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.

Glycosphingolipids are ubiquitous cell surface molecules undertaking fundamental cellular processes. Lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) are the representative core structures for lacto- and neolacto-series glycosphingolipids. These glycolipids are the carriers to the blood group antigen and human natural killer antigens mainly found on blood cells, and are also principal components in human milk, contributing to infant health. The beta-galactoside recognising galectins mediate various cellular functions of these glycosphingolipids. We report crystallographic structures of the galectin-8 N-terminal domain (galectin-8N) in complex with LNT and LNnT. We reveal the first example in which the non-reducing end of LNT binds to the primary binding site of a galectin, and provide a structure-based rationale for the significant ten-fold difference in binding affinities of galectin-8N toward LNT compared to LNnT, such a magnitude of difference not being observed for any other galectin. In addition, the LNnT complex showed that the unique Arg59 has ability to adopt a new orientation, and comparison of glycerol-and lactose-bound galectin-8N structures reveals a minimum atomic framework for ligand recognition. Overall, these results enhance our understanding of glycosphingolipids interactions with galectin-8N, and highlight a structure-based rationale for its significantly different affinity for components of biologically relevant glycosphingolipids.

Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins.

Pro-inflammatory cytokines induce death of beta cells and hamper engraftment of transplanted islet mass. Our aim was to reveal novel genes involved in this process, as a platform for innovative therapeutic approaches. Small interfering RNA (siRNA) high-throughput screening (HTS) of primary human islets was employed to identify novel genes involved in cytokine-induced beta cell apoptosis. Dispersed human islets from nine human donors, treated with a combination of TNF-alpha, IL-1 beta and IFN-gamma were transfected with 730 different siRNAs. Caspase-3/7 activity was measured, results were analysed and potential anti- and pro-apoptotic genes were identified. Dispersed human pancreatic islets appeared to be suitable targets for performance of siRNA HTS. Using this methodology we found a number of potential pro- and anti-apoptotic target hits that have not been previously associated with pancreatic beta cell death. One such hit was the de-ubiquitinating enzyme otubain 2 (OTUB2). OTUB2 knockdown increased caspase-3/7 activity in MIN6 cells and primary human islets and inhibited insulin secretion and increased nuclear factor-kappa B (NF-kappa B) activity both under basal conditions and following cytokine treatment. Use of dispersed human islets provides a new platform for functional HTS in a highly physiological system. Employing this technique enabled the identification of OTUB2 as a novel promoter of viability and insulin secretion in human beta cells. OTUB2 acts through the inhibition of NF-kappa B signalling, which is deleterious to beta cell survival. siRNA screens of human islets may therefore identify new targets, such as OTUB2, for therapeutic intervention in type 1 diabetes and islet transplantation.

Upon exposure, TiO2 nanoparticles (NPs) have been recovered in internal organs such as the liver, and are proposed to cause cellular/organ dysfunction, particularly in the liver and lungs. We hypothesized that despite being considered "inert" as bulk material, TiO2 NPs may impair insulin responses in liver-derived cells, either indirectly by inflammatory activation of macrophages, and/or by directly interfering with insulin signaling. Using qRT-PCR and conditioned medium (CM) approaches, we show that exposure to TiO2 NPs activates macrophages' expression of TNF-alpha, IL-6, IL-8, IL-1 alpha and IL-1 beta and the resulting CM induces insulin resistance in Fao cells. Furthermore, direct exposure of Fao cells to TiO2 results in activation of the stress kinases JNK and p38MAP kinase, and in induction of insulin resistance at the signaling and metabolic levels. Collectively, our findings provide a proof-of-concept for the ability of man-made NPs to induce insulin resistance in liver-derived cells, an endocrine abnormality underlying some of the most common human diseases.

