Insulin-Like Growth Factor I Induces Preferential Degradation of Insulin Receptor Substrate-2 through the Phosphatidylinositol 3-Kinase Pathway in Hum
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《内分泌学杂志》
University of Michigan, Department of Neurology Ann Arbor, Michigan 48109
Abstract
Insulin receptor substrate (IRS) signaling is regulated through serine/threonine phosphorylation, with subsequent IRS degradation. This study examines the differences in IRS-1 and IRS-2 degradation in human neuroblastoma cells. SH-EP cells are glial-like, express low levels of the type I IGF-I receptor (IGF-IR) and IRS-2 and high levels of IRS-1. SH-SY5Y cells are neuroblast-like, with high levels of IGF-IR and IRS-2 but virtually no IRS-1. When stimulated with IGF-I, IRS-1 expression remains constant in SH-EP cells; however, IRS-2 in SH-SY5Y cells shows time- and concentration-dependent degradation, which requires IGF-IR activation. SH-EP cells transfected with IRS-2 and SH-SY5Y cells transfected with IRS-1 show that only IRS-2 is degraded by IGF-I treatment. When SH-EP cells are transfected with IGF-IR or suppressor of cytokine signaling, IRS-1 is degraded by IGF-I treatment. IRS-1 and -2 degradation are almost completely blocked by phosphatidylinositol 3-kinase inhibitors and partially by proteasome inhibitors. In summary, 1) IRS-2 is more sensitive to IGF-I-mediated degradation; 2) IRS degradation is mediated by phosphatidylinositol 3-kinase and proteasome sensitive pathways; and 3) high levels of IGF-IR, and possibly the subsequent increase in Akt phosphorylation, are required for efficient IRS degradation.
Introduction
IGF-I AND INSULIN regulate growth and metabolism in many cell types (1). Insulin and IGF-I stimulate the tyrosine autophosphorylation of receptor tyrosine kinases. Autophosphorylation of the IGF-I receptor (IGF-IR) or insulin receptor (IR) subsequently initiates the binding of specific docking molecules to the phosphorylated receptor. One such docking molecule is insulin receptor substrate (IRS), which binds to phosphotyrosine (pTyr) residues on receptor -subunits. Receptor-IRS binding is required for insulin and IGF-I-mediated signal transduction (2). Four members of this family have been identified (IRS-1 through -4), each containing characteristic N-terminal pleckstrin homology and pTyr binding domains (3). IRS proteins lack intrinsic kinase activity; however, upon binding to activated receptors, IRS proteins are tyrosine phosphorylated and further bind downstream signaling molecules, including the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K), Fyn, Grb2, Nck, and Shb (2, 4, 5).
Along with many tyrosine residues, IRS-1 and IRS-2 also have more than 30 serine/threonine phosphorylation sites. Serine/threonine phosphorylation of IRS-1 and -2 prevents tyrosine phosphorylation, thereby inhibiting the ability of insulin or IGF-I to stimulate subsequent downstream signaling (6, 7). Multiple proteins have been suggested as possible serine/threonine kinases for IRS-1 and -2, including c-Jun N-terminal kinase (8), the inhibitor B kinase complex (9), Rho kinase (10), protein kinase C (11), and the PI 3-K/Akt/mammalian target of rapamycin (mTOR) pathway (12, 13). Once serine/threonine phosphorylated, the IRS proteins are degraded via the ubiquitin-proteasome degradation pathway (14, 15). Ubiquitin-proteasome-mediated degradation regulates a variety of cellular functions including cell cycle control, gene transcription, and apoptosis (16, 17). Inhibition of proteasome activation by MG132 or lactacystin prevents insulin or IGF-I-induced degradation of IRS-1 and IRS-2 (15, 18, 19). IRS degradation is also blocked by the inhibition of PI 3-K by LY294002 or its downstream effector mTOR by rapamycin (12). Furthermore, expression of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10), which antagonizes PI 3-K, up-regulates IRS-2 expression in breast cancer cells (20). These results suggest that PI 3-K mediated pathways involving Akt and mTOR are responsible for the proteasome-mediated degradation of IRS proteins.
The suppressor of cytokine signaling (SOCS) family of proteins is implicated in the regulation of insulin and IGF-I signaling. SOCS family members (SOCS1 through SOCS7 and CIS) target proteins for ubiquitination and subsequent degradation by the proteasome (21). SOCS-1, -2, and -3 bind both the IR and IGF-IR (22, 23, 24) and inhibit their signaling. Proteasome-mediated degradation of IRS-1 and -2 requires SOCS-1 and -3 in some cell types. Expression of SOCS-1 or -3 in primary cultured hepatocytes or targeted overexpression in liver by adenoviral-mediated gene transfer in mice induces IRS-1 and IRS-2 degradation (25, 26). In contrast, expression of mutant SOCS-1, which cannot bind ubiquitin ligase fails to induce IRS protein degradation (26).
Relatively few reports investigate IRS-2 degradation (19, 26, 27). Differential regulation of IRS-2 degradation occurs depending on cell type. For instance, in Chinese hamster ovary (CHO) cells expressing IR (CHO/IR) cells transfected with IRS-1 or IRS-2, only IRS-1 is degraded upon insulin treatment, and the N-terminal region of IRS-1 is responsible for its degradation by the proteasome/ubiquitin pathway (14). In contrast, IRS-2, but not IRS-1, is degraded upon insulin or IGF-I treatment in 3T3-L1 preadipocytes and MEF cells (19). Interestingly, when 3T3-L1 preadipocytes are differentiated to adipocytes, IRS-1 is then degraded by insulin treatment (19). These reports suggest that IRS protein degradation depends upon the cell type used and/or differentiation status. However, there are virtually no reports concerning the regulation of IRS degradation in neuronal cells. In our laboratory, we have been studying IGF-I signaling in primary and transformed neuronal cells for over 15 yr. Our data suggest that neuronal cells respond to IGF-I in a unique manner that cannot be predicted by IGF-I mediated signaling in other cell types (28).
In this report, we compare IRS-1 and IRS-2 degradation in response to IGF-I in human neuroblastoma cells. We have previously shown that more neuroblast-like N-type neuroblastoma cells (e.g. SH-SY5Y cells) have increased expression of the IGF-IR compared with more glial like S cells (e.g. SH-EP cells) (29). Interestingly S cells exclusively express IRS-1 that undergoes sustained phosphorylation by IGF-I, whereas N cells express IRS-2 that is transiently phosphorylated by IGF-I (29). The pattern of IRS expression holds true in primary cells from the peripheral nervous system as well; Schwann cells express IRS-1 (30), whereas spinal cord motor neurons express IRS-2 (31). We report here that IGF-I stimulation of SH-SY5Y cells results in the degradation of IRS-2; in contrast IGF-I treatment does not affect IRS-1 expression levels in SH-EP cells. Inhibition of the PI 3-K pathway completely blocks IGF-I-induced IRS-2 degradation, and IRS-2 degradation is tightly correlated with changes in Akt phosphorylation, indicating regulation through the PI 3-K pathway. Inhibition of the proteosome using proteosome inhibitors or inhibition of IGF-IR activation by the IGF-IR kinase-specific inhibitor NVP-AEW541 prevents IGF-I-induced IRS-2 degradation. When IRS-2 is transfected into SH-EP cells, IGF-I stimulation results in the degradation of IRS-2; however, when IRS-1 is transfected into SH-SY5Y cells, expression is not changed by IGF-I treatment. Both IGF-IR overexpression and SOCS-1 transfection in SH-EP cells result in IGF-I-induced IRS-1 degradation, but over a longer time course than IRS-2 degradation. Based upon these data, we conclude that IRS-2 is more sensitive to IGF-I-induced degradation. Furthermore, high IGF-IR expression is required for the efficient assembly of degradation machinery, an event which requires PI 3-K/Akt and proteasome activation.
