Insulin receptor substrate 4 supports insulin- and interleukin 4-stimulated proliferation of hematopoietic cells.

Signaling from the activated insulin receptor is initiated by its tyrosine phosphorylation of the insulin receptor substrates (IRSs). The IRSs then act as docking/effector proteins for various signaling proteins containing src homology 2 domains. Four members of the IRS family, designated IRS-1 through IRS-4, have been identified. Although these IRSs show considerable structural homology, the extent to which they overlap in functions has not been explored in detail. The 32D hematopoietic cell line, which contains no detectable amounts of any IRS, provides a system in which to determine whether an IRS supports cell proliferation. Previous studies have shown that introduction of IRS-1 or -2 into 32D cells overexpressing the insulin and IL-4 receptors (32D-R cells) enables the cells to undergo mitogenesis in response to insulin and IL-4. In the present study, we have examined IRS-4, a member of the IRS family that we recently discovered, in this system. Expression of IRS-4 in 32D-R cells permitted the cells to undergo mitogenesis and continuous proliferation in response to insulin and IL-4. Immunoblotting of phosphotyrosine proteins showed that insulin and IL-4 elicited the tyrosine phosphorylation of IRS-4 in these cells. Thus, IRS-4, like IRS-1 and -2, can function in the signal transduction pathways linking insulin and IL-4 receptors to cell proliferation.     

Differential roles of the insulin and insulin-like growth factor-I (IGF-I) receptors in response to insulin and IGF-I

Insulin and insulin-like growth factor-I (IGF-I) receptors are highly homologous tyrosine kinase receptors that share many common steps in their signaling pathways and have ligands that can bind to either receptor with differing affinities. To define precisely the signaling specific to the insulin receptor (IR) or the IGF-I receptor, we have generated brown preadipocyte cell lines that lack either receptor (insulin receptor knockout (IRKO) or insulin-like growth factor receptor knockout (IGFRKO)). Control preadipocytes expressed fewer insulin receptors than IGF-I receptors (20,000 versus 60,000), but during differentiation, insulin receptor levels increased so that mature adipocytes expressed slightly more insulin receptors than IGF-I receptors (120,000 versus 100,000). In these cells, insulin stimulated IR homodimer phosphorylation, whereas IGF-I activated both IGF-I receptor homodimers and hybrid receptors. Insulin-stimulated IRS-1 phosphorylation was significantly impaired in IRKO cells but was surprisingly elevated in IGFRKO cells. IRS-2 phosphorylation was unchanged in either cell line upon insulin stimulation. IGF-I-dependent phosphorylation of IRS-1 and IRS-2 was ablated in IGFRKO cells but not in IRKO cells. In control cells, both insulin and IGF-I produced a dose-dependent increase in phosphorylated Akt and MAPK, although IGF-I elicited a stronger response at an equivalent dose. In IRKO cells, the insulin-dependent increase in phospho-Akt was completely abolished at the lowest dose and reached only 20% of the control stimulation at 10 nm. Most interestingly, the response to IGF-I was also impaired at low doses, suggesting that IR is required for both insulin- and IGF-I-dependent phosphorylation of Akt. Most surprisingly, insulin- or IGF-I-dependent phosphorylation of MAPK was unaltered in either receptor-deficient cell line. Taken together, these results indicate that the insulin and IGF-I receptors contribute distinct signals to common downstream components in response to both insulin and IGF-I.     

Insulin-like growth factor-I (IGF-I)-dependent activation of pp42/44 mitogen-activated protein kinase occurs independently of IGF-I receptor kinase activation and IRS-1 tyrosine phosphorylation.

The proliferation and metabolism of H4IIE hepatoma cells is apparently mediated through the insulin receptor. These cells, however, also have high-affinity binding sites for insulin-like growth factor-I (IGF-I). Addition of insulin to H4IIE cells increased RNA synthesis, DNA synthesis and cell number. IGF-I, on the other hand, was ineffective at concentrations equivalent to the lowest effective insulin dose, although stimulation was observed with concentrations 100-fold higher. Similar results were obtained when glucose uptake was measured. Western blot analysis demonstrated that tyrosine phosphorylation patterns produced by insulin and IGF-I differed. In particular, phosphorylation of insulin receptor substrate-1 (IRS-1) was evident after treatment with insulin, but not after treatment with IGF-I. Correspondingly, insulin, but not IGF-I, stimulated receptor tyrosine kinase activity. In contrast with these results, both insulin and IGF-I induced mitogen-activated protein (MAP) kinase phosphorylation and activity at a concentration of 10 nM. The correlation between insulin-dependent and IGF-I-dependent MAP kinase activation was confirmed by Western blot analysis of phosphorylated MAP kinase kinase (MEK). These results suggest that phosphorylation of IRS-1 is essential for both cell proliferation and glucose metabolism, but is uncoupled from the MAP kinase cascade. Furthermore, stimulation of MEK and MAP kinase is independent of receptor tyrosine kinase activity.     