Pro-inflammatory cytokines induce death of pancreatic beta cells, leading to the development of type 1 diabetes. We sought to identify novel players and the underlying mechanisms involved in this process. A high-throughput screen of 3,850 mouse small interfering RNAs (siRNAs) was performed in cytokine-treated MIN6 beta cells. Cells were transfected with the different siRNAs and then treated with a combination of TNF alpha, IL-1 beta and IFN gamma. Cellular apoptosis (caspase-3/7 activity), and changes in cellular reducing power and cell morphology were monitored. The resulting data were analysed and the corresponding z scores calculated. Several gene families were identified as promoting cytokine-induced beta cell apoptosis, the most prominent being those encoding ubiquitin ligases and serine/threonine kinases. Conversely, deubiquitinating enzymes appeared to reduce apoptosis, while protein phosphatases were mainly associated with lowering cellular reducing power. The screen suggested with high confidence the involvement of several novel genes in cytokine-induced beta cell death, including Camkk2, Epn3, Foxp3 and Tm7sf3, which encodes an orphan seven transmembrane receptor. siRNAs to Tm7sf3 promoted cytokine-induced death of MIN6 cells and human pancreatic islets, and abrogated insulin secretion in these cells. These findings implicate transmembrane 7 superfamily member 3 as a potential new player in the inhibition of cytokine-induced death and in the promotion of insulin secretion from pancreatic beta cells. The signalling pathways and novel genes that we identified in this screen and that mediate beta cell death offer new possible targets for therapeutic intervention in diabetes and its adverse complications.

Keywords: Endocrinology & Metabolism

Protein kinase C delta (PKC delta) is induced by insulin to rapidly associate with insulin receptor (IR) and upregulates insulin signaling. We utilized specific JM and CT receptor domains and chimeras of PKC alpha and PKC delta regulatory and catalytic domains to elucidate which components of PKC delta are responsible for positive regulatory effects of PKC delta on IR signaling. Studies were performed on L6 and L8 skeletal muscle myoblasts and myotubes. PKC delta was preferentially bound to the JM domain of IR, and insulin stimulation increased this binding. Both PKC delta/alpha and PKC alpha/delta chimeras (regulatory/catalytic) were bound preferentially to the JM but not to the CT domain of IR. Although IR-PKC delta binding was higher in cells expressing either the PKC delta/alpha or PKC alpha/delta chimera than in control cells, upregulation of IR signaling was observed only in PKC delta/alpha cells. Thus, in response to insulin increases in tyrosine phosphorylation of IR and insulin receptor substrate-1, downstream signaling to protein kinase B and glycogen synthase kinase 3 (GSK3) and glucose uptake were greater in cells overexpressing PKC delta/alpha and the PKC delta/delta domains than in cells expressing the PKC alpha/delta domains. Basal binding of Src to PKC delta was higher in both PKC delta/alpha- and PKC alpha/delta-expressing cells compared to control. Binding of Src to IR was decreased in PKC alpha/delta cells but remained elevated in the PKC delta/alpha cells in response to insulin. Finally, insulin increased Src activity in PKC delta/alpha-expressing cells but decreased it in PKC alpha/delta-expressing cells. Thus, the regulatory domain of PKC delta via interaction with Src appears to determine the role of PKC delta as a positive regulator of IR signaling in skeletal muscle.

Signaling of insulin and insulin-like growth factor-1 (IGF-1) at target tissues is essential for growth, 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-feed back control mechanism, whereby downstream components inhibit upstream elements along the insulin and IGF-1 signaling pathway or by signals from other pathways that inhibit insulin/IGF-1 signaling thus leading to insulin/IGF-1 resistance. Phosphorylation of insulin receptor substrates (IRS) proteins on serine residues has emerged as a key step in these control processes both under physiological and pathological conditions. The list of IRS kinases 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 selected domains. The specificity of this reaction is discussed and its characteristic as an "array" phosphorylation is suggested. Finally, its implications on insulin/IGF-1 signaling, insulin/IGF-1 resistance and diabetes, an emerging epidemic of the twenty-first century are outlined. (C) 2009 Elsevier Inc.

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 (I h) of rat hepatoma Fan cells with the SSRls 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 RM. This inhibition correlated with a rapid phosphorylation and activation of a number of Ser/Thr IRS-1 kinases including JNK, S6K1, ERK and p38 NIAPK, but not PKB (Akt). JNK appears as a key player activated by SSRls because specific JNK inhibitors partially eliminated the effects of these drugs. The SSRIs induced the phosphorylation of IRS-I 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. (c) 2007 Elsevier Inc. All rights reserved.