Materials and Methods
Materials
Anti-IRS-1, anti-IRS-2, anti-IGF-IR subunit, and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Shc antibody was purchased from Transduction Laboratory (Lexington, KY). Anti-pTyr and anti-EGF (epidermal growth factor) receptor antibodies and antibodies against phosphorylated forms of Akt and ERK1/2 were from Cell Signaling Technology (Beverly, MA). All inhibitors and EGF were purchased from Calbiochem (La Jolla, CA). IGF-IR kinase inhibitor, NVP-AEW541, was provided by Novartis Institute of Biomedical Research, Inc. (Cambridge, MA). Recombinant human IGF-I was kindly provided by Cephalon, Inc. (West Chester, PA) and des(1–3)IGF-I was purchased from GroPrep (Adelaide, Australia). IRS-1 and IRS-2 containing vectors were kindly provided by Dr. D. Yee of the University of Minnesota (15). SOCS-1 in pcDNA vector was provided by Dr. L. Rui of the University of Michigan (26).
Cell culture
SH-SY5Y and SH-EP human neuroblastoma cells were maintained in DMEM containing 10% calf serum. IRS-1, IRS-2, and SOCS-1 containing vectors or empty pcDNA vectors were transfected into neuroblastoma cells using Lipofectamine 2000 reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. SH-EP cells transfected with pSFFV or IGF-IR were described previously (32). Transfected cells were maintained in DMEM containing 10% calf serum and 0.25 mg/ml G418 (Invitrogen Life Technologies). The cells were serum starved 4–6 h before treatment. The inhibitors were added 1 h before IGF-I treatment.
Immunoprecipitation and immunoblotting
Western immunoblotting and immunoprecipitation were performed as described previously (33). All experiments were repeated at least three times. Typical representative results are presented in the figures.
Results
IRS-2, but not IRS-1, is degraded by IGF-I treatment in neuroblastoma cells
We have previously reported the detailed analysis of IGF-I:IGF-IR signaling among N and S type neuroblastoma cells (29) including differential expression of IGF-IR, IRS-1, and IRS-2. In this report we investigate the regulation of IRS-1 and -2, the major signaling molecules of IGF-IR, in more detail. First, serum-starved SH-EP and SH-SY5Y human neuroblastoma cells are treated with 10 nM IGF-I for 0–2 h. Equal amounts of whole-cell lysates are immunoblotted with anti-pTyr antibody (Fig. 1A). In agreement with our previous results (29, 34) treatment of the cells with 10 nM IGF-I results in strong tyrosine phosphorylation of a 97-kDa protein in SH-SY5Y cells, which represents IGF-IR (Fig. 1A, upper panel). We cannot detect IGF-IR phosphorylation in SH-EP cells due to very low expression of IGF-IR (Fig. 1A, middle panel). The protein levels of IRS-1 in SH-EP cells are not changed with IGF-I treatment. In contrast, IRS-2 in SH-SY5Y cells shows a time-dependent mobility shift along with a decrease in its expression level (Fig. 1A, lower panel). Various studies have suggested that these changes represent serine/threonine phosphorylation and degradation of IRS proteins (6, 7). As can be seen in Fig. 1, A and B, the degradation of IRS-2 starts within 30 min of IGF-I treatment and continued up to 2 h. IRS-2 degradation is also dependent on IGF-I concentration. IGF-I treatment results in the concentration-dependent tyrosine phosphorylation of IGF-IR, beginning at 1 nM (Fig. 1C, top panel). Interestingly, the mobility shift and degradation of IRS-2 starts at a much lower concentration, as low as 1 pM IGF-I (Fig. 1C, middle panel). IGF-IR protein expression is not changed (Fig. 1C, bottom panel).
Several investigators reported an interaction between IGF-IR and EGF receptor (EGFR) signaling (35, 36). We have shown that EGF treatment results in ERK1/2 phosphorylation in SH-SY5Y cells (37). Anti-EGFR immunoblotting reveals that both SH-EP and SH-SY5Y cells express comparable amounts of EGFR (Fig. 2A). Similar to IGF-I treatment, EGF stimulation results in IRS-2 degradation in SH-SY5Y cells but does not affect IRS-1 expression in SH-EP cells (Fig. 2B). Unlike IGF-I treatment, however, EGF-induced IRS-2 degradation is transient; IRS-2 expression decreases at 30 min, then returns to basal levels at 2 h. We confirmed that IGF-I-mediated IRS-2 degradation is not mediated by EGFR by using the EGFR kinase-specific inhibitor AG1478 (38). EGF-induced IRS-2 degradation is blocked by the pretreatment of the cells with 0.25 μM AG1478; however, AG1478 has no effect on IGF-I-induced IRS-2 degradation (Fig. 2C).
IRS-2 degradation correlates with the activation of PI 3-K pathway
Next we investigated signaling pathways downstream from the IGF-IR involved in IRS-2 degradation. For this experiment, we used pharmacological inhibitors of ERK1/2 (PD98059), PI 3-K (LY294002), and proteasome (MG132) pathways (Fig. 3A). Treatment of SH-SY5Y cells with PD98059 completely blocks ERK1/2 phosphorylation but has no effect on IGF-I-induced IRS-2 degradation. In contrast, LY294002 almost completely blocks both the degradation and mobility shift of IRS-2. The proteasome inhibitor MG132 slightly prevents IRS-2 degradation but does not inhibit the observed mobility shift. Other proteasome inhibitors, such as epoxomycin and PS1, have similar effects (data not shown). These results suggest that IGF-I-induced IRS-2 degradation is mediated by both PI 3-K and proteasome-dependent pathways.
One of the major signaling molecules downstream of PI 3-K is Akt (39). Akt is directly involved in IRS degradation in Fao hepatocytes (40). In our system, Akt is highly phosphorylated in SH-SY5Y cells; there is, however, only weak phosphorylation of Akt in SH-EP cells (Fig. 3B). IRS-2 degradation and Akt phosphorylation show similar concentration-dependent changes by IGF-I (Fig. 3C). Similar to changes in IRS-2 degradation (Fig. 1C) Akt phosphorylation starts as low as 1 pM IGF-I treatment (Fig. 3C). In contrast, ERK1/2 phosphorylation begins at concentrations of 0.1 nM IGF-I. The time course of Akt phosphorylation by IGF-I and EGF is also consistent with IRS-2 degradation. IGF-I induces sustained phosphorylation of Akt, whereas EGF-induced Akt phosphorylation is transient (Fig. 3D). Therefore, Akt phosphorylation status and IRS-2 degradation are strongly correlated, supporting a potential role for Akt in IRS-2 degradation.
