JC virus large T-antigen and IGF-I signaling system merge to affect DNA repair and genomic integrity

The progression of cancer is often associated with genomic instability, which may develop as a result of compromised defense mechanisms responsible for the maintenance of chromosomal integrity. These include defects in telomere preservation, chromosomal segregation, and DNA repair. In this review, we discuss molecular interactions between viral and cellular signaling components, which interfere with DNA repair mechanisms, and possibly contribute to the development of a mutagenic phenotype. Our studies indicate that large T-antigen from the human polyomavirus JC (JCV T-antigen) inhibits homologous recombination directed DNA repair (HRR)-causing accumulation of mutations in the affected cells (JCP 2005, in press). Surprisingly, T-antigen does not operate directly, but utilizes insulin receptor substrate 1 (IRS-1), which is the major signaling molecule for insulin-like growth factor I receptor (IGF-IR). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with an artificial nuclear localization signal. The interplay described between the IGF-IR signaling system and JCV T-antigen in the process of DNA repair could be relevant, since nearly 90% of the human population is seropositive for JC virus, JCV T-antigen transforms cells in vitro, is tumorigenic in experimental animals, and the presence of JC virus has been shown in an increasing number of biopsies of human cancer.     

Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by beta1 integrins

We show here that beta1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The beta1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, beta1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, beta1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the beta1 cytodomain plays an important role in mediating beta1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, beta1A associates with IRS-1 and beta1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.     

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.     

T-antigen of the human polyomavirus JC attenuates faithful DNA repair by forcing nuclear interaction between IRS-1 and Rad51

JC polyomavirus (JCV), which infects 90% of the human population, is detectable in human tumors. Its early protein, JCV T-antigen, transforms cells in vitro and is tumorigenic in experimental animals. Although T-antigen-mediated transformation involves genetic alterations of the affected cells, the mechanism underlying this genomic instability is not known. We show that JCV T-antigen inhibits homologous recombination DNA repair (HRR), which results in an accumulation of mutations. T-antigen does not operate directly but utilizes a cytosolic molecule, insulin receptor substrate 1 (IRS-1). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with artificial nuclear localization signal. Our observations define a new mechanism by which viral protein utilizes cytosolic molecule to inhibit faithful DNA repair, and suggest how polyomaviruses could compromise stability of the genome. (c) 2005 Wiley-Liss, Inc.     

Inhibition of ERβ induces resistance to cisplatin by enhancing Rad51-mediated DNA repair in human medulloblastoma cell lines

Cisplatin is one of the most widely used and effective anticancer drugs against solid tumors including cerebellar tumor of the childhood, Medulloblastoma. However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug. In this study, we demonstrate that human medulloblastoma cell lines develop acute resistance to cisplatin in the presence of estrogen receptor (ER) antagonist, ICI182,780. This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation. We have previously reported that ERβ, which is highly expressed in medulloblastomas, translocates insulin receptor substrate 1 (IRS-1) to the nucleus, and that nuclear IRS-1 binds to Rad51 and attenuates homologous recombination directed DNA repair (HRR). Here, we demonstrate that in the presence of ICI182,780, cisplatin-treated medulloblastoma cells show recruitment of Rad51 to the sites of damaged DNA and increase in HRR activity. This enhanced DNA repair during the S phase preserved also clonogenic potential of medulloblastoma cells treated with cisplatin. In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin-induced cytotoxicity in human medulloblastoma cell lines.     

The Reciprocal Regulation of γ-Synuclein and IGF-I Receptor Expression Creates a Circuit That Modulates IGF-I Signaling

Insulin-like growth factor (IGF) system plays important roles in carcinogenesis and maintenance of the malignant phenotype. Signaling through the IGF-I receptor (IGF-IR) has been shown to stimulate the growth and motility of a wide range of cancer cells. γ-Synuclein (SNCG) is primarily expressed in peripheral neurons but also overexpressed in various cancer cells. Overexpression of SNCG correlates with tumor progression. In the present study we demonstrated a reciprocal regulation of IGF-I signaling and SNCG expression. IGF-I induced SNCG expression in various cancer cells. IGF-IR knockdown or IGF-IR inhibitor repressed SNCG expression. Both phosphatidylinositol 3-kinase and mitogen-activated protein kinase were involved in IGF-I induction of SNCG expression. Interestingly, SNCG knockdown led to proteasomal degradation of IGF-IR, thereby decreasing the steady-state levels of IGF-IR. Silencing of SNCG resulted in a decrease in ligand-induced phosphorylation of IGF-IR and its downstream signaling components, including insulin receptor substrate (IRS), Akt, and ERK1/2. Strikingly, SNCG physically interacted with IGF-IR and IRS-2. Silencing of IRS-2 impaired the interaction between SNCG and IGF-IR. Finally, SNCG knockdown suppressed IGF-I-induced cell proliferation and migration. These data reveal that SNCG and IGF-IR are mutually regulated by each other. SNCG blockade may suppress IGF-I-induced cell proliferation and migration. Conversely, IGF-IR inhibitors may be of utility in suppressing the aberrant expression of SNCG in cancer cells and thereby block its pro-tumor effects.     