The Insulin Receptor Substrate (IRS) proteins are key players in insulin signal transduction and are the best studied targets of the insulin receptor. Ser/Thr phosphorylation of IRS proteins negatively modulates insulin signaling; therefore, the identification of IRS kinases and their target Ser phosphorylation sites is of physiological importance. Here we show that in Fao rat hepatoma cells, the I kappa B kinase beta (IKK beta) is an IRS-1 kinase activated by selected inducers of insulin resistance, including sphingomyelinase, ceramide, and free fatty acids. Moreover, IKK beta shares a repertoire of seven potential target sites on IRS-1 with protein kinase C zeta( PKC zeta), an IRS-1 kinase activated both by insulin and by inducers of insulin resistance. We further show that mutation of these seven sites (Ser-265, Ser-302, Ser-325, Ser-336, Ser-358, Ser-407, and Ser-408) confers protection from the action of IKK beta and PKC beta when they are overexpressed in Fao cells or primary hepatocytes. This enables the mutated IRS proteins to better propagate insulin signaling. These findings suggest that insulin-stimulated IRS kinases such as PKC zeta overlap with IRS kinases triggered by inducers of insulin resistance, such as IKK beta, to phosphorylate IRS-1 on common Ser sites.

Galectin-8, a mammalian β-galactoside binding lectin, functions as an extracellular matrix protein that forms high affinity interactions with integrins. Here we demonstrated that soluble galectin-8 inhibits cell cycle progression and induces growth arrest. These effects cannot be attributed to interference with cell adhesion but can be attributed to a 4-5-fold increase in the cellular content of the cyclin-dependent kinase inhibitor p21, which was already evident following a 4-h incubation of H1299 cells with galectin-8. The increase in p21 levels was preceded by a 3-5-fold increase in JNK and protein kinase B (PKB) activities. Accordingly, SP600125, the inhibitor of JNK, and wortmannin, the inhibitor of phosphatidylinositol 3-kinase, which is the upstream activator of PKB, inhibited the increase in the cellular content of p21. Furthermore, overexpression of a dominant inhibitory form of SEK1, the upstream kinase regulator of JNK, inhibited both JNK activation and p21 accumulation. When p21 expression was inhibited by cycloheximide, galectin-8 directed the cells toward apoptosis, which involves induction of poly(ADP-ribose) polymerase cleavage. Indeed, galectin-8-induced apoptosis was 2-fold higher in HTC (p21-null) cells when compared with parental HTC cells. Because overexpression of galectin-8 attenuates the rate of DNA synthesis, stable colonies that overexpress and secrete galectin-8 can be generated only in cells overexpressing a growth factor receptor, such as the insulin receptor. These results implicate galectin-8 as a modulator of cellular growth through up-regulation of p21. This process involves activation of JNK, which enhances the synthesis of p21, combined with the activation of PKB, which inhibits p21 degradation. These effects of the lectin depended upon protein-sugar interactions and were induced when galectin-8 was present as a soluble ligand or when it was overexpressed in cells.

Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-1(7A)), unlike wild-type IRS-1 (IRS-1(WT)), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-17A to remain complexed with the insulin receptor (IR), unlike IRS-1(WT), which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-17A and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.

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

Galectin-8, a member of the galectin family of mammalian lectins, is a secreted protein that promotes cell adhesion and migration upon binding to a subset of integrins through sugar-protein interactions. Ligation of integrins by galectin-8 triggers a distinct pattern of cytoskeletal organization, including formation of F-actin-containing microspikes. This is associated with activation of integrin-mediated signaling cascades (ERK and phosphatidylinositol 3 kinase (PI3K)) that are much more robust and are of longer duration than those induced upon cell adhesion to fibronectin. Indeed, formation of microspikes is enhanced 40% in cells that overexpress protein kinase B, the downstream effector of PI3K. Inhibition of PI3K activity induced by wortmannin partially inhibits cell adhesion and spreading while largely inhibiting microspike formation in cells adherent to galectin-8. Furthermore, the inhibitory effects of wortmannin are markedly accentuated in cells overexpressing PKB or p70S6K (CHOPKB and CHOp70S6K cells), whose adhesion and spreading on galectin-8 (but not on fibronectin) is inhibited similar to25-35% in the presence of wortmannin. The above results suggest that galectin-8 is an extracellular matrix protein that triggers a unique repertoire of integrin-mediated signals, which leads to a distinctive cytoskeletal organization and microspike formation. They further suggest that downstream effectors of PI3K, including PKB and p70 S6 kinase, in part mediate cell adhesion, spreading, and microspike formation induced by galectin-8.