IGF-IR activation is required for IGF-I-induced IRS-2 degradation and Akt phosphorylation
To confirm the requirement of IGF-IR activation for IRS-2 degradation in SH-SY5Y cells, we used NVP-AEW541, a potent and specific inhibitor of IGF-IR kinase activity (41). SH-SY5Y cells are incubated for 1 h with increasing concentrations of NVP-AEW541 and then treated with 10 nM IGF-I for 30 min. NVP-AEW541 treatment results in concentration-dependent inhibition of the tyrosine phosphorylation of a 97-kDa protein (Fig. 4A, upper panel). To confirm that this 97-kDa protein is IGF-IR, we immunoprecipitated IGF-IR followed by anti-pTyr immunoblotting (Fig. 4A, bottom panel). Once again, NVP-AEW541 prevents tyrosine phosphorylation in a concentration-dependent fashion (Fig. 4A, bottom panel). Inhibition of IGF-IR activation by NVP-AEW541 also prevents IRS-2 degradation in a concentration-dependent manner (Fig. 4B). NVP-AEW541 alone has no effect on IGF-IR phosphorylation, IGF-I-mediated IRS-2 degradation, and EGF-induced IRS-2 degradation (Fig. 4B). Expression of Shc, another major signaling molecule of IGF-IR, is not affected by any treatment (Fig. 4B). To exclude the possibility of IGF binding protein involvement, we treated cells with desIGF-I, which has very low affinity for the IGF binding proteins (42). Treatment of SH-SY5Y cells with 10 nM desIGF-I has a similar effect on IRS-2 degradation as IGF-I (Fig. 4C). NVP-AEW541 also induced concentration-dependent inhibition of Akt phosphorylation (Fig. 4D), which correlates well with IRS-2 degradation. Total Akt expression is not affected by NVP-AEW541 treatment (Fig. 4D, bottom panel).
Differential degradation of IRS-1 and -2
The results described above illustrate differential regulation of IRS-1 and IRS-2 degradation upon IGF-I treatment. This differential regulation is due to either intrinsic differences between the IRS-1 and IRS-2 proteins or differences in the cell types used. We therefore transfected IRS-2 into SH-EP cells (SH-EP/IRS-2) and IRS-1 into SH-SY5Y cells (SH-SY5Y/IRS-1) to attempt to distinguish between these two possibilities, using an empty pcDNA vector (SH-EP/pcDNA and SH-SY5Y/pcDNA) transfection as a control. IRS-2 transfection does not affect the basal or IGF-I-induced expression level of endogenous IRS-1 level in SH-EP/IRS-2 cells (Fig. 5A). When SH-EP/IRS-2 cells are treated with 10 nM IGF-I there is a time-dependent degradation of IRS-2 similar to that seen in SH-SY5Y cells. IRS-1 transfection does not affect the basal expression level of IRS-2 in SH-SY5Y cells, and IGF-I treatment results in IRS-2 degradation in both vector and IRS-1 transfected cells (Fig. 5B). In contrast, IGF-I has little effect on IRS-1 expression in SH-SY5Y/IRS-1 cells. IGF-IR and Shc expression remains unchanged by IRS transfection and IGF-I treatment in all cell types. The tyrosine phosphorylation of IRS-1 and IRS-2 in both cell types follows similar patterns as the protein levels. These results demonstrate that IRS-2 degradation occurs regardless of the cell type; therefore, there are likely intrinsic differences within the IRS-1 and IRS-2 proteins affecting the IGF-I-induced degradation.
Next, we examined the effect of increased IGF-IR expression on IRS-1 degradation in SH-EP cells. SH-EP or vector (pSFFV) transfected SH-EP (SH-EP/pSFFV) cells express about one fifth of the IGF-IR detected in SH-SY5Y cells (Fig. 6A). When transfected with IGF-IR, SH-EP/IGF-IR cells express about 15 times more receptor compared with SH-EP/pSFFV cells and three times more than SH-SY5Y cells. SH-EP/IGF-IR cells show increased IGF-IR tyrosine phosphorylation upon IGF-I exposure compared with the weak phosphorylation detected in SH-EP/pSFFV cells (Fig. 6B, upper panel). SH-EP/IGF-IR cells also have increased basal phosphorylation of IGF-IR. Consistent with the activation of IGF-IR, basal and IGF-I-induced Akt phosphorylation is increased in SH-EP/IGF-IR cells (Fig. 6B, middle panel). Compared with SH-EP/pSFFV, SH-EP/IGF-IR cells show reduced basal expression of IRS-1 (Fig. 6C). When treated with 10 nM IGF-I, IRS-1 is further degraded; however, IRS-1 degradation is delayed compared with IRS-2 degradation in SH-SY5Y or SH-EP/IRS-2 cells. IGF-IR expression remains unchanged. IGF-I treatment of SH-EP/pSFFV cells has little effect on IRS-1 expression level, although there is a slight decrease in mobility (Fig. 6D). LY294002, but not PD98059 or MG132, treatment prevents this mobility shift (Fig. 6D). In SH-EP/IGF-IR cells, IGF-I exposure induces IRS-1 mobility shift and degradation, which are both unaffected by PD98059 treatment. In contrast, LY294002 almost completely blocks both the degradation and mobility shift of IRS-1, and MG132 slightly blocks the degradation but has no effect on mobility shift (Fig. 6D).
Previous reports demonstrate that SOCS-1 and -3 bind to IRS proteins and promote their ubiquitination and subsequent proteosomal degradation (25, 26). Therefore, SOCS-1 was transfected into SH-EP cells and IGF-I-mediated IRS degradation analyzed. SOCS-1 transfection itself does not affect IRS-1 levels in SH-EP cells (Fig. 7A). However, when SH-EP/SOCS-1 cells are treated with 10 nM IGF-I for 24 h, a 42% reduction in IRS-1 protein levels occur compared with an 11% decrease in vector (pcDNA) transfected SH-EP cells (Fig. 7, A and B). Transfection of SOCS-1 does not affect the basal and IGF-I-induced expression levels of Shc in SH-EP cells or IRS-2 in SH-SY5Y cells (data not shown).
Discussion
Our laboratory is interested in understanding the role of IGF-I signaling in the nervous system (43, 44, 45). We have used neuroblastoma cells as an in vitro model and recently reported a detailed analysis of IGF-I signaling pathways in different human neuroblastoma cells (29). The most striking difference between N type (neuroblast-like) cells and S type (glial-like) cells is the expression pattern of IRS; neuroblast-like cells express high levels of IRS-2 with minimal expression of IRS-1, whereas glial-like cells exclusively express IRS-2. IGF-I stimulation results in sustained tyrosine phosphorylation of IRS-1 in S cells; however, IRS-2 tyrosine phosphorylation in N cells is transient. Therefore, this report examines specifically the regulation of the IRS proteins in N and S cells.
IGF-I stimulation results in the degradation of IRS-2 in N type SH-SY5Y cells but has no effect on IRS-1 expression level in S-type SH-EP cells. IGF-IR activation is required for this effect because the IGF-IR kinase-specific inhibitor NVP-AEW541 effectively blocks IRS-2 degradation. Even though several reports suggest an interaction between IGF-IR and EGFR in nonneural cells (35, 36), in N and S cells, the inhibitors of the two receptors are ineffective in preventing the opposite ligands’ effect. These results are in contrast with previous reports in which EGF induces increased IRS-1 expression (46) and prevents IGF-I-induced IRS-1 degradation in nonneural cells (47). The EGF-induced increase in IRS-1 expression is mediated by the ERK1/2 pathway (46). Therefore, specific downstream signaling pathways may be responsible for the cell type-specific regulation of IRS proteins by different growth factors.
