycle' could enhance the development of a 'cytokine storm' which is a key contributor to the poor prognosis of COVID-19 patients.

Galectin-8 is a β-galactoside-recognizing protein having an important role in the regulation of bone remodeling and cancer progression and metastasis. Methyl β-d-galactopyranoside malonyl aromatic esters have been designed to target and engage with particular amino acid residues of the galectin-8N extended carbohydrate-binding site. The chemically synthesized compounds had in vitro binding affinity toward galectin-8N in the range of 5-33 μM, as evaluated by isothermal titration calorimetry. This affinity directly correlated with the compounds' ability to inhibit galectin-8-induced expression of chemokines and proinflammatory cytokines in the SUM159 breast cancer cell line. X-ray crystallographic structure determination revealed that these monosaccharide-based compounds bind galectin-8N by engaging its unique arginine (Arg59) and simultaneously cross-linking to another arginine (Arg45) located across the carbohydrate-binding site. This structure-based drug design approach has led to the discovery of novel monosaccharide galactose-based antagonists, with the strongest-binding compound (Kd 5.72 μM) holding 7-fold tighter than the disaccharide lactose.

Galectin-8 is a -galactoside-recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3-O-[1-carboxyethyl]--d-galactopyranoside (compound 6) as a ligand to target the N-terminal domain of galectin-8 (galectin-8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3-O-sialylated lactose (3-SiaLac), with galectin-8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin-8N for 6 was 32.8m, whereas no significant affinity was detected for the C-terminal domain of galectin-8 (galectin-8C). The crystal structure of the galectin-8N-6 complex validated the predicted binding conformation and revealed the exact protein-ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin-8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide-based scaffold as a binder of galectin-8.

Galectin-8 (Gal-8) is a member of the galectin family of animal lectins that regulate a myriad of biological processes including cell growth, cell transformation, embryogenesis, apoptosis, cell adhesion and immune responses. Gal-8 expression increases in several, though not all, cancerous tissues including lung, bladder, kidney, prostate, and breast tissues. Based on its prevalence, an estimated ~500,000 newly diagnosed cancer patients/year are expected to possess an amplified Gal-8 gene. Yet, the molecular mechanisms underlying its role in cancer growth and metastasis remain incompletely understood. Here we describe potential modes of action of Gal-8 that might account for its central role in cancer biology. The evidence, gathered thus far, implicates Gal-8 as a driver of a vicious cycle, whereby cancer cells that overexpress and secrete Gal-8, benefit from its potential to promote their own growth; potentiate epithelial mesenchymal transition, and induce secretion of metastasis-promoting agents at the metastatic niche that induce further recruitment and seeding of cancer cells. Further in-depth studies related to its mode of action, are expected to support ongoing efforts aimed at implementing Gal-8-targeted therapies for the treatment of cancer patients.

Earlier reported small interfering RNA (siRNA) high-throughput screens, identified seven-transmembrane superfamily member 3 (TM7SF3) as a novel inhibitor of pancreatic β-cell death. Here we show that TM7SF3 maintains protein homeostasis and promotes cell survival through attenuation of ER stress. Overexpression of TM7SF3 inhibits caspase 3/7 activation. In contrast, siRNA-mediated silencing of TM7SF3 accelerates ER stress and activation of the unfolded protein response (UPR). This involves inhibitory phosphorylation of eukaryotic translation initiation factor 2α activity and increased expression of activating transcription factor-3 (ATF3), ATF4 and C/EBP homologous protein, followed by induction of apoptosis. This process is observed both in human pancreatic islets and in a number of cell lines. Some of the effects of TM7SF3 silencing are evident both under basal conditions, in otherwise untreated cells, as well as under different stress conditions induced by thapsigargin, tunicamycin or a mixture of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1 beta and interferon gamma). Notably, TM7SF3 is a downstream target of p53: activation of p53 by Nutlin increases TM7SF3 expression in a time-dependent manner, although silencing of p53 abrogates this effect. Furthermore, p53 is found in physical association with the TM7SF3 promoter. Interestingly, silencing of TM7SF3 promotes p53 activity, suggesting the existence of a negative-feedback loop, whereby p53 promotes expression of TM7SF3 that acts to restrict p53 activity. Our findings implicate TM7SF3 as a novel p53-regulated pro-survival homeostatic factor that attenuates the development of cellular stress and the subsequent induction of the UPR.

High-throughput siRNA screening was employed to identify novel genes that regulate cytokine-induced death of pancreatic β-cells. One of the 'hits' was Nedd4 family interacting protein 1 (Ndfip1), an adaptor and activator of Nedd4-family ubiquitin ligases. Silencing of Ndfip1 inhibited cytokine-induced apoptosis of mouse and human pancreatic islets and promoted glucose-stimulated insulin secretion. These effects were associated with an increase in the cellular content of JunB, a potent inhibitor of ER stress and apoptosis. Silencing of Ndfip1 also increased the expression of ATF4, IRE-1α, and the spliced form of XBP that govern the unfolded protein response (UPR) and relieve cytokine-induced ER stress, while overexpression of Ndfip1 exerted opposite effects. These findings implicate Ndfip1 in the degradation of JunB; inhibition of the UPR and insulin secretion; and promotion of cytokine-induced death of pancreatic β-cells.

The periparturient period in dairy cows is associated with alterations in insulin action in peripheral tissues; however, the molecular mechanism underlying this process is not completely understood. The objective was to examine the response to a glucose tolerance test (GTT) and to analyze insulin signaling in liver and adipose tissues in pre- and postpartum dairy cows. Liver and adipose tissue biopsies were taken before and after GTT, at 17. d prepartum and again at 3 to 5. d postpartum from 8 high-yielding Israeli Holstein dairy cows. Glucose clearance rate after GTT was similar pre- and postpartum. Basal insulin concentrations and the insulin response to GTT were approximately 4-fold higher prepartum than postpartum. In accordance, phosphorylation of the hepatic insulin receptor after GTT was higher prepartum than postpartum. Across periods, a positive correlation was observed between the basal and peak plasma insulin and phosphorylated insulin receptor after GTT in the liver. Hepatic phosphorylation of protein kinase B after GTT was elevated pre- and postpartum. Conversely, in adipose tissue, phosphorylation of protein kinase B after GTT pre- and postpartum was increased only in 4 out of 8 cows that lost less body weight postpartum. Our results demonstrate that hepatic insulin signaling is regulated by plasma insulin concentrations as part of the homeorhetic adjustments toward calving, and do not support a model of hepatic insulin resistance in periparturient cows. Nevertheless, we suggest that specific insulin resistance in adipose tissue occurs pre- and postpartum only in cows prone to high weight loss. The different responses among these cows imply that genetic background may affect insulin responsiveness in adipose tissue pre- and postpartum.