The cellular pathways involved in the impairment of insulin signaling by cellular stress, triggered by the inflammatory cytokine tumor necrosis factor-alpha (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 NDFbeta1, 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.

Owing to their relevance for growth regulation and cell adhesion monitoring the expression of galectins (endogenous beta-galactoside-binding lectins) and their binding sites has relevance for tumor biology. Using galectin-type-specific reagents (non-crossreactive antibodies for proto-type galectin-1, chimera-type galectin-3 and tandem-repeat-type galectins-4 and -8, and labeled galectins-1, -3, and -4) we determined galectin expression in cutaneous T cell lymphomas (CTCL) and controls. In addition to commonly studied galectins-1 and -3, tandem-repeat-type galectins could be detected, i.e., galectin-8 in six from 15 cases and galectin-4 in one of 15 cases. In view of relevant ligands such as bcl-2 or integrins the presence of galectins-3 and -8 seems to be possibly related to loss of proliferation control and change in cell adhesion properties that are involved in clonal expansion and epidermal spread of malignant T cell clones. Successful chemotherapy of CTCL alters galectin expression selectively as shown for liposomal doxorubicin.

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 21 st 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.

The interaction of cells with the extracellular matrix regulates cell adhesion and motility. Here we demonstrate that different cell types adhere and spread when cultured in serum-free medium on immobilized galectin-8, a mammalian 13-galactoside-binding protein. At maximal doses, galectin-8 is equipotent to fibronectin in promoting cen adhesion and spreading. Cell adhesion to immobilized galectin-8 is mediated by sugar-protein interactions with integrins, and galectin-8 triggers integrin-mediated signaling cascades including Tyr phosphorylation of focal adhesion kinase and paxillin. Cell adhesion is potentiated in the presence of Mn2+, whereas it is interrupted in the presence of soluble galectin-8, integrin beta (1), inhibitory antibodies, EDTA, or thiodigalactoside but not by RGD peptides. Furthermore, cells readily adhere onto immobilized monoclonal galectin-8 antibodies, which are equipotent to integrin antibodies in promoting cell adhesion. Cell adhesion to immobilized galectin-8 is partially inhibited by serum proteins, suggesting that complex formation between immobilized galectin-8 and serum components generates a matrix that is less supportive of cell adhesion. Accordingly, cell motility on immobilized galectin-8 readily takes place in the presence of serum. Truncation of the C-terminal half of galectin-8, including one of its two carbohydrate recognition domains, largely abolishes its ability to modulate cell adhesion, indicating that both carbohydrate recognition domains are required to maintain a functional form of galectin-8. Collectively, our findings implicate galectin-8 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 cell surface integrin receptors. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Because of

Cholesteatoma is a benign disease characterized by the presence of an unrestrained growth and the accumulation of keratin debris in the middle ear cavity. This often recurs, even when surgical resection is thought to be complete. In a previous study we showed that cholesteatomas with the highest apoptotic indices recurred more rapidly and also exhibited a high level of p53 immunopositive cells. In view of their relevance to the characterization of the cell differentiation status, the present study focuses on the expression of retinoid acid receptors (RARs) and galectins in human cholesteatomas. Retinoids control the differentiation processes in keratinocytes while galectins play strikingly modulatory roles at apoptosis and cell adhesion levels in a wide variety of tissue (embryonic, normal and neoplastic). To clarify the expression of these two protein families in human cholesteatomas we examined and quantified the levels of immunohistochemical expression of RAR alpha, beta and gamma, and also galectin-1, -3 and -8 in a series of 70 human cholesteatomas. Our data show clearly that predominantly RAR beta and galectin-1 were expressed. The RAR gamma concentration was significantly lower than that of the RAR alpha; this was also observed for the galectin-8 concentration in comparison with the galectin-3 one. Furthermore, the level of RAR beta expression correlated highly (P = 0.00001) with the level of galectin-8 expression, which also correlated significantly with the level of RAR alpha and RAR gamma expression. In addition, this parameter also correlated with the level of galectin-1 and galectin-3 expression. These data suggest that cholesteatomas may originate in an undifferentiated population of keratinocytes, and that a relation may exist between retinoid activity and galectins. (C) 2001 Elsevier Science B.V. All rights reserved.