Our data indicate that IRS degradation is mediated by PI 3-K and proteasome-dependent pathways. IRS degradation is tightly correlated with Akt phosphorylation in both SH-SY5Y and SH-EP/IGF-IR cells, and inhibition of the PI 3-K/Akt pathway by LY294002 completely blocks the mobility shift and degradation. In contrast, inhibition of the ERK1/2 pathway is ineffective in blocking IRS degradation. These results are in agreement with previous reports that demonstrate PI 3-K pathway involvement in IRS degradation (15, 40). Proteasome inhibitors are less effective in inhibiting IRS degradation than LY294002 and do not block the IRS mobility shift. These results suggest that serine/threonine phosphorylation occurs before proteasome-mediated degradation. Indeed, prior studies have shown that IRS-1 serine phosphorylation is required for its recognition by ubiquitin machinery (48, 49, 50). Our results also suggest that other proteases, such as calpain or other lysosomal proteases (51, 52), may be operational for IRS degradation. Caspase inhibitors, however, are ineffective against IGF-I mediated IRS degradation (data not shown), even though these inhibitors block mannitol-induced IRS-1 degradation in SH-EP cells (53).
Our data show differential regulation of IRS-1 and IRS-2, likely due to intrinsic differences within the proteins. IRS-1 and IRS-2 have different cellular localization patterns. IRS-1 is 2-fold more concentrated in intracellular membranes than in the cytosol, whereas IRS-2 is 2-fold more concentrated in the cytosol than in intracellular membranes (54). Therefore, IRS-2 may be more accessible to proteasome complexes, which reside primarily in the cytosol (55). Another possible explanation for the differential degradation between IRS-1 and IRS-2 could be structural differences. IRS-1 and IRS-2 share N-terminal pleckstrin homology and pTyr binding domains and show overall 43% amino acid identity (56). The middle section of IRS-2 (amino acids 591–786) is called the kinase regulatory loop binding (KRLB) domain and is unique to IRS-2 (57, 58). The KRLB domain is required for the initial binding of IRS-2 to IR; IRS-2:IR binding results in the phosphorylation of two critical tyrosine residues within the KRLB, which leads to decreased binding to the IR. The interaction between the IRS-2 KRLB domain and the IGF-IR has not been demonstrated. However, if the same mechanism applies to an IRS-2:IGF-IR interaction, faster dissociation of IRS-2 from the receptor may lead to easier access by the proteasome complex, resulting in degradation. This early dissociation may also explain the transient IRS-2 phosphorylation in SH-SY5Y cells compared with the sustained IRS-1 phosphorylation in SH-EP cells (29).
An additional alternative explanation is a potential differential sensitivity of IRS-1 and IRS-2 for ubiquitination enzymes. IGF-I induced a slight decrease in IRS-1 mobility in SH-EP and SH-EP/pSFFV cells without affecting the protein expression level. This mobility shift is blocked by the inhibition of the PI 3-K pathway but not by the inhibitors of ERK1/2 or of the proteasome. These results suggest that IRS-1 is serine/threonine phosphorylated by IGF-I treatment, but the subsequent degradation signal is impaired in SH-EP cells. Because transfected IRS-2 in SH-EP cells is degraded, we can assume that the degradation machinery is intact in SH-EP cells. IRS-1 and IRS-2 therefore could have different sensitivity to ubiquitination enzymes, especially the E3 enzyme. Ubiquitination of proteins requires three enzyme complexes; E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme and E3 ubiquitin ligase complex (59). E3 is responsible for substrate recognition in vivo and is required for recruitment of the E2 enzyme to the vicinity of the substrate. SOCS-1 and -3 bind elongin BC E3 complex through their SOCS box domain (26). SOCS-1 binds directly to both IRS-1 and IRS-2 and promotes their ubiquitination and degradation (26). However, the sensitivity of the E3 enzyme to IRS-1 and IRS-2 may be different. Indeed when we overexpressed SOCS-1 in SH-EP cells, IGF-I treatment resulted in IRS-1 degradation; however, it took a much longer time than the degradation of IRS-2. Most E3 ligases target phosphorylated substrates (59). Therefore E3 may only recognize IRS-1 or IRS-2 phosphorylated on specific sites. The ability of IGF-I to promote phosphorylation on those sites may be different for IRS-1 and IRS-2, thus contributing to the differential effects on IRS-1 and IRS-2.
Zhande et al. (14) have previously shown that insulin stimulation of CHO/IR cells transfected with IRS-1 or IRS-2 results in the degradation of only IRS-1, but not IRS-2. Furthermore, by switching the N-terminal region, they can reverse the degradation characteristics of IRS-1 and IRS-2 (14), indicating that intrinsic differences within the protein regulate differential protein degradation. Other evidence suggests that IRS-1 and IRS-2 degradation are cell type specific. IRS-1 degradation occurs in CHO/IR (14), MCF-7 breast cancer (15), and 293 embryonic kidney cells (7) and in skeletal muscle and white adipose tissue (60). Degradation of both IRS-1 and IRS-2 is detected in Fao and H4IIE hepatoma cells (19, 48) and L6 rat myoblasts (27). The degradation of IRSs in 3T3-L1 adipocytes is controversial; Rui et al. (19) reported that both IRS-1 and IRS-2 are degraded by insulin and IGF-I treatment in adipocytes, whereas only IRS-2 is degraded in preadipocytes. However others show that only IRS-1, but not IRS-2, is degraded in adipocytes (50, 60, 61). Therefore, the degradation of IRS proteins appears cell type or culture condition specific.
Our results indicate that IRS-2 is the preferential signaling molecule over IRS-1 in neuronal cells. IRS-2 KO, but not IRS-1-deficient mice, develop diabetes (62, 63), and disruption of the IRS-2 gene results in reduced neuronal proliferation and impaired brain growth (64). In IRS-2 –/– mice, an accumulation of phosphorylated tau occurs in neurofibrillary tangles present at an old age. These results suggest that IRS-2 is the molecular link between diabetes and neurodegeneration.
In summary, we report the differential regulation of IRS proteins in an in vitro model of nervous system development. Our data suggest that IRS-2, expressed in neuroblast-like cells, is more sensitive to IGF-I-mediated degradation, a process regulated by PI 3-K and proteasome pathways. In contrast, IRS-1, expressed in glial-like cells, is resistant to IGF-I mediated degradation. Collectively, our data support the idea that the differential degradation of IRS proteins by IGF-I in specific cells, representing different nervous system cell types, is a key pathway used by IGF-I to modulate activation of the IGF-IR. These results suggest that IRS-2 regulation is important for normal neuronal functions, and perturbance of IRS-2 regulation may contribute to neurodegenerative disease.
Acknowledgments
The authors thank Ms. Judy Boldt for expert secretarial assistance.
Footnotes
This work was supported by grants from the National Institutes of Health (NS38849 and NS36778), the Juvenile Diabetes Research Foundation Center for the Study of Complications in Diabetes, and the Program for Understanding Neurological Diseases (PFUND).
First Published Online September 8, 2005
Abbreviations: CHO, Chinese hamster ovary; EGF, epidermal growth factor; EGFR, EGF receptor; IGF-IR, IGF-I receptor; IR, insulin receptor; IRS, insulin receptor substrate; KRLB, kinase regulatory loop binding; mTOR, mammalian target of rapamycin; PI 3-K, phosphatidylinositol 3-kinase; pTyr, phosphotyrosine; SOCS, suppressor of cytokine signaling.