Background: Selective serotonin reuptake inhibitors (SSRIs) are used for the treatment of mood and anxiety disorders. Results: SSRIs inhibit insulin action and secretion, promote the unfolded protein response, and induce apoptosis of pancreatic β cells. Conclusion: SSRIs inhibit insulin signaling and beta cell function. Significance: SSRIs might accelerate the transition from an insulin-resistant state to overt diabetes.

Chronic infection with hepatitis C virus (HCV), mainly genotype 1, has been shown to be associated with insulin resistance and type 2 diabetes. The mechanisms underlying this association are partly understood. Increased levels of tumor necrosis factor (TNF)-α occurring in HCV infection have an important role in HCV-mediated insulin resistance; however, other direct effects of HCV core protein on disrupting insulin signalling have been suggested. The insulin receptor substrate (IRS) proteins are key players in insulin signal transduction and are the major substrates of the insulin receptor. To further elucidate the direct effect of HCV core protein on insulin signalling. We studied the direct effects of HCV core protein in two cell lines transfected with HCV core protein. We found several impairments in the insulin signalling cascade which could be attributed to a significant proteasomal degradation of IRS-1 protein, in a dose-dependent way. In addition, our data show that liver cells transfected by HCV core protein show a marked attenuation of the regulatory inhibitory role of insulin on insulin growth factor binding protein-1 (IGFBP-1) expression. Since IGFBP-1 may have a role in glucose regulation and hepatic insulin sensitivity, this effect of HCV core protein can contribute to insulin resistance in chronic HCV infection. Our data suggest that the degradation of IRS-1 by HCV core protein translates to impaired ability of insulin to inhibit the expression of the target gene IGFBP-1 in the liver and may serve as a novel mechanism for insulin resistance and hyperglycaemia.

Insulin receptor substrate-1 (IRS-1) plays a pivotal role in insulin signaling, therefore its degradation is exquisitely regulated. Here, we show that insulin-stimulated degradation of IRS-1 requires the presence of a highly conserved Ser/Thr-rich domain that we named domain involved in degradation of IRS-1 (DIDI). DIDI (amino acids 386-430 of IRS-1) was identified by comparing the intracellular degradation rate of several truncated forms of IRS-1 transfected into CHO cells. The isolated DIDI domain underwent insulin-stimulated Ser/Thr phosphorylation, suggesting that it serves as a target for IRS-1 kinases. The effects of deletion of DIDI were studied in Fao rat hepatoma and in CHO cells expressing Myc-IRS-1^WT or Myc-IRS-1^Δ386-430. Deletion of DIDI maintained the ability of IRS-1^Δ386-434 to undergo ubiquitination while rendering it insensitive to insulin-induced proteasomal degradation, which affected IRS-1^WT (80% at 8 h). Consequently, IRS-1^Δ386-434 mediated insulin signaling (activation of Akt and glycogen synthesis) better than IRS-1^WT. IRS-1^Δ386-434 exhibited a significant greater preference for nuclear localization, compared with IRS-1^WT. Higher nuclear localization was also observed when cells expressing IRS-1 ^WT were incubated with the proteasome inhibitor MG-132. The sequence of DIDI is conserved more than 93% across species, from fish to mammals, as opposed to approximately 40% homology of the entire IRS-1. These findings implicate DIDI as a novel, highly conserved domain of IRS-1, which mediates its cellular localization, rate of degradation, and biological activity, with a direct impact on insulin signal transduction.

OBJECTIVE - Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-1 signaling. Here, we examined the role of Ser phosphorylation of IRS-2 in mediating the inhibitory effects of proinflammatory cytokines and cellular stress on β-cell function. RESEARCH DESIGN AND METHODS - Five potential inhibitory Ser sites located proximally to the P-Tyr binding domain of IRS-2 were mutated to Ala. These IRS-2 mutants, denoted IRS-2^5A, and their wild-type controls (IRS-2^WT) were introduced into adenoviral constructs that were infected into Min6 cells or into cultured murine islets. RESULTS - When expressed in cultured mouse islets, IRS-2^5A was better than IRS-2^WT in protecting β-cells from apoptosis induced by a combination of IL-1β, IFN-γ, TNF-α, and Fas ligand. Cytokine-treated islets expressing IRS2 ^5A secreted significantly more insulin in response to glucose than did islets expressing IRS-2^WT. This could be attributed to the higher transcription of Pdx1 in cytokine-treated islets that expressed IRS-2 ^5A. Accordingly, transplantation of 200 islets expressing IRS2 ^5A into STZ-induced diabetic mice restored their ability to respond to a glucose load similar to naïve mice. In contrast, mice transplanted with islets expressing IRS2^WT maintained sustained hyperglycemia 3 days after transplantation. CONCLUSIONS - Elimination of a physiological negative feedback control mechanism along the insulin-signaling pathway that involves Ser/Thr phosphorylation of IRS-2 affords protection against the adverse effects of proinflammatory cytokines and improves β-cell function under stress. Genetic approaches that promote IRS25A expression in pancreatic β-cells, therefore, could be considered a rational treatment against β-cell failure after islet transplantation.

Insulin signaling at target tissues is essential for growth and development and for normal homeostasis of glucose, fat, and protein metabolism. Control over this process is therefore tightly regulated. It can be achieved by a negative feedback control mechanism whereby downstream components inhibit upstream elements along the insulin-signaling pathway (autoregulation) or by signals from apparently unrelated pathways that inhibit insulin signaling thus leading to insulin resistance. Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues has emerged as a key step in these control processes under both physiological and pathological conditions. The list of IRS kinases implicated in the development of insulin resistance is growing rapidly, concomitant with the list of potential Ser/Thr phosphorylation sites in IRS proteins. Here, we review a range of conditions that activate IRS kinases to phosphorylate IRS proteins on "hot spot" domains. The flexibility vs. specificity features of this reaction is discussed and its characteristic as an "array" phosphorylation is suggested. Finally, its implications on insulin signaling, insulin resistance and type 2 diabetes, an emerging epidemic of the 21st century are outlined.