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-l function are downstream effectors of PI3K. PKC zeta fulfills this criterion, being an insulin-activated downstream effector of PI3K, Overexpression of PKC zeta in Fao cells, by infection of the cells with adenovirus-based PKC zeta 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 PKC zeta was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC alpha, delta, or eta. 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 PKC zeta has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKC zeta inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKC zeta itself. These findings implicate PKC zeta as a key element in a multistep negative feedback control mechanism of IRS-1 functions.

Galectins, a family of mammalian lectins with specificity to beta -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 revel 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.

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 beta-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 alpha(3)beta(1) 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 alpha(3)beta(1) integrins expressed on the surface of 1299 cells. Galectin-8 also interacts with other members of the integrin family, like alpha(6)beta(1) integrins, In contrast, galectin-8 only minimally interacts with alpha(4) or beta(3) 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-S 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-l. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-l; 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-I, 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-I 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 PHB as a positive regulator of IRS-l 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 reg

Tumor necrosis factor alpha (TNF alpha) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (Hi) 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 (amino acids 1245-1331) of IR expressed in bacteria as His(6) fusion peptides, Moreover, incubation of rat hepatoma Fao cells with TNF alpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-I 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 TNF alpha 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-S, which impairs their interaction with the JIVE 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 TNF alpha 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 TNF alpha 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.

Annexin II is a Ca2+-, phospholipid-, and actin- binding protein that was implicated in the regulation of vesicular traffic and endosome fusion. It is a known substrate for protein kinases including the platelet-derived growth factor receptor, src protein-tyrosine kinase, and protein kinase C. In the present study we investigated the possible involvement of annexin II in insulin signal transduction. Phosphorylation of annexin II in response to insulin treatment of intact Chinese hamster ovary (CHO)-T cells was detected by 5 min and reached maximal levels after a 2-3-h incubation with the hormone, However, unlike other receptor substrates, annexin II failed to undergo insulin induced Tyr phosphorylation under conditions where receptor internalization was inhibited. This was evident in CHO cells, overexpressing the insulin receptor, in which internalization was inhibited either by tyrosine kinase inhibitors or by lowering the temperature to 4 degrees C, and in CHO cells overexpressing various insulin receptor mutants in which normal internalization was impaired. Hence, Tyr phosphorylation of annexin II could be part of the internalization and sorting mechanism of the insulin receptor.

AG-18, an inhibitor of protein-tyrosine kinases, was employed to study the role of tyrosine-phosphorylated proteins in insulin- and phorbol ester-induced signaling cascades. When incubated with Chinese hamster ovary cells overexpressing the insulin receptor, AG-18 reversibly inhibited insulin-induced tyrosine phosphorylation of insulin receptor substrate-1, with minimal effects either on receptor autophosphorylation or on phosphorylation of Shc64, Under these conditions, AG-18 inhibited insulin-stimulated phosphorylation of the ribosomal protein S6, while no inhibition of insulin-induced activation of mitogen-activated protein kinase (MAPK) kinase or MAPK was detected, In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of MAPK kinase and MAPK and phosphorylation of S6 were inhibited by AG-18, This correlated with inhibition of TPA-stimulated tyrosine phosphorylation of several proteins, the most prominent ones being pp114 and pp120. We conclude that Tyr-phosphorylated insulin receptor substrate-1 is the main upstream regulator of insulin-induced S6 phosphorylation by p70(s6k), whereas MAPK signaling seems to be activated in these cells primarily through the adaptor molecule She. In contrast, TPA induced S6 phosphorylation is mediated by the MAPK/p90(rsk) cascade, A key element of this TPA-stimulated signaling pathway is an AG-18-sensitive protein-tyrosine kinase.