Accepted for publication August 25, 2005.
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Abstract
Insulin receptor substrate (IRS) signaling is regulated through serine/threonine phosphorylation, with subsequent IRS degradation. This study examines the differences in IRS-1 and IRS-2 degradation in human neuroblastoma cells. SH-EP cells are glial-like, express low levels of the type I IGF-I receptor (IGF-IR) and IRS-2 and high levels of IRS-1. SH-SY5Y cells are neuroblast-like, with high levels of IGF-IR and IRS-2 but virtually no IRS-1. When stimulated with IGF-I, IRS-1 expression remains constant in SH-EP cells; however, IRS-2 in SH-SY5Y cells shows time- and concentration-dependent degradation, which requires IGF-IR activation. SH-EP cells transfected with IRS-2 and SH-SY5Y cells transfected with IRS-1 show that only IRS-2 is degraded by IGF-I treatment. When SH-EP cells are transfected with IGF-IR or suppressor of cytokine signaling, IRS-1 is degraded by IGF-I treatment. IRS-1 and -2 degradation are almost completely blocked by phosphatidylinositol 3-kinase inhibitors and partially by proteasome inhibitors. In summary, 1) IRS-2 is more sensitive to IGF-I-mediated degradation; 2) IRS degradation is mediated by phosphatidylinositol 3-kinase and proteasome sensitive pathways; and 3) high levels of IGF-IR, and possibly the subsequent increase in Akt phosphorylation, are required for efficient IRS degradation.
Introduction
IGF-I AND INSULIN regulate growth and metabolism in many cell types (1). Insulin and IGF-I stimulate the tyrosine autophosphorylation of receptor tyrosine kinases. Autophosphorylation of the IGF-I receptor (IGF-IR) or insulin receptor (IR) subsequently initiates the binding of specific docking molecules to the phosphorylated receptor. One such docking molecule is insulin receptor substrate (IRS), which binds to phosphotyrosine (pTyr) residues on receptor -subunits. Receptor-IRS binding is required for insulin and IGF-I-mediated signal transduction (2). Four members of this family have been identified (IRS-1 through -4), each containing characteristic N-terminal pleckstrin homology and pTyr binding domains (3). IRS proteins lack intrinsic kinase activity; however, upon binding to activated receptors, IRS proteins are tyrosine phosphorylated and further bind downstream signaling molecules, including the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K), Fyn, Grb2, Nck, and Shb (2, 4, 5).
Along with many tyrosine residues, IRS-1 and IRS-2 also have more than 30 serine/threonine phosphorylation sites. Serine/threonine phosphorylation of IRS-1 and -2 prevents tyrosine phosphorylation, thereby inhibiting the ability of insulin or IGF-I to stimulate subsequent downstream signaling (6, 7). Multiple proteins have been suggested as possible serine/threonine kinases for IRS-1 and -2, including c-Jun N-terminal kinase (8), the inhibitor B kinase complex (9), Rho kinase (10), protein kinase C (11), and the PI 3-K/Akt/mammalian target of rapamycin (mTOR) pathway (12, 13). Once serine/threonine phosphorylated, the IRS proteins are degraded via the ubiquitin-proteasome degradation pathway (14, 15). Ubiquitin-proteasome-mediated degradation regulates a variety of cellular functions including cell cycle control, gene transcription, and apoptosis (16, 17). Inhibition of proteasome activation by MG132 or lactacystin prevents insulin or IGF-I-induced degradation of IRS-1 and IRS-2 (15, 18, 19). IRS degradation is also blocked by the inhibition of PI 3-K by LY294002 or its downstream effector mTOR by rapamycin (12). Furthermore, expression of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10), which antagonizes PI 3-K, up-regulates IRS-2 expression in breast cancer cells (20). These results suggest that PI 3-K mediated pathways involving Akt and mTOR are responsible for the proteasome-mediated degradation of IRS proteins.
The suppressor of cytokine signaling (SOCS) family of proteins is implicated in the regulation of insulin and IGF-I signaling. SOCS family members (SOCS1 through SOCS7 and CIS) target proteins for ubiquitination and subsequent degradation by the proteasome (21). SOCS-1, -2, and -3 bind both the IR and IGF-IR (22, 23, 24) and inhibit their signaling. Proteasome-mediated degradation of IRS-1 and -2 requires SOCS-1 and -3 in some cell types. Expression of SOCS-1 or -3 in primary cultured hepatocytes or targeted overexpression in liver by adenoviral-mediated gene transfer in mice induces IRS-1 and IRS-2 degradation (25, 26). In contrast, expression of mutant SOCS-1, which cannot bind ubiquitin ligase fails to induce IRS protein degradation (26).
Relatively few reports investigate IRS-2 degradation (19, 26, 27). Differential regulation of IRS-2 degradation occurs depending on cell type. For instance, in Chinese hamster ovary (CHO) cells expressing IR (CHO/IR) cells transfected with IRS-1 or IRS-2, only IRS-1 is degraded upon insulin treatment, and the N-terminal region of IRS-1 is responsible for its degradation by the proteasome/ubiquitin pathway (14). In contrast, IRS-2, but not IRS-1, is degraded upon insulin or IGF-I treatment in 3T3-L1 preadipocytes and MEF cells (19). Interestingly, when 3T3-L1 preadipocytes are differentiated to adipocytes, IRS-1 is then degraded by insulin treatment (19). These reports suggest that IRS protein degradation depends upon the cell type used and/or differentiation status. However, there are virtually no reports concerning the regulation of IRS degradation in neuronal cells. In our laboratory, we have been studying IGF-I signaling in primary and transformed neuronal cells for over 15 yr. Our data suggest that neuronal cells respond to IGF-I in a unique manner that cannot be predicted by IGF-I mediated signaling in other cell types (28).
In this report, we compare IRS-1 and IRS-2 degradation in response to IGF-I in human neuroblastoma cells. We have previously shown that more neuroblast-like N-type neuroblastoma cells (e.g. SH-SY5Y cells) have increased expression of the IGF-IR compared with more glial like S cells (e.g. SH-EP cells) (29). Interestingly S cells exclusively express IRS-1 that undergoes sustained phosphorylation by IGF-I, whereas N cells express IRS-2 that is transiently phosphorylated by IGF-I (29). The pattern of IRS expression holds true in primary cells from the peripheral nervous system as well; Schwann cells express IRS-1 (30), whereas spinal cord motor neurons express IRS-2 (31). We report here that IGF-I stimulation of SH-SY5Y cells results in the degradation of IRS-2; in contrast IGF-I treatment does not affect IRS-1 expression levels in SH-EP cells. Inhibition of the PI 3-K pathway completely blocks IGF-I-induced IRS-2 degradation, and IRS-2 degradation is tightly correlated with changes in Akt phosphorylation, indicating regulation through the PI 3-K pathway. Inhibition of the proteosome using proteosome inhibitors or inhibition of IGF-IR activation by the IGF-IR kinase-specific inhibitor NVP-AEW541 prevents IGF-I-induced IRS-2 degradation. When IRS-2 is transfected into SH-EP cells, IGF-I stimulation results in the degradation of IRS-2; however, when IRS-1 is transfected into SH-SY5Y cells, expression is not changed by IGF-I treatment. Both IGF-IR overexpression and SOCS-1 transfection in SH-EP cells result in IGF-I-induced IRS-1 degradation, but over a longer time course than IRS-2 degradation. Based upon these data, we conclude that IRS-2 is more sensitive to IGF-I-induced degradation. Furthermore, high IGF-IR expression is required for the efficient assembly of degradation machinery, an event which requires PI 3-K/Akt and proteasome activation.