Insulin signalling at target tissues results in a large array of biological responses. These events are essential for normal growth and development, and for normal homeostasis of glucose, fat and protein metabolism. Elucidating the intracellular events following activation of the insulin receptor and the interactions between the insulin and IGF-1 signalling systems has been the main focus of a large number of investigators, and for excellent reasons. Improved understanding of the signalling pathways involved in insulin action and the impact of IGF-1 on these processes could lead to a better understanding of the pathophysiology of insulin resistance associated with obesity and type 2 diabetes and the identification of key molecules that could lead to newer and more effective therapeutic agents for treating these common disorders that are already an uprising epidemic of the 21st century. This chapter will summarize our current understanding of the molecular basis of insulin action, beginning with outlining key that constitute the insulin signalling pathways. Then, impairments in insulin signalling pathways and new paradigms regarding the molecular basis of insulin and IGF-1 resistance will be analysed.

Certain selective serotonin reuptake inhibitors (SSRIs) induce the clinical and biochemical manifestations of a metabolic syndrome by as yet unknown mechanism. Here we demonstrate that incubation (1 h) of rat hepatoma Fao cells with the SSRIs paroxetine and sertraline, but not with the atypical antipsychotic drug olanzapine, inhibited the insulin-stimulated Tyr phosphorylation of the insulin receptor substrate-1 (IRS-1) with half-maximal effects at ∼ 10 μM. This inhibition correlated with a rapid phosphorylation and activation of a number of Ser/Thr IRS-1 kinases including JNK, S6K1, ERK and p38 MAPK, but not PKB (Akt). JNK appears as a key player activated by SSRIs because specific JNK inhibitors partially eliminated the effects of these drugs. The SSRIs induced the phosphorylation of IRS-1 on S307 and S408, which inhibits IRS-1 function and insulin signaling. These results implicate selected SSRIs as inhibitors of insulin signaling and as potential inducers of cellular insulin resistance.

The synovial fluid (SF) cells of rheumatoid arthritis (RA) patients express a specific CD44 variant designated CD44vRA. Using a cellular model of this autoimmune disease, we show in this study that the mammalian lectin, galectin-8 (gal-8), is a novel high-affinity ligand of CD44vRA. By affinity chromatography, flow cytometry, and surface plasmon resonance, we demonstrate that gal-8 interacts with a high affinity (K_d, 6 × 10^-9 M) with CD44vRA. We further demonstrate that SF cells from RA patients express and secrete gal-8, to a concentration of 25-65 nM, well within the concentration of gal-8 required to induce apoptosis of SF cells. We further show that not all gal-8 remains freely soluble in the SF and at least part forms triple complexes with CD44 and fibrinogen that can be detected, after fibrinogen immunoprecipitation, with Abs against fibrinogen, gal-8 and CD44. These triple complexes may therefore increase the inflammatory reaction by sequestering the soluble gal-8, thereby reducing its ability to induce apoptosis in the inflammatory cells. Our findings not only shed light on the receptor-ligand relationships between CD44 and gal-8, but also underline the biological significance of these interactions, which may affect the extent of the autoimmune inflammatory response in the SF of RA patients.

Galectin-8, a member of the galectin family of mammalian lectins, is made of two carbohydrate-recognition domains (CRDs), joined by a "hinge" region. Ligation of integrins by galectin-8 induces a distinct cytoskeletal organization, associated with activation of the extracellular-regulated kinase (ERK) and phosphatidylinositol 3-kinase signaling cascades. We show that these properties of galectin-8 are mediated by the concerted action of its two CRDs and involve both protein-sugar and protein-protein interactions. Accordingly, the isolated N - or C -CRD domains of galectin-8 or galectin-8 mutated at selected residues implicated in sugar binding (E251Q; W85Y, W248Y, W[85,248]Y) exhibited reduced sugar binding, which was accompanied by severe impairment in the capacity of these mutants to promote the adhesive, spreading, and signaling functions of galectin-8. Other mutations that did not impair sugar binding (e.g. E88Q) still impeded the signaling and cell-adherence functions of galectin-8. Deletion of the "hinge" region similarly impaired the biological effects of galectin-8. These results provide evidence that cooperative interactions between the two CRDs and the "hinge" domain are required for the proper functioning of galectin-8.

Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative feedback control mechanisms otherwise utilized by insulin itself to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article focuses on the Ser/Thr protein kinases which phosphorylate insulin receptor substrates and the major Ser sites that are phosphorylated, as key elements in the uncoupling of insulin signalling and the induction of an insulin resistance state.

Primary olfactory neurons project axons from the olfactory neuroepithelium lining the nasal cavity to the olfactory bulb in the brain. These axons grow within large mixed bundles in the olfactory nerve and then sort out into homotypic fascicles in the nerve fiber layer of the olfactory bulb before terminating in topographically fixed glomeruli. Carbohydrates expressed on the cell surface have been implicated in axon sorting within the nerve fiber layer. We have identified two novel subpopulations of primary olfactory neurons that express distinct α-extended lactoseries carbohydrates recognised by monoclonal antibodies LA4 and KH10. Both carbohydrate epitopes are present on novel glycoforms of the neural cell adhesion molecule, which we have named NOC-7 and NOC-8. Primary axon fasciculation is disrupted in vitro when interactions between these cell surface lactoseries carbohydrates and their endogenous binding molecules are inhibited by the LA4 and KH10 antibodies or lactosamine sugars. We report the expression of multiple members of the lactoseries binding galectin family in the primary olfactory system. In particular, galectin-3 is expressed by ensheathing cells surrounding nerve fascicles in the submucosa and nerve fiber layer, where it may mediate cross-linking of axons. Galectin-4, -7, and -8 are expressed by the primary olfactory axons as they grow from the nasal cavity to the olfactory bulb. A putative role for NOC-7 and NOC-8 in axon fasciculation and the expression of multiple galectins in the developing olfactory nerve suggest that these molecules may be involved in the formation of this pathway, particularly in the sorting of axons as they converge towards their target.

Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. It is associated with the development of type II diabetes, a 21st century epidemic. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative feedback control mechanisms otherwise utilized by insulin itself, to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article focuses on the cardinal role of Ser/Thr protein kinases, which phosphorylate insulin receptor substrates, as key players in the uncoupling of insulin signaling and the induction of an insulin resistance state.