A protein of 35 kDa which has the characteristic properties of galectins (S-type lectins) was cloned from rat liver cDNA expression library. Since names for galectins 1-7 were already assigned, this new protein was named galectin-8. Three lines of evidence demonstrate that galectin-8 is indeed a novel galectin: (i) its deduced amino acid sequence contains two domains with conserved motifs that are implicated in the carbohydrate binding of galectins, (ii) in vitro translation products of galectin-8 cDNA or bacterially expressed recombinant galectin-8 are biologically active and possess sugar binding and hemagglutination activity, and (iii) a protein of the expected size (34 kDa) that binds to lactosyl-Sepharose and reacts with galectin-8-specific antibodies is present in rat liver and comprises similar to 0.025% of the total Triton X-100-soluble hepatic proteins. Overall, galectin-8 is structurally related (34% identity) to galectin-4, a soluble rat galectin with two carbohydrate-binding domains in the same polypeptide chain, joined by a link peptide. Nonetheless, several important features distinguish these two galectins: (i) Northern blot analysis revealed that, unlike galectin-4 that is confined to the intestine and stomach, galectin-8 is expressed in liver, kidney, cardiac muscle, lung, and brain; (ii) unlike galectin-4, but similar to galectins-1 and -2, galectin-8 contains 4 Cys residues; (iii) the link peptide of galectin-8 is unique and bears no similarity to any known protein; (iv) the N-terminal carbohydrate-binding region of galectin-8 contains a unique WG-E-I motif instead of the consensus WG-E-R/K motif implicated as playing an essential role in sugar-binding of all galectins. Together with galectin-4, galectin-8 therefore represents a subfamily of galectins consisting of a tandem repeat of structurally different carbohydrate recognition domains within a single polypeptide chain.

Insulin resistance is very common in the elderly, and may be associated with glucose intolerance or frank diabetes. In previous studies we demonstrated that insulin resistance in old Wistar rats is associated with decreased autophosphorylation and activation of the hepatic insulin receptor kinase (IRK) in vivo. We now show that this defect can be reproduced in vitro, where the extent of insulin-induced activation of IRK in liver membranes of old rats was decreased by similar to 50% compared with young controls. The defect could be largely abolished after solubilization of the membranes with Triton X-100. We also show that: (a) the viscosity of membranes from the old rats was significantly (P

H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate or H2O2 alone. We have previously shown that a combination of H2O2 and vanadate, when added to intact cells, rapidly stimulates protein tyrosine phosphorylation, owing to the inhibitory effects of these agents on intracellular protein tyrosine phosphatases (PTPases). Employing Western blotting with anti-phosphotyrosine antibodies, we have now identified in Chinese-hamster ovary (CHO) cells transfected with a wild-type insulin-receptor gene (CHO. T cells) several proteins (e.g. pp 180, 125, 100, 60 and 52) whose phosphotyrosine content is rapidly increased upon treatment of the cells with a combination of insulin and 3 mm-H2O2. Tyrosine phosphorylation of these and additional proteins was further potentiated when 100 muM-sodium orthovanadate was added together with H2O2. The effects of insulin, insulin/H2O2, and H2O2/vanadate on tyrosine phosphorylation were markedly decreased in CHO cells transfected with an insulin-receptor gene where the twin tyrosines 1162 and 1163 were replaced with phenylalanine (CHO. YF-3 cells). Similarly, most of these proteins failed to undergo enhanced tyrosine phosphorylation in parental CHO cells incubated in the presence of insulin or the insulinomimetic agents. Our findings suggest that inhibition of PTPase activity by H2O2/vanadate augments the autophosphorylation of tyrosines 1162 and 1163 of the insulin receptor kinase, leading to its activation in an insulin-independent manner. As a result, tyrosine phosphorylation of potential targets for this enzyme takes place. Failure of H2O2/vanadate to induce phosphorylation of these proteins in receptor mutants lacking these twin tyrosine residues supports this hypothesis.

The major cytosolic substrate of the insulin receptor is a 185-kDa phosphoprotein (IRS-1) that contains multiple putative attachment sites for the p85-alpha regulatory subunit of phosphatidylinositol 3'-kinase (PI3K). To examine the possible interaction of pp185 with p85-alpha in vivo, we injected insulin or insulinomimetic agents (a combination of H2O2 and vanadate (H/V)) into the portal vein of anesthetized rats. In this model system, H/V treatment and, to a lesser extent, injection of insulin resulted in rapid and sustained tyrosine phosphorylation of multiple cellular proteins, including pp185/IRS-1. The latter was found to undergo specific association with the p85-alpha regulatory subunit of PI3K but not with two other proteins that contain src homology domains. As p85-alpha was not detectably phosphorylated on tyrosine residues and did not appear to interact directly with the insulin receptor, we conclude that tyrosine phosphorylation of pp185 promotes its association with p85-alpha and the catalytic subunit of PI3K. The recruitment of the holoenzyme may also involve its enzymatic activation and thus constitute an important step in the transduction of insulin signals.