Materials and Methods
Materials
Anti-IRS-1, anti-IRS-2, anti-IGF-IR subunit, and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Shc antibody was purchased from Transduction Laboratory (Lexington, KY). Anti-pTyr and anti-EGF (epidermal growth factor) receptor antibodies and antibodies against phosphorylated forms of Akt and ERK1/2 were from Cell Signaling Technology (Beverly, MA). All inhibitors and EGF were purchased from Calbiochem (La Jolla, CA). IGF-IR kinase inhibitor, NVP-AEW541, was provided by Novartis Institute of Biomedical Research, Inc. (Cambridge, MA). Recombinant human IGF-I was kindly provided by Cephalon, Inc. (West Chester, PA) and des(1–3)IGF-I was purchased from GroPrep (Adelaide, Australia). IRS-1 and IRS-2 containing vectors were kindly provided by Dr. D. Yee of the University of Minnesota (15). SOCS-1 in pcDNA vector was provided by Dr. L. Rui of the University of Michigan (26).
Cell culture
SH-SY5Y and SH-EP human neuroblastoma cells were maintained in DMEM containing 10% calf serum. IRS-1, IRS-2, and SOCS-1 containing vectors or empty pcDNA vectors were transfected into neuroblastoma cells using Lipofectamine 2000 reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. SH-EP cells transfected with pSFFV or IGF-IR were described previously (32). Transfected cells were maintained in DMEM containing 10% calf serum and 0.25 mg/ml G418 (Invitrogen Life Technologies). The cells were serum starved 4–6 h before treatment. The inhibitors were added 1 h before IGF-I treatment.
Immunoprecipitation and immunoblotting
Western immunoblotting and immunoprecipitation were performed as described previously (33). All experiments were repeated at least three times. Typical representative results are presented in the figures.
Results
IRS-2, but not IRS-1, is degraded by IGF-I treatment in neuroblastoma cells
We have previously reported the detailed analysis of IGF-I:IGF-IR signaling among N and S type neuroblastoma cells (29) including differential expression of IGF-IR, IRS-1, and IRS-2. In this report we investigate the regulation of IRS-1 and -2, the major signaling molecules of IGF-IR, in more detail. First, serum-starved SH-EP and SH-SY5Y human neuroblastoma cells are treated with 10 nM IGF-I for 0–2 h. Equal amounts of whole-cell lysates are immunoblotted with anti-pTyr antibody (Fig. 1A). In agreement with our previous results (29, 34) treatment of the cells with 10 nM IGF-I results in strong tyrosine phosphorylation of a 97-kDa protein in SH-SY5Y cells, which represents IGF-IR (Fig. 1A, upper panel). We cannot detect IGF-IR phosphorylation in SH-EP cells due to very low expression of IGF-IR (Fig. 1A, middle panel). The protein levels of IRS-1 in SH-EP cells are not changed with IGF-I treatment. In contrast, IRS-2 in SH-SY5Y cells shows a time-dependent mobility shift along with a decrease in its expression level (Fig. 1A, lower panel). Various studies have suggested that these changes represent serine/threonine phosphorylation and degradation of IRS proteins (6, 7). As can be seen in Fig. 1, A and B, the degradation of IRS-2 starts within 30 min of IGF-I treatment and continued up to 2 h. IRS-2 degradation is also dependent on IGF-I concentration. IGF-I treatment results in the concentration-dependent tyrosine phosphorylation of IGF-IR, beginning at 1 nM (Fig. 1C, top panel). Interestingly, the mobility shift and degradation of IRS-2 starts at a much lower concentration, as low as 1 pM IGF-I (Fig. 1C, middle panel). IGF-IR protein expression is not changed (Fig. 1C, bottom panel).
Several investigators reported an interaction between IGF-IR and EGF receptor (EGFR) signaling (35, 36). We have shown that EGF treatment results in ERK1/2 phosphorylation in SH-SY5Y cells (37). Anti-EGFR immunoblotting reveals that both SH-EP and SH-SY5Y cells express comparable amounts of EGFR (Fig. 2A). Similar to IGF-I treatment, EGF stimulation results in IRS-2 degradation in SH-SY5Y cells but does not affect IRS-1 expression in SH-EP cells (Fig. 2B). Unlike IGF-I treatment, however, EGF-induced IRS-2 degradation is transient; IRS-2 expression decreases at 30 min, then returns to basal levels at 2 h. We confirmed that IGF-I-mediated IRS-2 degradation is not mediated by EGFR by using the EGFR kinase-specific inhibitor AG1478 (38). EGF-induced IRS-2 degradation is blocked by the pretreatment of the cells with 0.25 μM AG1478; however, AG1478 has no effect on IGF-I-induced IRS-2 degradation (Fig. 2C).
IRS-2 degradation correlates with the activation of PI 3-K pathway
Next we investigated signaling pathways downstream from the IGF-IR involved in IRS-2 degradation. For this experiment, we used pharmacological inhibitors of ERK1/2 (PD98059), PI 3-K (LY294002), and proteasome (MG132) pathways (Fig. 3A). Treatment of SH-SY5Y cells with PD98059 completely blocks ERK1/2 phosphorylation but has no effect on IGF-I-induced IRS-2 degradation. In contrast, LY294002 almost completely blocks both the degradation and mobility shift of IRS-2. The proteasome inhibitor MG132 slightly prevents IRS-2 degradation but does not inhibit the observed mobility shift. Other proteasome inhibitors, such as epoxomycin and PS1, have similar effects (data not shown). These results suggest that IGF-I-induced IRS-2 degradation is mediated by both PI 3-K and proteasome-dependent pathways.
One of the major signaling molecules downstream of PI 3-K is Akt (39). Akt is directly involved in IRS degradation in Fao hepatocytes (40). In our system, Akt is highly phosphorylated in SH-SY5Y cells; there is, however, only weak phosphorylation of Akt in SH-EP cells (Fig. 3B). IRS-2 degradation and Akt phosphorylation show similar concentration-dependent changes by IGF-I (Fig. 3C). Similar to changes in IRS-2 degradation (Fig. 1C) Akt phosphorylation starts as low as 1 pM IGF-I treatment (Fig. 3C). In contrast, ERK1/2 phosphorylation begins at concentrations of 0.1 nM IGF-I. The time course of Akt phosphorylation by IGF-I and EGF is also consistent with IRS-2 degradation. IGF-I induces sustained phosphorylation of Akt, whereas EGF-induced Akt phosphorylation is transient (Fig. 3D). Therefore, Akt phosphorylation status and IRS-2 degradation are strongly correlated, supporting a potential role for Akt in IRS-2 degradation.