Internalization of the insulin receptor (IR) is a highly regulated multi-step process whose underlying molecular basis is not fully understood. Here we undertook to study the role of extracellular matrix (ECM) proteins in the modulation of IR internalization. Employing Chinese hamster ovary cells that overexpress IR (CHO-T cells), our results indicate that IR internalization proceeds unaffected even when Tyr phosphorylation of IR substrates, such as IRS-1, is impaired (e.g. in CHO-T cells overexpressing IRS-1 whose pleckstrin-homology domain has been deleted or in CHO-T cells that overexpress the PH/PTB domain of IRS-1). In contrast, IR internalization is affected by the context of the ECM proteins to which the cells adhere. Hence, IR internalization was inhibited 40-60% in CHO-T cells adherent onto galectin-8 (an ECM protein and an integrin ligand of the galectin family) when compared with cells adherent onto fibronectin, collagen, or laminin. Cells adherent to galectin-8 manifested a unique cytoskeletal organization, which involved formation of cortical actin and generation of F-actin microspikes that contrasted with the prominent stress-fibers formed when cells adhered to fibronectin. To better establish a role for actin filament organization in IR endocytosis, this process was assayed in CHO-T cells (adherent onto fibronectin), whose actin filaments were disrupted upon treatment with latrunculin B. Latrunculin B did not affect insulin-induced Tyr phosphorylation of IR or its ability to phosphorylate its substrates; still, a 30-50% reduction in the rate of IR internalization was observed in cells treated with latrunculin B. Treatment of cells with nocodazole, which disrupts formation of microtubules, did not affect IR internalization. These results indicate that proper actin, but not microtubular, organization is a critical requirement for IR internalization and suggest that integrin-mediated signaling pathways emitted upon cell adhesion to different extracell

BACKGROUND. Knowledge of the expression of the galectins in human colon carcinomas is mainly restricted to galectin-3 and, to a lesser extent, galectin-1. The current study analyzed the prognostic values contributed by galectin-1, galectin-3, galectin-4, and galectin-8 in cases of colon carcinoma. METHODS. The authors selected 55 colon carcinomas (including 10 Dukes A, 16 Dukes B, 15 Dukes C, and 14 metastatic tumors that the authors labeled "Stage D"). The immunohistochemical levels of expression of the four galectins were determined quantitatively by means of computer-assisted microscopy. RESULTS. The data from the current study indicate that the four galectins under study are associated with significant and separate prognostic values that depend on the Dukes stage of the colon tumor. In particular, the authors observed a significant prognostic value associated with galectins-1, -3, and -4 in Dukes A and B colon tumors. In addition, significant prognostic value also was associated with galectin-8 in Dukes C and D colon tumors. The prognostic values associated with the levels of expression of galectin-1 and galectin-4 in Dukes A and B tumors appear to be independent of the Dukes stage. The same feature was observed when galectin-4 and galectin-8 were analyzed in the complete series. CONCLUSIONS. The data from the current study strongly suggest that galectins-1, -3, and -4 may be involved in the early stages of human colon carcinoma development and that galectin-8 is involved in the later stages.

The cellular pathways involved in the impairment of insulin signaling by cellular stress, triggered by the inflammatory cytokine tumor necrosis factor-α (TNF) or by translational inhibitors like cycloheximide and anisomycin were studied. Similar to TNF, cycloheximide and anisomycin stimulated serine phosphorylation of IRS-1 and IRS-2, reduced their ability to interact with the insulin receptor, inhibited the insulin-induced tyrosine phosphorylation of IRS proteins, and diminished their association with phosphatidylinositol 3-kinase (PI3K). These defects were partially reversed by wortmannin and LY294002, indicating that a PI3K-regulated step is critical for the impairment of insulin signaling by cellular stress. Induction of cellular stress resulted in complex formation between PI3K and ErbB2/ErbB3 and enhanced PI3K activity, implicating ErbB proteins as downstream effectors of stress-induced insulin resistance. Indeed, stimulation of ErbB2/ErbB3 by NDFβ1, the ErbB3 ligand, inhibited IRS protein tyrosine phosphorylation and recruitment of downstream effectors. Specific inhibitors of the ErbB2 tyrosine kinase abrogated the activation of ErbB2/ErbB3 and in parallel prevented the reduction in IRS protein functions. Taken together, our results suggest a novel mechanism by which cellular stress induces cross-talk between two different signaling pathways. Stress-dependent transactivation of ErbB2/ErbB3 receptors triggers a PI3K cascade that induces serine phosphorylation of IRS proteins culminating in insulin resistance.

Galectin-8 belongs to the family of tandem-repeat type galectins. It consists as several isoforms, each made of two domains of similar to140 amino-acids, both having a carbohydrate recognition domain (CRD). These domains are joined by a 'link peptide' of variable length. The human galectin-8 gene covers 33 kbp of genomic DNA. It is localized on chromosome 1 (1q42.11) and contains 11 exons. The gene produces by alternative splicing 14 different transcripts, altogether encoding 6 proteins. Galectin-8, like other galectins, is a secreted protein. Upon secretion galectin-8 acts as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of a selective subset of cell surface integrin receptors. Complex formation between galectin-8 and integrins involves sugar-protein interactions and triggers integrin-mediated signaling cascades such as Tyr phosphorylation of FAK and paxillin. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Such a mechanism allows local signals emitted by secreted galectin-8 to specify territories available for cell adhesion and migration. Due to its dual effects on the adhesive properties of cells and its association with fibronectin, galectin-8 might be considered as a novel type of a matricellular protein. Galectin-8 levels of expression positively correlate with certain human neoplasms, prostate cancer being the best example studied thus far. The overexpressed lectin might give these neoplasms some growth and metastasis related advantages due to its ability to modulate cell adhesion and cellular growth. Hence, galectin-8 may modulate cell-matrix interactions and regulate cellular functions in a variety of physiological and pathological conditions.

Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. It is associated with the development of type 2 diabetes - an ever-increasing epidemic of the 21st century. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative-feedback control mechanisms, otherwise utilized by insulin itself, to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article describes recent findings that present novel viewpoints of the molecular basis of insulin resistance, focusing on the cardinal role of Ser/Thr protein kinases as emerging key players in this arena.

Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCζ fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCζ in Fao cells, by infection of the cells with adenovirus-based PKCζ construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR-IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCζ was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC α, δ, or η. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCζ, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCζ inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCζ itself. These findings implicate PKCζ as a key element in a multistep negative feedback control mechanism of IRS-1 functions.