RACK1, an insulin-like growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with extracellular matrix

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gbeta homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and beta1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible beta1 integrin-associated kinase activity and association of Crk with p130(CAS) were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G(1) or G(1)/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21(Cip1/WAF1) and p27(Kip1), and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.     

Growth hormone receptor gene deficiency causes delayed insulin responsiveness in skeletal muscles without affecting compensatory islet cell overgrowth in obese mice

Growth hormone (GH), acting through its receptor (GHR), is essential for somatic growth and development and maintaining metabolic homeostasis. GHR gene-deficient (GHR(-/-)) mice exhibit drastically diminished insulin-like growth factor-I (IGF-I) levels, proportional growth retardation, elevated insulin sensitivity, and reduced islet beta-cell mass. Unlike the liver, which is mostly unaffected by changes in IGF-I level, skeletal muscles express high levels of IGF-I receptor (IGF-IR). The net result of a concurrent deficiency in the actions of both GH and IGF-I, which exert opposite influences on insulin responsiveness, has not been evaluated. We studied insulin-stimulated early responses in the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and p85 subunit of phosphatidylinositol 3-kinase. Upon in vivo insulin stimulation, skeletal muscles of GHR(-/-) mice exhibit transient delayed responses in IR and IRS-1 phosphorylation but normal levels of p85 association with IRS-1. This is in contrast to normal/elevated insulin responses in hepatocytes and indicates tissue-specific effects of GHR gene deficiency. In addition to stimulating normal islet cell growth, GH may participate in islet cell overgrowth, which compensates for insulin resistance induced by obesity. To determine whether the islet cell overgrowth is dependent on GH signaling, we studied the response of male GHR(-/-) mice to high-fat diet (HFD)-induced obesity. After 17 wk on a HFD, GHR(-/-) mice became more significantly obese than wild-type mice and exhibited increased beta-cell mass to a slightly higher extent. These data demonstrate that GH signaling is not required for compensatory islet growth. Thus, in both muscle insulin responsiveness and islet growth compensation, normal levels of GH signals do not seem to play a dominant role.     

Insulin receptor substrate-1, p70S6K, and cell size in transformation and differentiation of hemopoietic cells

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70(S6K), which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70(S6K) and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70(S6K) seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70(S6K) in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70(S6K) activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.     

Role of pescadillo and upstream binding factor in the proliferation and differentiation of murine myeloid cells

Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.     

Decorin antagonizes IGF receptor I (IGF-IR) function by interfering with IGF-IR activity and attenuating downstream signaling

We have recently discovered that the insulin-like growth factor receptor I (IGF-IR) is up-regulated in human invasive bladder cancer and promotes migration and invasion of transformed urothelial cells. The proteoglycan decorin, a key component of the tumor stroma, can positively regulate the IGF-IR system in normal cells. However, there are no available data on the role of decorin in modulating IGF-IR activity in transformed cells or in tumor models. Here we show that the expression of decorin inversely correlated with IGF-IR expression in low and high grade bladder cancers (n = 20 each). Decorin bound with high affinity IGF-IR and IGF-I at distinct sites and negatively regulated IGF-IR activity in urothelial cancer cells. Nanomolar concentrations of decorin promoted down-regulation of IRS-1, one of the critical proteins of the IGF-IR pathway, and attenuated IGF-I-dependent activation of Akt and MAPK. This led to decorin-evoked inhibition of migration and invasion upon IGF-I stimulation. Notably, decorin did not cause down-regulation of the IGF-IR in bladder, breast, and squamous carcinoma cells. This indicates that decorin action on the IGF-IR differs from its known activity on other receptor tyrosine kinases such as the EGF receptor and Met. Our results provide a novel mechanism for decorin in negatively modulating both IGF-I and its receptor. Thus, decorin loss may contribute to increased IGF-IR activity in the progression of bladder cancer and perhaps other forms of cancer where IGF-IR plays a role.     