IGF-IR activation is required for IGF-I-induced IRS-2 degradation and Akt phosphorylation
To confirm the requirement of IGF-IR activation for IRS-2 degradation in SH-SY5Y cells, we used NVP-AEW541, a potent and specific inhibitor of IGF-IR kinase activity (41). SH-SY5Y cells are incubated for 1 h with increasing concentrations of NVP-AEW541 and then treated with 10 nM IGF-I for 30 min. NVP-AEW541 treatment results in concentration-dependent inhibition of the tyrosine phosphorylation of a 97-kDa protein (Fig. 4A, upper panel). To confirm that this 97-kDa protein is IGF-IR, we immunoprecipitated IGF-IR followed by anti-pTyr immunoblotting (Fig. 4A, bottom panel). Once again, NVP-AEW541 prevents tyrosine phosphorylation in a concentration-dependent fashion (Fig. 4A, bottom panel). Inhibition of IGF-IR activation by NVP-AEW541 also prevents IRS-2 degradation in a concentration-dependent manner (Fig. 4B). NVP-AEW541 alone has no effect on IGF-IR phosphorylation, IGF-I-mediated IRS-2 degradation, and EGF-induced IRS-2 degradation (Fig. 4B). Expression of Shc, another major signaling molecule of IGF-IR, is not affected by any treatment (Fig. 4B). To exclude the possibility of IGF binding protein involvement, we treated cells with desIGF-I, which has very low affinity for the IGF binding proteins (42). Treatment of SH-SY5Y cells with 10 nM desIGF-I has a similar effect on IRS-2 degradation as IGF-I (Fig. 4C). NVP-AEW541 also induced concentration-dependent inhibition of Akt phosphorylation (Fig. 4D), which correlates well with IRS-2 degradation. Total Akt expression is not affected by NVP-AEW541 treatment (Fig. 4D, bottom panel).
Differential degradation of IRS-1 and -2
The results described above illustrate differential regulation of IRS-1 and IRS-2 degradation upon IGF-I treatment. This differential regulation is due to either intrinsic differences between the IRS-1 and IRS-2 proteins or differences in the cell types used. We therefore transfected IRS-2 into SH-EP cells (SH-EP/IRS-2) and IRS-1 into SH-SY5Y cells (SH-SY5Y/IRS-1) to attempt to distinguish between these two possibilities, using an empty pcDNA vector (SH-EP/pcDNA and SH-SY5Y/pcDNA) transfection as a control. IRS-2 transfection does not affect the basal or IGF-I-induced expression level of endogenous IRS-1 level in SH-EP/IRS-2 cells (Fig. 5A). When SH-EP/IRS-2 cells are treated with 10 nM IGF-I there is a time-dependent degradation of IRS-2 similar to that seen in SH-SY5Y cells. IRS-1 transfection does not affect the basal expression level of IRS-2 in SH-SY5Y cells, and IGF-I treatment results in IRS-2 degradation in both vector and IRS-1 transfected cells (Fig. 5B). In contrast, IGF-I has little effect on IRS-1 expression in SH-SY5Y/IRS-1 cells. IGF-IR and Shc expression remains unchanged by IRS transfection and IGF-I treatment in all cell types. The tyrosine phosphorylation of IRS-1 and IRS-2 in both cell types follows similar patterns as the protein levels. These results demonstrate that IRS-2 degradation occurs regardless of the cell type; therefore, there are likely intrinsic differences within the IRS-1 and IRS-2 proteins affecting the IGF-I-induced degradation.
Next, we examined the effect of increased IGF-IR expression on IRS-1 degradation in SH-EP cells. SH-EP or vector (pSFFV) transfected SH-EP (SH-EP/pSFFV) cells express about one fifth of the IGF-IR detected in SH-SY5Y cells (Fig. 6A). When transfected with IGF-IR, SH-EP/IGF-IR cells express about 15 times more receptor compared with SH-EP/pSFFV cells and three times more than SH-SY5Y cells. SH-EP/IGF-IR cells show increased IGF-IR tyrosine phosphorylation upon IGF-I exposure compared with the weak phosphorylation detected in SH-EP/pSFFV cells (Fig. 6B, upper panel). SH-EP/IGF-IR cells also have increased basal phosphorylation of IGF-IR. Consistent with the activation of IGF-IR, basal and IGF-I-induced Akt phosphorylation is increased in SH-EP/IGF-IR cells (Fig. 6B, middle panel). Compared with SH-EP/pSFFV, SH-EP/IGF-IR cells show reduced basal expression of IRS-1 (Fig. 6C). When treated with 10 nM IGF-I, IRS-1 is further degraded; however, IRS-1 degradation is delayed compared with IRS-2 degradation in SH-SY5Y or SH-EP/IRS-2 cells. IGF-IR expression remains unchanged. IGF-I treatment of SH-EP/pSFFV cells has little effect on IRS-1 expression level, although there is a slight decrease in mobility (Fig. 6D). LY294002, but not PD98059 or MG132, treatment prevents this mobility shift (Fig. 6D). In SH-EP/IGF-IR cells, IGF-I exposure induces IRS-1 mobility shift and degradation, which are both unaffected by PD98059 treatment. In contrast, LY294002 almost completely blocks both the degradation and mobility shift of IRS-1, and MG132 slightly blocks the degradation but has no effect on mobility shift (Fig. 6D).
Previous reports demonstrate that SOCS-1 and -3 bind to IRS proteins and promote their ubiquitination and subsequent proteosomal degradation (25, 26). Therefore, SOCS-1 was transfected into SH-EP cells and IGF-I-mediated IRS degradation analyzed. SOCS-1 transfection itself does not affect IRS-1 levels in SH-EP cells (Fig. 7A). However, when SH-EP/SOCS-1 cells are treated with 10 nM IGF-I for 24 h, a 42% reduction in IRS-1 protein levels occur compared with an 11% decrease in vector (pcDNA) transfected SH-EP cells (Fig. 7, A and B). Transfection of SOCS-1 does not affect the basal and IGF-I-induced expression levels of Shc in SH-EP cells or IRS-2 in SH-SY5Y cells (data not shown).
Discussion
Our laboratory is interested in understanding the role of IGF-I signaling in the nervous system (43, 44, 45). We have used neuroblastoma cells as an in vitro model and recently reported a detailed analysis of IGF-I signaling pathways in different human neuroblastoma cells (29). The most striking difference between N type (neuroblast-like) cells and S type (glial-like) cells is the expression pattern of IRS; neuroblast-like cells express high levels of IRS-2 with minimal expression of IRS-1, whereas glial-like cells exclusively express IRS-2. IGF-I stimulation results in sustained tyrosine phosphorylation of IRS-1 in S cells; however, IRS-2 tyrosine phosphorylation in N cells is transient. Therefore, this report examines specifically the regulation of the IRS proteins in N and S cells.
IGF-I stimulation results in the degradation of IRS-2 in N type SH-SY5Y cells but has no effect on IRS-1 expression level in S-type SH-EP cells. IGF-IR activation is required for this effect because the IGF-IR kinase-specific inhibitor NVP-AEW541 effectively blocks IRS-2 degradation. Even though several reports suggest an interaction between IGF-IR and EGFR in nonneural cells (35, 36), in N and S cells, the inhibitors of the two receptors are ineffective in preventing the opposite ligands’ effect. These results are in contrast with previous reports in which EGF induces increased IRS-1 expression (46) and prevents IGF-I-induced IRS-1 degradation in nonneural cells (47). The EGF-induced increase in IRS-1 expression is mediated by the ERK1/2 pathway (46). Therefore, specific downstream signaling pathways may be responsible for the cell type-specific regulation of IRS proteins by different growth factors.