Objectives: To investigate whether galectins 1, 3, and 8 are expressed in human cholesteatomas and whether any such expression does correlate with the level of apoptosis, which is, as we have previously shown, predictive of recurrence. Study Design: The analysis of 52 cholesteatomas resected by the same surgeon by means of canal wall up and canal wall down procedures. Methods: The immunohistochemical levels of expression of galectins 1, 3, and 8 were quantitatively determined (using computer-assisted microscopy) on conventional histological slides by means of specific anti-galectin-1, anti-galectin-3, and anti-galectin-8 antibodies. The level of apoptosis in each cholesteatoma under study had already been determined by means of the in situ labeling of nuclear DNA fragmentation (Tolt-mediated dUTP nick end labeling [TUNEL] staining). Results: Galectin-1 was expressed markedly in both the epithelial and the connective tissue areas of all the cholesteatomas under study. The levels of expression of galectin-3 and galectin-8 were considerably lower than that of galectin-1. The level of expression of galectin-3 correlated both highly and positively with the level of apoptosis. Conclusions: An upregulation of galectin-3 (known to have an antiapoptotic and antianoikis effect in certain model systems) expression, which is associated with pronounced apoptotic activity, could have a physiologically protective effect against the characteristically substantial apoptotic features occurring in recurrent cholesteatomas.

Galectins, a family of mammalian lectins with specificity to β-galactosides, are involved in growth-regulatory mechanisms and cell adhesion. A relationship is assumed to exist between the levels of expression of galectins and the level of malignancy in human gliomas. A comparative study of this aspect in the same series of clinical samples is required to prove this hypothesis. Using computer-assisted microscopy, we quantitatively characterized by immunohistochemistry the levels of expression of galectins-1,-3 and -8 in 116 human astrocytic tumors of grades I to IV. Extent of transcription of galectins-1, -3, and -8 genes was investigated in 8 human glioblastoma cell lines by means of RT-PCR techniques. Three of these cell lines were grafted into the brains of nude mice in order to characterize in vivo the galectins-1, -3 and -8 expression in relation to the patterns of the tumor invasion of the brain. The role of galectin-1, -3 and -8 in tumor astrocyte migration was quantitatively determined in vitro by means of computer-assisted phase-contrast videomicroscopy. The data indicate that the levels of galectin-1 and galectin-3 expression significantly change during the progression of malignancy in human astrocytic tumors, while that of galectin-8 remains unchanged. These three galectins are involved in tumor astrocyte invasion of the brain parenchyma since their levels of expression are higher in the invasive parts of xenografted glioblastomas than in their less invasive parts. Galectin-3, galectin-1, and to a lesser extent galectin-8, markedly stimulate glioblastoma cell migration in vitro. Since bands for the transcripts of human galectins-2, -4 and -9 were apparently less frequent and intense in the 8 human glioblastoma cell lines, this system provides an excellent model to assign defined roles to individual galectins and delineate overlapping and distinct functional aspects.

Objectives: To investigate whether the expression of the macrophage migration inhibitory factor (MIF) 1) is detectable, 2) changes in relation to recurrence and infection status, and 3) relates to the levels of expression of growth regulators/differentiation markers, including galectin-1, -3, and -8, retinoid acid receptors (RAR)]-α, -β, and -γ, binding sites for sarcolectin, and invasion markers (cathepsins -B and -D, and matrix metalloproteinases [MMP]-2, -3, and -9) in human cholesteatomas. Study Design: An analysis of 56 cholesteatomas resected by the same surgeon using canal wall up and canal wall down surgical procedures. Methods: The immunohistochemical levels of expression of MIF and the proteases were quantitatively determined (using computer-assisted microscopy) on routine histologic slides by specific antibodies, and statistically correlated to parameters of the other markers determined previously in conjunction with data on apoptosis/proliferation. Results: MIF expression was detected. It was significantly higher in the epithelium (P = .002) and vessels (P = .04) of the connective tissues (but not in the connective tissue itself) of recurrent as opposed to non-recurrent cholesteatomas. The MIF expression is significantly correlated (P = .006) to the RARβ expression in non-infected cholesteatomas, and to MMP-3 (P

The interaction of cells with the extracellular matrix regulates cell adhesion, motility, growth, survival and differentiation through integrin-mediated signal transduction. Here we demonstrate that galectin-8, a secreted mammalian β-galactoside binding protein, inhibits adhesion of human carcinoma (1299) cells to plates coated with integrin ligands, and induces cell apoptosis. Pretreatment of the cells with Mn²⁺, which increases the affinity of integrins for their ligands, abolished the inhibitory effects of galectin-8. The inhibitory effects of galectin-8 were specific and were not mimicked by plant lectins or other galectins (galectin-1 and galectin-3). In accordance with its anti-adhesive effects, transfection of galectin-8 cDNA into 1299 cells significantly reduced (by 75%) colony formation, when compared to the number of colonies formed by cells transfected with an empty vector. Affinity chromatography over immobilized galectin-8 indicated that few membrane proteins interacted with galectin-8 in a sugar-dependent manner. Microsequencing and western immunoblotting revealed that α₃β₁ integrin derived from 1299 as well as other cells (e.g. HeLa and human endothelial cells) is a major galectin-8 binding-protein. Furthermore, immunoprecipitation and immunohistochemical studies suggested that endogenous galectin-8, secreted from 1299 cells, forms complexes with α₃β₁ integrins expressed on the surface of 1299 cells. Galectin-8 also interacts with other members of the integrin family, like α₆β₁ integrins. In contrast, galectin-8 only minimally interacts with α₄ or β₃ integrins. We propose that galectin-8 is an integrin binding-protein that interacts to a different extent with several, but not all members of the integrin family. Binding of galectin-8 modulates integrin interactions with the extracellular matrix and thus regulates cell adhesion and cell survival.

Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin- stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr- phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. Mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)₆-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.

Tumor necrosis factor α (TNFα) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS- 2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amine acids 1245-1331) of IR expressed in bacteria as His₆ fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFα, bacterial sphingomyelinase, or other Ser(P)/Thr(P)- elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFα for periods as short as 30 min reversed its inhibitory effects on IR- IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin- induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFα effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFα on insulin action.

The combination of H₂O₂ and vanadate generates aqueous peroxovanadium (pV) species, which are effective cell-permeable oxidants, and potent inhibitors of protein-tyrosine phosphatases. As a result, treatment of intact cells with pV compounds significantly enhances protein Tyr phosphorylation. Here we demonstrate that treatment of intact rat hepatoma Fao cells with pV markedly enhances Tyr phosphorylation of a 75-kDa protein, termed pp75. Amino-terminal sequencing of pp75 revealed that this protein is a member of the 70-75-kDa heat shock protein family, which includes PBP-74, glucose- related protein (GRP)-75, and mortalin. Tyr phosphorylation of pp75 is selective, because other proteins that belong to the heat shock protein 70 family, such as GRP-72, Bip (GRP-78), and HSC-70 fail to undergo Tyr phosphorylation when cells are treated with pV. Our findings suggest that heat shock proteins such as pp75 may undergo tyrosine phosphorylation when intact cells are subjected to oxidative stress induced by pV compounds.