IRS-2, but not IRS-1, can sustain proliferation and rescue UBF stabilization in InR or InR defective signaling of 32D myeloid cells

Despite both IRS-1 and IRS-2 are two important adaptor molecules essential for intracellular signaling of insulin and IGF-I, the distinct biological pattern of IRS-2 versus IRS-1 remains as a concernful issue to be clarified. We demonstrated here an important evidence that in 32D myeloid cells expressing the insulin receptor (InR) or selected mutants of the InR, IRS-2, but not IRS-1, is required for promoting the proliferation and inhibiting the granulocytic differentiation, thus restore ERKs phosphorylation and UBF1 stabilization. In addition, unlike IRS-1, IRS-2 can effectively compensate the InR defective signaling and lead to the activation of cell cycle progression and proliferation genes in 32D myeloid cells. These results indicate a predominant role of IRS-2 involved in InR signaling of 32 D myeloid cells.     

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.     

IRS-4 mediated mitogenic signalling by insulin and growth hormone in LB cells,a murine T-cell lymphoma devoid of IGF-I receptors

Insulin and growth hormone (GH) induce mitogenic and metabolic signals in cells, GH either directly or indirectly via IGF-I production. We have studied a spontaneous murine T-cell lymphoma (LB cells) devoid of IGF-1 receptors in which proliferation is maintained by insulin [Int. J. Cancer 50 (1992) 80], and show that GH is more potent than insulin, with both GH and insulin dose-response curves for thymidine incorporation being bell-shaped. Binding showed somatogenic rather than lactogenic GH receptors. Insulin stimulated phosphorylation of the insulin receptor and of a 160-kDa protein, identified as the IRS-4 protein. This phosphorylated IRS-4 associated with PI3-kinase, which was activated along with the downstream p70(S6) kinase, whereas the Ras-MAPK pathway was not. Using selective inhibitors, the PI3-kinase, but not p70(S6) kinase or MEK, was found to be involved in insulin-stimulated DNA synthesis. GH induced tyrosine phosphorylation of IRS-4 and nuclear translocation of STAT5. The LB cells constitute a new model for studying GH and insulin signalling without interference of IGF-1 receptors.     

Differential phosphorylation of IRS-1 and IRS-2 by insulin and IGF-I receptors

The specific contribution of insulin and IGF-I receptors to IRS-protein activation remains elusive. We studied the signalling properties of AspB10-insulin, an analog with enhanced affinity for the IGF-I receptor, in comparison to native insulin using primary human skeletal muscle cells. In myoblasts regular insulin and AspB10-insulin were equipotent in stimulating the IRS cascade, whereas this analog induced a significantly higher Shc phosphorylation. Phosphorylation of IRS-1 in response to insulin was inhibited equally by blocking either the insulin or the IGF-I receptor. IRS-1 activation by AspB10-insulin was only inhibited by blocking the IGF-I receptor. IRS-2 phosphorylation induced by both insulin and AspB10-insulin was nearly insensitive to blocking the insulin receptor, being predominantly mediated by the IGF-I receptor. We conclude that in myoblasts IRS-2, but not IRS-1, functions as preferred substrate for the IGF-I receptor. These data suggest a specific role for IRS-2 in growth and differentiation of human skeletal muscle.     

TGF-beta 1 modulation of IGF-I signaling pathway in rat thyroid epithelial cells

Transforming growth factor beta 1 (TGF-beta 1) and insulin-like growth factor I (IGF-I) have contrasting effects on cell cycle regulation in thyroid cells and TGF-beta 1 induces a dramatic decrease in IGF-I-induced cell proliferation. The aim of the present study was to investigate the molecular mechanism of cross-talk between TGF-beta 1 and IGF-I in FRTL-5 cells. TGF-beta 1 affected IGF-I-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with Grb2 protein. Moreover, TGF-beta 1 decreased the IGF-I-induced tyrosine phosphorylation of the adaptor protein CrkII and its association with the IGF-I receptor. These results were accompanied by TGF-beta 1 inhibition of IGF-I-stimulated mitogen-activated protein kinase phosphorylation and activation. Conversely, TGF-beta 1 did not alter IGF-I-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, IGF-I-induced tyrosine phosphorylation of Shc, and its binding to Grb2. Taken together, these findings provide a molecular basis for the growth-inhibitory action of TGF-beta 1 on the IGF-I-induced mitogenic effect.     