Our data indicate that IRS degradation is mediated by PI 3-K and proteasome-dependent pathways. IRS degradation is tightly correlated with Akt phosphorylation in both SH-SY5Y and SH-EP/IGF-IR cells, and inhibition of the PI 3-K/Akt pathway by LY294002 completely blocks the mobility shift and degradation. In contrast, inhibition of the ERK1/2 pathway is ineffective in blocking IRS degradation. These results are in agreement with previous reports that demonstrate PI 3-K pathway involvement in IRS degradation (15, 40). Proteasome inhibitors are less effective in inhibiting IRS degradation than LY294002 and do not block the IRS mobility shift. These results suggest that serine/threonine phosphorylation occurs before proteasome-mediated degradation. Indeed, prior studies have shown that IRS-1 serine phosphorylation is required for its recognition by ubiquitin machinery (48, 49, 50). Our results also suggest that other proteases, such as calpain or other lysosomal proteases (51, 52), may be operational for IRS degradation. Caspase inhibitors, however, are ineffective against IGF-I mediated IRS degradation (data not shown), even though these inhibitors block mannitol-induced IRS-1 degradation in SH-EP cells (53).
Our data show differential regulation of IRS-1 and IRS-2, likely due to intrinsic differences within the proteins. IRS-1 and IRS-2 have different cellular localization patterns. IRS-1 is 2-fold more concentrated in intracellular membranes than in the cytosol, whereas IRS-2 is 2-fold more concentrated in the cytosol than in intracellular membranes (54). Therefore, IRS-2 may be more accessible to proteasome complexes, which reside primarily in the cytosol (55). Another possible explanation for the differential degradation between IRS-1 and IRS-2 could be structural differences. IRS-1 and IRS-2 share N-terminal pleckstrin homology and pTyr binding domains and show overall 43% amino acid identity (56). The middle section of IRS-2 (amino acids 591–786) is called the kinase regulatory loop binding (KRLB) domain and is unique to IRS-2 (57, 58). The KRLB domain is required for the initial binding of IRS-2 to IR; IRS-2:IR binding results in the phosphorylation of two critical tyrosine residues within the KRLB, which leads to decreased binding to the IR. The interaction between the IRS-2 KRLB domain and the IGF-IR has not been demonstrated. However, if the same mechanism applies to an IRS-2:IGF-IR interaction, faster dissociation of IRS-2 from the receptor may lead to easier access by the proteasome complex, resulting in degradation. This early dissociation may also explain the transient IRS-2 phosphorylation in SH-SY5Y cells compared with the sustained IRS-1 phosphorylation in SH-EP cells (29).
An additional alternative explanation is a potential differential sensitivity of IRS-1 and IRS-2 for ubiquitination enzymes. IGF-I induced a slight decrease in IRS-1 mobility in SH-EP and SH-EP/pSFFV cells without affecting the protein expression level. This mobility shift is blocked by the inhibition of the PI 3-K pathway but not by the inhibitors of ERK1/2 or of the proteasome. These results suggest that IRS-1 is serine/threonine phosphorylated by IGF-I treatment, but the subsequent degradation signal is impaired in SH-EP cells. Because transfected IRS-2 in SH-EP cells is degraded, we can assume that the degradation machinery is intact in SH-EP cells. IRS-1 and IRS-2 therefore could have different sensitivity to ubiquitination enzymes, especially the E3 enzyme. Ubiquitination of proteins requires three enzyme complexes; E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme and E3 ubiquitin ligase complex (59). E3 is responsible for substrate recognition in vivo and is required for recruitment of the E2 enzyme to the vicinity of the substrate. SOCS-1 and -3 bind elongin BC E3 complex through their SOCS box domain (26). SOCS-1 binds directly to both IRS-1 and IRS-2 and promotes their ubiquitination and degradation (26). However, the sensitivity of the E3 enzyme to IRS-1 and IRS-2 may be different. Indeed when we overexpressed SOCS-1 in SH-EP cells, IGF-I treatment resulted in IRS-1 degradation; however, it took a much longer time than the degradation of IRS-2. Most E3 ligases target phosphorylated substrates (59). Therefore E3 may only recognize IRS-1 or IRS-2 phosphorylated on specific sites. The ability of IGF-I to promote phosphorylation on those sites may be different for IRS-1 and IRS-2, thus contributing to the differential effects on IRS-1 and IRS-2.
Zhande et al. (14) have previously shown that insulin stimulation of CHO/IR cells transfected with IRS-1 or IRS-2 results in the degradation of only IRS-1, but not IRS-2. Furthermore, by switching the N-terminal region, they can reverse the degradation characteristics of IRS-1 and IRS-2 (14), indicating that intrinsic differences within the protein regulate differential protein degradation. Other evidence suggests that IRS-1 and IRS-2 degradation are cell type specific. IRS-1 degradation occurs in CHO/IR (14), MCF-7 breast cancer (15), and 293 embryonic kidney cells (7) and in skeletal muscle and white adipose tissue (60). Degradation of both IRS-1 and IRS-2 is detected in Fao and H4IIE hepatoma cells (19, 48) and L6 rat myoblasts (27). The degradation of IRSs in 3T3-L1 adipocytes is controversial; Rui et al. (19) reported that both IRS-1 and IRS-2 are degraded by insulin and IGF-I treatment in adipocytes, whereas only IRS-2 is degraded in preadipocytes. However others show that only IRS-1, but not IRS-2, is degraded in adipocytes (50, 60, 61). Therefore, the degradation of IRS proteins appears cell type or culture condition specific.
Our results indicate that IRS-2 is the preferential signaling molecule over IRS-1 in neuronal cells. IRS-2 KO, but not IRS-1-deficient mice, develop diabetes (62, 63), and disruption of the IRS-2 gene results in reduced neuronal proliferation and impaired brain growth (64). In IRS-2 –/– mice, an accumulation of phosphorylated tau occurs in neurofibrillary tangles present at an old age. These results suggest that IRS-2 is the molecular link between diabetes and neurodegeneration.
In summary, we report the differential regulation of IRS proteins in an in vitro model of nervous system development. Our data suggest that IRS-2, expressed in neuroblast-like cells, is more sensitive to IGF-I-mediated degradation, a process regulated by PI 3-K and proteasome pathways. In contrast, IRS-1, expressed in glial-like cells, is resistant to IGF-I mediated degradation. Collectively, our data support the idea that the differential degradation of IRS proteins by IGF-I in specific cells, representing different nervous system cell types, is a key pathway used by IGF-I to modulate activation of the IGF-IR. These results suggest that IRS-2 regulation is important for normal neuronal functions, and perturbance of IRS-2 regulation may contribute to neurodegenerative disease.
Acknowledgments
The authors thank Ms. Judy Boldt for expert secretarial assistance.
Footnotes
This work was supported by grants from the National Institutes of Health (NS38849 and NS36778), the Juvenile Diabetes Research Foundation Center for the Study of Complications in Diabetes, and the Program for Understanding Neurological Diseases (PFUND).
First Published Online September 8, 2005
Abbreviations: CHO, Chinese hamster ovary; EGF, epidermal growth factor; EGFR, EGF receptor; IGF-IR, IGF-I receptor; IR, insulin receptor; IRS, insulin receptor substrate; KRLB, kinase regulatory loop binding; mTOR, mammalian target of rapamycin; PI 3-K, phosphatidylinositol 3-kinase; pTyr, phosphotyrosine; SOCS, suppressor of cytokine signaling.
Accepted for publication August 25, 2005.
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