We have recently cloned and expressed a novel mammalian lectin of the galectin family and named it galectin-8 [Hadari et al. (1995) J. Biol. Chem. 270, 3447-3453], Galectin-8 is a 35 kDa protein, made of two domains of ∼140 amino-acids, each containing a single carbohydrate recognition domain (CRD). These domains are joined by a ∼30 amino acids 'link peptide'. Galectin-8 is a widely expressed protein present in liver, heart, muscle, kidney and brain. Native galectin-8 exists as a monomer, that is tightly associated to yet undefined cellular constituent. A conserved Arg residue which forms part of the sugar-binding site of all galectins, including the C-terminal CRD of galectin-8, is replaced with Ile90 at the N-terminal CRD of galectin-8. This substitution markedly changes the predicted surface of the N-terminal CRD, creating an extended hydrophobic pocket that can accommodate hydrophobic glycoconjugates. As such, the two CRDs of galectin-8 are expected to be structurally different and to interact with two different types of carbohydrates. Hence, galectin-8 is a naturally-occurring, ubiquitous, bifunctional mammalian lectin that might complex glycoconjugates of different types.

A structural analysis has been carried out to determine which part of the intracellular domain of the insulin receptor (IR) β subunit is involved in direct interaction with the receptor substrates IRS-1 and Shc. Toward this end, the juxtamembrane (JM) domain (amino acids 943-984) and the carboxyl-terminal (CT) region (amino acids 1245-1331) of IR were expressed in bacteria as (His)₆-fusion peptides, and their interaction with IRS-1 and Shc was studied. We could demonstrate that the CT region of IR was sufficient to bind Shc, although significant, but much lower binding of Shc to the JM region could be detected as well. Furthermore, in vitro Tyr phosphorylation of the CT region potentiated its interactions with Shc 2-fold. In contrast, the JM region, but not the CT domain of the IR, was sufficient to mediate interactions between the IR and IRS-1. These interactions did not involve the pleckstrin homology (PH) region of IRS-1, since an IRS-1 mutant, in which four "blocks" of the PH domain (Pro⁵-Pro⁶⁵) were deleted, interacted with the JM region of IR with the same efficiency as native IRS-1. These results suggest that the ER interacts with its downstream effectors through distinct receptor regions, and that autophosphorylation of Tyr residues located at the CT domain of the IR can modulate these interactions.

To characterize the structural basis for the interactions between the insulin receptor (IR) and its major substrate, insulin receptor substrate-1 (IRS-1), a segment of the NH₂-terminal region of IRS-1 (Pro⁵-Pro⁶⁵) was deleted. This region contains the first four conserved boxes of a pleckstrin homology (PH) domain, located at the NH₂-terminal part of IRS-1. COS-7 cells were then cotransfected with the genes coding for IR and a wild-type (WT) or a mutated form of IRS-1. IRS-1(ΔPH) underwent significantly reduced insulin- dependent tyrosine phosphorylation compared with WT IRS-1. The reduced in vivo tyrosine phosphorylation of IRS-1(ΔPH) was accompanied by reduced association between IRS-1(ΔPH) and its downstream effector p85 regulatory subunit of phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and IRS- 1(ΔPH) underwent comparable insulin-dependent tyrosine phosphorylation in vitro when incubated with partially purified insulin receptor kinase. These findings suggest that the overall structure of IRS-1 is not altered by deletion of its PH domain and that the PH domain is not the main site for protein-protein interactions between the insulin receptor and IRS-1, at least in vitro. In conclusion, the PH region might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas the actual interactions with the IR are presumably mediated through other domains of the IRS-1 molecule. This could account for the fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions remain unaffected.

Protein tyrosine phosphorylation is a major signal transduction pathway involved in cellular metabolism, growth, and differentiation. Recent data indicate that tyrosine phosphorylation also plays a role in neuronal plasticity, We are using conditioned taste aversion, a fast and robust associative learning paradigm subserved among other brain areas by the insular cortex, to investigate molecular correlates of learning and memory in the rat cortex, In conditioned taste aversion, rats learn to associate a novel taste (e.g., saccharin) with delayed poisoning (e.g., by LiCl injection), Here we report that after conditioned taste aversion training, there is a rapid and marked increase in tyrosine phosphorylation of a set of proteins in the insular cortex but not in other brain areas. A major protein so modulated, of 180 kDa, is abundant in a membrane fraction and remains modulated for more than an hour after training, Exposure of the rats to the novel taste alone results in only a small modulation of the aforementioned proteins whereas administration of the malaise-inducing agent per se has no effect, To the best of our knowledge, this is the first demonstration of modulation of protein tyrosine phosphorylation in the brain after a behavioral experience.

Injection of a combination of H₂O₂ and vanadate (H/V) into the portal vein of rat livers resulted in inhibition of protein tyrosine phosphatase activity and led to a dramatic enhanced in vivo protein tyrosine phosphorylation. Some of the phosphorylated proteins were identified as the β-subunit of the insulin receptor, the insulin receptor substrate 1 (ppl85), PLC-γ (pp145), and a 100 kDa PLC-γ-associated protein. Immunofluorescense and immune electron microscopy of frozen liver sections with anti-P-Tyr antibodies revealed that most of the tyrosine-phosphorylated proteins are localized in close proximity to the plasma membrane in intercellular adherence junctions and tight junction regions. This close in vivo association between membranal protein tyrosine kinases, their target proteins, and cytoskeletal elements could enable formation of 'signaling complexes' which may play a role in transmembrane signal transduction. By affinity chromatography over immobilized anti-P-Tyr antibodies, a large number of these tyrosine-phosphorylated proteins were partially purified.

Acute (10-30 min) treatment of intact rat hepatoma (Fao) cells with H₂O₂, inhibits in vivo protein tyrosine phosphatase activity. Vanadate markedly potentiates this effect although it has only trivial effects of its own. Here we show that H₂O₂ inhibits a protein tyrosine phosphatase activity, but not a p-nitro phenyl phosphate hydrolysing activity, in cytosolic extracts of these cells. This effect is completely reversed by 10 mM dithiothreitol. Other oxidants have similar inhibitory effects. Vanadate inhibits the protein tyrosine phosphatase activity in vitro, and its effects are additive with those of H₂O₂. These findings suggest that H₂O₂ and vanadate interact with the protein tyrosine phosphatases at two independent sites. They also suggest that in intact cells H₂O₂ has a direct inhibitory effect on protein tyrosine phosphatase activity and an indirect effect of facilitating the entry of vanadate.