Expression profiles of micro RNA in proliferating and differentiating 32D murine myeloid cells

32D cells are murine myeloid cells that grow indefinitely in Interleukin-3 (IL-3). In these cells, the type 1 insulin-like growth factor (IGF-I) and granulocytic-colony stimulating factor (G-CSF) induce differentiation to granulocytes. 32D cells do not express insulin receptor substrate-1 (IRS-1) or IRS-2, docking proteins of the IGF-I receptor. Ectopic expression of IRS-1 in these cells inhibits differentiation, the cells become IL-3 independent and IGF-1 dependent and can form tumors in mice. 32D and 32D-derived cells offer a good model in which to study the expression profiles of Micro Rna (miR) related to sustained proliferation or differentiation. We present here the data obtained with miR micro-arrays and identify the miR that are regulated by IGF-1 or G-CSF and are associated with either differentiation or indefinite cell proliferation of 32D murine myeloid cells.     

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.     

Expression and function of insulin-like growth factor receptors on anti-CD3-activated human T lymphocytes.

The pattern of expression of receptors for insulin-like growth factors (IGF-I and IGF-II) and insulin was studied on monocyte-depleted human peripheral blood T cells activated via anti-CD3. Binding assays demonstrated the sequential appearance of receptors for IGF-I, IGF-II, and insulin on activated T cells. IGF-IR appeared early, their expression reaching maximum levels at or before the peak of cellular proliferation. IGF-IIR expression generally followed that of the IGF-IR and was more transient, with increases and decreases in expression paralleling the rise and decline of cellular proliferation. Insulin receptor expression remained low throughout the activation time course. The identity of the IGFR on anti-CD3-activated T cells was confirmed in affinity cross-linking experiments. These data demonstrated a 135,000 Mr peptide that specifically binds radiolabeled IGF-I and corresponds to the alpha subunit of the type I IGF-IR, and a 260,000 Mr peptide that specifically binds radiolabeled IGF-II and corresponds to the type II IGFR. We have additionally found that IGF-I and IGF-II (in nanomolar concentrations) produce as much as a threefold enhancement of T cell proliferation early in the activation process, correlating with the early appearance of IGF-IR. The effect of both IGF appeared to be mediated through the type I receptor, since an antibody (alpha IR3), which blocks binding to the alpha subunit of this receptor, inhibited enhancement by up to 83%. Furthermore, we have found expression of IGF-IR on T cells after activation to be associated with both CD4+ and CD8+ T cell subpopulations. These observations provide a foundation for investigating the contribution of IGF in regulating T cell proliferation, differentiation, and effector function.     

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.     

Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts

We have recently shown that insulin induced myogenesis in the mouse C2C12 skeletal muscle cell line by activation of phosphatidylinositol (PI) 3-kinase/p70S6-kinase and p38-mitogen-activated protein kinase (MAPK) and downregulation of p42/p44-MAPK. This study investigated the insulin-signaling pathways involved in mitogenesis, survival, and membrane ruffling in C2C12 myoblasts, a cellular system that besides IGF-I receptors, expressed a high number of functional insulin receptors. Insulin (10 nM) rapidly stimulated beta-chain insulin receptor and IRS-1 tyrosine phosphorylation, IRS-2 being poorly and SHC not phosphorylated at all. However, an association of SHC with IRS-1 was found under insulin stimulation. Insulin stimulated IRS-1 association with p85alpha leading to the activation of PI3-kinase, and, subsequently AKT and p70S6-kinases. Moreover, both p42/p44- and p38-MAPKs resulted in phosphorylation after insulin stimulation. Insulin treatment for 24 h produced mitogenesis, as demonstrated by the increase in ((3)H)-thymidine incorporation, DNA content, the expression of PCNA and cyclin D1 proteins, and the proportion of cells in S + G2/M phases of the cell cycle. This mitogenic effect of insulin was precluded by inhibition of p70S6-kinase (either by rapamycin or by the PI3-kinase inhibitor LY294002) as well as by inhibition of p44/p42-MAPK with PD098059, but was not affected by inhibition of p38-MAPK. Serum deprivation of C2C12 myoblasts resulted in growth arrest at the GO/G1 phases of the cell cycle and apoptosis, as detected either by DNA laddering or by increase in the percentage of hypodiploid cells. Insulin rescued serum-deprived cells from apoptosis in an AKT-dependent manner, as demonstrated by the inhibition of AKT-activity by the use of LY294002 and ML-9, meanwhile neither inhibition of p70S6-kinase, nor MAPK affected insulin-induced survival. Finally, we evaluated the capacity of insulin to modulate actin cytoskeleton rearrangement. Insulin stimulation of myoblasts produced membrane ruffling and decreased actin stress fibers; this biological response being dependent of p38-MAPK, as demonstrated by the use of the p38-MAPK inhibitors SB203580 or PD169316, but independent of PI3-kinase and p42/p44-MAPK.     