Differential regulation of insulin receptor substrate-1 degradation during mannitol and okadaic acid induced apoptosis in human neuroblastoma cells

Insulin receptor substrate (IRS) proteins are major docking molecules for the type I insulin like growth factor (IGF) receptor (IGF-IR) and mediate their effects on downstream signaling molecules. In this report, we investigated IRS-1 regulation during apoptosis in human neuroblastoma SH-EP cells. Treatment of SH-EP cells with mannitol or okadaic acid (OA) induces apoptosis with the typical characteristics of anoikis. Mannitol treatment results in IRS-1 degradation with concomitant appearance of smaller fragments, likely representing caspase cleavage products. In contrast OA-induced IRS-1 degradation is accompanied by a mobility shift in IRS-1, suggesting IRS-1 serine/threonine phosphorylation. Mannitol-induced, but not OA-induced, degradation is blocked by IGF-I. Pretreatment of the cells with caspase or proteasome inhibitors also partially blocks mannitol-induced IRS-1 degradation. These results suggest two independent pathways are involved in IRS-1 degradation; one pathway is dependent on caspase activation and is blocked by IGF-I, while a second pathway is caspase-independent and IGF-I-insensitive.     

Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2.

Activation of insulin-like growth factor I receptor (IGF-IR) kinase is an important site of control of IGF-I-linked intracellular signaling pathways. One potentially important regulatory variable is IGF-IR dephosphorylation. It has been shown that SHP-2, a tyrosine phosphatase, can bind to the activated IGF-IR in vitro; however, its role in IGF-IR dephosphorylation in whole cells is unknown. These studies were undertaken to determine whether SHP-2 was a candidate for mediating IGF-IR dephosphorylation. The IGF-IR in smooth muscle cells was dephosphorylated rapidly beginning 10 min after ligand addition, and this was temporally associated with SHP-2 binding to the receptor. IGF-I stimulated SHPS-1 phosphorylation and the subsequent recruitment of SHP-2. In cells expressing a SHPS-1 mutant that did not bind SHP-2 there was no recruitment of SHP-2 to the IGF-IR. Cells expressing a catalytically inactive form of SHP-2 showed SHP-2 recruitment to SHPS-1, but this did not result in SHPS-1 dephosphorylation, and there was a prolonged IGF-IR phosphorylation response after IGF-I stimulation. These studies indicate that IGF-IR stimulates phosphorylation of SHPS-1 which is critical for SHP-2 recruitment to the plasma membrane and for its recruitment to the IGF-IR. Recruitment of SHP-2 to the receptor then results in receptor dephosphorylation. The regulation of this process may be an important determinant of IGF-IR-mediated signaling.     

The Rad51 paralog Rad51B promotes homologous recombinational repair

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.     

Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment.     

Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling

Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser(302) to study IRS-1-mediated signaling during insulin and insulin-like growth factor IGF-I stimulation. Insulin or IGF-I stimulated phosphorylation of Ser(302) in various cell backgrounds and in murine muscle. Wortmannin or rapamycin inhibited Ser(302) phosphorylation, and amino acids or glucose stimulated Ser(302) phosphorylation, suggesting a role for the mTOR cascade. The Ser(302) kinase associates with IRS-1 during immunoprecipitation, but its identity is unknown. The NH(2)-terminal c-Jun kinase did not phosphorylate Ser(302). Replacing Ser(302) with alanine significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and p85 binding and reduced insulin-stimulated phosphorylation of p70(S6K), ribosomal S6 protein, and 4E-BP1; however, this mutation had no effect on insulin-stimulated Akt or glycogen synthase kinase 3beta phosphorylation. Replacing Ser(302) with alanine reduced insulin/IGF-I-stimulated DNA synthesis. We conclude that Ser(302) phosphorylation integrates nutrient availability with insulin/IGF-I signaling to promote mitogenesis and cell growth.     

Insulin receptor substrate (IRS)-2, not IRS-1, protects human neuroblastoma cells against apoptosis

Insulin receptor substrates (IRS)-1 and -2 are major substrates of insulin and type I insulin-like growth factor (IGF-I) receptor (IGF-IR) signaling. In this study, SH-EP human neuroblastoma cells are used as a model system to examine the differential roles of IRS-1 and IRS-2 on glucose-mediated apoptosis. In the presence of high glucose, IRS-1 underwent caspase-mediated degradation, followed by focal adhesion kinase (FAK) and Akt degradation and apoptosis. IRS-2 expression blocked all these changes whereas IRS-1 overexpression had no effect. In parallel, IRS-2, but not IRS-1, overexpression enhanced IGF-I-mediated Akt activation without affecting extracellular regulated kinase signaling. While IRS-1 was readily degraded by caspases, hyperglycemia-mediated IRS-2 degradation was unaffected by caspase inhibitors but blocked by proteasome and calpain inhibitors. Our data suggest that the differential degradation of IRS-1 and IRS-2 contributes to their distinct modes of action and the increased neuroprotective effects of IRS-2 in this report are due, in part, to its resistance to caspase-mediated degradation.