Adherens-type junctions (AJs) are major subcellular targets for tyrosine specific protein phosphorylation [Volberg et al. (1991) Cell Regul., 2, 105-120]. Here we report on the apparent effect of such phosphorylation events on the assembly and integrity of AJs. We show that incubation of MDCK cells with potent inhibitors of tyrosine-specific phosphatases (FTP), namely H₂O₂ and vanadate, leads to a dramatic increase in AJ-associated phosphotyrosine which was apparent already within 2-5 min of treatment and progressed upon further incubation. Examination of H₂O₂ vanadate treated cells at later time points indicated that intercellular AJs rapidly deteriorated, concomitantly with a marked increase in the number and size of vinculin and actin containing focal contacts. In parallel, major changes were observed in cell structure and topology, as revealed by electron microscopy. These were manifested by rapid rounding-up of the cells followed by reorganization of the cell monolayer. Other intercellular junctions, including desmosomes and tight junctions, visualized by staining with desmoplakin and ZO-I antibodies, were not significantly affected. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we have carred out reciprocal experiments in which Rovs Sarcoma virus (RSV) transformed chick lens cells, expressing high levels of pp60^src kinase, were treated with inhibitors of tyrosine kinases, (tyrphostins). We show that following such treatment, intercellular AJs which were deteriorated in the transformed cells, were reformed. Based on these observations, we propose that specific tyrosine phosphorylation of AJ components is involved in the downregulation of these cellular contacts.

The effects of cationic polyamino acids on insulin binding to soluble insulin receptor preparations were studied. Incubation of partially or fully purified receptor preparations with polylysine (pLys) increased by several-fold the amount of [¹²⁵I]insulin that remained associated with the receptor, as determined both by precipitation of receptor-insulin complexes by polyethylene glycol or by separation of the complexes from the free hormone by gel filtration. This elevation in the amount of bound insulin resulted from increased number of insulin binding sites, and could not be attributed to an increased affinity of the receptors to insulin. In fact, pLys reduced 2 - 3-fold the affinity of insulin binding to its receptor as determined by equilibrium binding studies, and by monitoring the rate of exchange of bound [¹²⁵I]insulin with unlabeled hormone. pLys induced specific interactions between insulin and its native receptor since other basic compounds such as histone, spermidine, polymixin B, compound 48/80, lysine, and arginine failed to reproduce its effects. pLys did not interact with the free ligand, nor did it promote interactions between insulin and denatured receptor forms. Furthermore, pLys did not induce binding of insulin to other proteins present in the partially purified receptor preparations. The effects of pLys were time and dose-dependent and were proportional to the pLys chain length. The longer the chain, the greater was the effect. Enhanced insulin binding and receptor β-subunit autophosphorylation (in the presence of insulin) exhibited a similar dependency on the chain length of pLys. pLys effects on insulin binding were associated with formation of large protein aggregates that remained trapped at the top of Sephacryl S-300 columns. These aggregates contained substantial amounts of receptor-insulin complexes. Our results suggest that pLys induces formation of receptor clusters that create de novo insulin binding sites among adjacent receptor tetramers. Alternatively, formation of receptor aggregates might facilitate insulin binding to a soluble receptor subfraction that otherwise fails to bind the hormone.

The human promyeloid cell line HL-60 differentiates toward monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) or dibutyryl cAMP, respectively. When nondifferentiated cells were incubated for 20 min with 2 mM H₂O₂ and 0.1 mM sodium orthovanadate to inhibit their protein-tyrosine-phosphatase activity (Heffetz, D., Bushkin, I., Dror, R., and Zick, Y. (1990) J. Biol. Chem. 265, 2896-2902), we found marked tyrosine phosphorylation of a single major protein of 53 kDa. Induction of differentiation of HL-60 cells was accompanied by the appearance of an additional major cytosolic tyrosine-phosphorylated protein of 75 kDa (pp75). In dibutyryl cAMP-treated cells, tyrosine phosphorylation of pp75 peaked after 24 h and then declined rapidly. In l,25(OH)₂D₃-treated cells, increased tyrosine phosphorylation was detected as early as 2 h and peaked after 3 days, whereas the presence of differentiated phenotypes, assessed by the capacity of the cells to reduce nitro blue tetrazolium, was detected no earlier than 24 h. Doses of 1,25(OH)₂D₃ as low as 1 nM induced the appearance of pp75 at a stage where almost no differentiation measured by nitro blue tetrazolium reduction was detected. Phosphorylation of pp75 was not stimulated by adriamycin, which induced growth arrest without initiation of differentiation. pp75 could also be detected in U-937, a monocytic cell line that is more advanced in its differentiation state, and also in terminally differentiated circulating human monocytes treated with H₂O₂/vanadate. pp75 underwent in vitro tyrosine phosphorylation in cytosolic extracts derived from l,25(OH)₂D₃-induced HL-60 cells, but not in extracts derived from uninduced cells. Our results raise the possibility that tyrosine phosphorylation of pp75 may be a common early event that heralds the differentiation of HL-60 cells into both the monocytic and granulocytic pathways.

Treatment of four cell lines [rat hepatoma (Fao), murine muscle (BC3H-1), Chinese hamster ovary (CHO), and rat basophilic leukemia (RBL)] with a combination of 3 mM H₂O₂ and 1 mM sodium orthovanadate markedly stimulates protein tyrosine phosphorylation, which is accompanied by a dramatic increase (5-15-fold) in inositol phosphate (InsP) formation. H₂O₂/vanadate stimulate best formation of inositol triphosphate while their effects on the mono and di derivatives are more moderate. In the presence of 3 mM H₂O₂, both protein tyrosine phosphorylation and InsP formation are highly correlated and manifest an identical dose-response relationship for vanadate. Half-maximal and maximal effects are obtained at 30 and 100 μM, respectively. This stimulatory effect of H₂O₂/vanadate is not mimicked by other oxidants such as spermine, spermidine, KMnO₄, and vitamin K₃. In RBL cells, the kinetics of inositol triphosphate formation correlate with tyrosine phosphorylation of a 67-kDa protein, while tyrosine phosphorylation of a 55-kDa protein is closely correlated with both inositol monophosphate formation and serotonin secretion from these cells. Taken together, these results suggest a causal relationship between tyrosine phosphorylation triggered in a nonhormonal manner and polyphosphoinositide breakdown. Furthermore, these results implicate protein tyrosine phosphorylation in playing a role in the stimulus-secretion coupling in RBL cells.