Aging is associated with a postbinding defect in insulin action, leading to increased glucose intolerance and occasional diabetes. To determine whether defects in insulin receptor kinase (IRK) activity or in the phosphorylation of its physiological substrates underlie this age-related phenomenon, young (2-3 months old) and old (24-27 months old) Wistar rats were studied. When assayed in vitro, the hepatic IRK activities of noninjected old and young rats were comparable. Thirty seconds after the injection of insulin, the hepatic IRK activity of young rats increased 7- to 10-fold in a dose-dependent manner, with maximal effects obtained in rats injected with 20 mg insulin. By contrast, old animals exhibited impaired in vivo activation, with a mean 50% reduction in maximal IRK activity. When the rats were grouped into animals with mild (20%), moderate (50%), and severe (80%) reductions in maximal IRK activity, it was found that the mild and moderate defects could be reversed once the receptors were subjected to extensive autophosphorylation in vitro. The severe form of the defect was essentially irreversible and could not be corrected by phosphorylation in vitro. Immunoblotting with anti P-Tyr antibodies revealed that the reduced IRK activity in the old animals correlated with reduced intrahepatic tyrosine phosphorylation of the beta-subunit of the insulin receptor and pp180, a putative substrate of IRK. We, therefore, conclude that glucose intolerance in aging could be attributed at least in part to acquired defects in the in vivo activation of the hepatic IRK, which results in reduced phosphorylation of its putative substrate pp180.

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 1,25(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 1,25(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.

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.

We have previously reported that the GDP-bound alpha-subunit of the GTP-binding protein transducin, present in outer segments of retinal rod cells (ROS), serves as a high affinity in vitro substrate (Km = 1 microM) for the insulin receptor kinase. The present study demonstrates that transducin also serves as in vitro substrate for an endogenous IGF-I receptor kinase isolated from ROS membranes. The presence of insulin-like growth factor I (IGF-I) receptors in ROS is evident from the high affinity and specific binding of 125I-IGF-I to ROS membranes (Kd = 3 nM) which contain 110 fmol of IGF-I binding sites/mg of membrane protein. Furthermore, cross-linking of 125I-IGF-I labels the 135-kDa alpha-subunit of this receptor. 125I-Insulin binding capacity to ROS membranes is less than 5% that of IGF-I. The IGF-I-stimulated tyrosine kinase activity in solubilized and partially purified receptors from ROS autophosphorylates its own 95-kDa beta-subunits as well as other substrates like transducin. Insulin, which is 200-fold less potent than IGF-I in competing for 125I-IGF-I binding, is only 5-fold less potent than IGF-I in stimulating the receptor kinase activity. This suggests that insulin is much more potent than IGF-I in coupling ligand binding with kinase activation. The previously reported presence of IGF-I in the vitreous, together with our present studies, strongly suggest that the IGF-I receptor kinase, through phosphorylation of endogenous proteins like transducin, could play a role in mediating transmembrane signal transduction in ROS.

Purified polyclonal human antibodies (B-8) against the receptor for insulin (anti-R IgG), and their F(ab')2 and Fab' fragments, were used to study a possible role of receptor aggregation in the process that couples insulin binding with the activation of the insulin receptor kinase. Anti-R IgG, F(ab')2, and Fab' fragments were shown to inhibit insulin binding to solubilized partially purified receptor preparations from rat liver. This suggests that the antibodies and fragments bind near or at the insulin-binding site. Only anti-R IgG and its bivalent F(ab')2 fragments were capable of stimulating the receptor kinase activity. Monovalent Fab' fragments were completely devoid of such activity. Cross-linking of anti-R Fab' with goat anti-human Fab' restored the capability of the Fab' fragments to activate the receptor kinase. These data strongly suggest that receptor cross-linking or aggregation constitutes a sufficient trigger to activate the insulin-receptor kinase and could, therefore, be an important step in the transmembrane signaling process. This step presumably precedes the activation of the receptor kinase and the resulting phosphorylation of its protein substrates.