Differentiation of the human promyelocytic leukemia cell line HL-60 into monocytes or macrophages is associated with increased expression of cell surface insulin receptors, while differentiation of these cells into granulocytes is associated with receptor loss. Here we demonstrate that differentiation of HL-60 cells into monocytes or granulocytes induced by 1;25(OH)₂vitD₃ or Bt₂cAMP, respectively, has no major effect on the specific activity of the insulin receptor kinase (IRK). By contrast, when HL-60 cells are incubated with a combination of 1;25(Oh)₂vitD₃ and Bt₂cAMP, their differentiation into adherent macrophages-like cells is accompanied by a 505 reduction in the specific activity of IRK. These findings suggest that acquisition or loss of insulin receptors during differentiation of HL-60 involves selective alterations in the functional aspects of these receptors. Our results also implicate the generation of specific regulatory signals that inhibit IRK activity when HL-60 cells are stimulated with a combination of 1;25(OH)₂vitD₃ and Bt₂cAMP.

Western blotting with anti-phosphotyrosine antibodies was employed in order to study insulin-dependent protein tyrosine phosphorylation in intact Fao cells. In insulin-treated cells, a prominent 180-kDa protein underwent tyrosine phosphorylation, which peaked at 45 s and then rapidly declined. Pretreatment of the cells with 1 mM Bt2cAMP or 0.16 microM 12-O-tetradecanoylphorbol-13-acetate inhibited the insulin-dependent phosphorylation of pp 180, while 1 mM vanadate or 3 mM H2O2 markedly potentiated it. These results indicate that phosphorylation of pp 180 is respectively regulated by agents that are known to synergize with or antagonize the action of the insulin receptor kinase. pp 180 is therefore likely to mediate physiological functions of this receptor kinase. Incubation of Fao cells with 3 mM H2O2 for 30 min prior to their treatment with insulin for 45 s allowed the detection of additional, previously undescribed, proteins pp 150, 114, 100, 85, 68, and 56 kDa that underwent insulin-dependent tyrosine phosphorylation. The potentiating effects of H2O2 were time- and dose-dependent and could be reversed by 2 mM dithiothreitol. Proteins phosphorylated in response to H2O2 plus insulin maintained their fully phosphorylated state for at least 20 min. We suggest that these phosphoproteins are potential physiological substrates for the insulin receptor kinase.

In the present study we demonstrate that TD-α undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-γ-S which activates TD, suggesting that it is only the inactive conformation of TD-α that serves as a substrate for PKC. Indeed, both vanadate and AlF₄, that confer an active conformation on TD-α-GDP, inhibit PKC-mediated phosphorylation of purified TD-α-GDP. We demonstrate that the purified β subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-α and β from high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the α and β subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor β subunits. The insulin receptor β subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor β subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.

Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulinlike growth factor1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4:1 ratio (Glu/Tyr, 4:1). The insulinstimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosinecontaining polymers Ala/Glu/Lys/Tyr (6:2:5:1) and Glu/Ala/Tyr (6:3:1) were also phosphorylated by the insulinstimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulinlike growth factor1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4:1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6:3:1) or Ala/Glu/Lys/Tyr (6:2:5:1). Glu/Tyr (4:1) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6:3:1). Three additional polymers: Glu/Tyr (1:1), Glu/Ala/Tyr (1:1:1), and Lys/Tyr (1:1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. (a) Hormonesensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. (b) Tyrosinecontaining polymer substrates are highly sensitive and convenient tools to study (hormonesensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.

Mouse thymocytes are characterized as a model cellular system for studying the onset of hormone-induced cellular refractoriness (desensitization). This system has the following combination of useful features. (a) The cells can be isolated without the use of digestive enzymes, avoiding possible damage to surface receptors or to other exposed membranal constituents. (b) They can be kept viable for several hours, a period during which both stimulation and desensitization get well under way. (c) They can be stimulated by a variety of hormones which function via cAMP (β-agonists, prostaglandin E₁ and specific thymic humoral factors). (d) Their desensitization is receptor-specific. (e) They can be readily ruptured under mild conditions so as to allow a physiologically relevant biochemical analysis of hormonal stimulation and desensitization. (f) The hormonal response of these cells can be monitored simultaneously by the activation of adenylate cyclase, by the intracellular level of cAMP, and by the activation of cAMP-dependent protein kinase (which functions as a metabolic sensor for cAMP). In this cellular system, desensitization does not involve processes such as the efflux of cAMP, the activation of cAMP-phosphodiesterase or the synthesis of a protein mediator. On the other hand, desensitization can be accounted for by a hormone-triggered inactivation of the adenylate cyclase system. The immediate desensitization of thymocytes is reversible and occurs without apparent loss of functional receptors. Continuous presence of hormone is shown to be required not only for triggering the chain of events which leads to the readily reversible desensitization, but also for the process which transfers the cells to the subsequent, 'locked' desensitized state.

Nα-Tosyl-L-lysine chloromethyl ketone (Tos-LysCH₂Cl) was found to inhibit irreversibly the onset of the hormone-induced refractory state in intact thymocytes. When thymocytes (≃2 x 10⁷ cells per ml) are treated with Tos-hysCh₂Cl (10^-4M, for 90 min at pH 7 and 37°C) the cells retain their viability, including a full capacity to recognize and respond to hormonal stimuli, yet they selectively lose their ability to become desensitized to persistent triggering by a hormone, as reflected in the state of activation of intracellular cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Whereas upon hormonal stimulation of untreated cells the immediate rise in the state of activation of this enzyme (up to an activity ratio of >0.85) is followed by an exponential decline to basal values within ≃60 min, in TosLysCH₂CL-treated cells the hormone-triggered elevation in the state of activation of the enzyme is maintained for >60 min. Evidence is presented to suggest that in thymocytes TosLysCH₂Cl inhibits the regulatory process that normally uncouples the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system without interfering with previous or subsequent molecular events connected with the transfer of hormonal signals across the cell membrane. This technique allows, therefore, the preparation of viable thymocytes with a limited and distinct regulatory defect introduced by chemical (covalent) means. As such, it is most useful for studies aimed at the elucidation of the mechanism of cell desensitization and for further characterization and localization of key components responsible for cellular refractoriness.