Insulin-like growth factor I (IGF-I) stimulates proliferation but also increases caspase-3 activity, Annexin-V binding, and DNA-fragmentation in human MG63 osteosarcoma cells: co-activation of pro- and anti-apoptotic pathways by IGF-I.

Insulin-like growth factor-I (IGF-I) was found to promote proliferation, cell survival, and inhibition of apoptosis. But in some instances, IGF-I was found to mildly induce apoptosis, i. e. Fas-mediated apoptosis in human MG63 osteosarcoma cells. In the present study, we intended to further investigate IGF-I dependent pathways leading either to proliferation and cell survival or to cell death. MG63 osteosarcoma cells were treated with serum free medium alone or in combination with IGF-I, a neutralizing antibody against the human IGF-I receptor (alphaIR-3) or non-immune control IgG (1) for two to six days. We investigated cell survival (cell count), proliferation (CD71-FACS), apoptosis (Annexin-V-FACS, Caspase-3 activity, PCD) and anti-apoptosis (112-Ser Bad phosphorylation), and regulation of IGF-I receptor surface expression (IGF-I receptor-FACS). We found that IGF-I treatment (48 h) stimulated cell growth and proliferation, but also mildly induced apoptosis. IGF-I activated specific apoptotic pathways (Caspase-3 activation, Annexin-V binding and DNA degradation), as well as anti-apoptotic signals (Bad phosphorylation at serine 112). alphaIR-3 blocked cell proliferation, strongly induced apoptosis, and inhibited Bad-phosphorylation. Thus, IGF-I treatment overall resulted in increased tumour cell mass, despite a detectable stimulation of apoptosis; in other words proliferation exceeded cell death. If IGF-I was first added on day 0, 2, or 4 of serum free culture, we found decreasing IGF-I specific effects on proliferation and apoptosis. In parallel, we found a down-regulation of IGF-I receptors (FACS) by serum withdrawal, which was partly reversed if IGF-I was added. Therefore receptor number might have an impact on IGF-I function in MG63 cells. In conclusion, co-activation of apoptosis and proliferation by IGF-I might result in higher cell turnover in MG63 osteosarcoma cells. Furthermore, in sarcomas or carcinomas showing clinical association to IGF-I levels and malignancy, IGF-I dependent apoptosis and proliferation could be a significant mechanism of malignant tumour growth.     

Regulation of breast cancer cell cycle progression by growth factors, steroids and steroid antagonists

The control of human breast cancer cell proliferation in vitro is known to involve complex interactions between steroid hormones, peptide hormones and growth factors. Little is known, however, of the mechanisms by which these factors, alone or in combination, control cell cycle progression and the expression of specific genes involved in cell cycle control. A pre-requisite for such studies is a cellular system in which non-proliferating or slowly proliferating cells can be maintained in a defined environment and stimulated to progress through the cell cycle by addition of hormones and growth factors. Such a system has been developed for T-47D human breast cancer cells: quiescent or slowly proliferating cells maintained in a serum-free medium can be stimulated to increase their rate of cell cycle progression upon a single addition of insulin, IGF-I, EGF, TGF or bFGF. Oestradiol alone was ineffective but caused a significant increase in % S phase cells when added in the presence of insulin. Progestins, in the presence or absence of insulin, had a biphasic effect with an initial increase in cell cycle progression followed by cell cycle arrest. Both antioestrogens and the antiprogestin, RU 486, in the absence of oestrogen or progestin, were potent inhibitors of insulin-induced proliferation. Increases in cell cycle progression were invariably accompanied by acute increases in c-fos and c-myc mRNA levels. Induction of c-myc by oestrogen and 3rogestin was inhibited by antioestrogens and RU 486, respectively. These data illustrate that the culture of breast cancer cells in a serum-free, chemically defined environment provides an excellent model in which to define the role of individual factors involved in breast cancer growth control. The biological data derived from this system provide a basis for identifying and characterizing genes involved in the control of cell cycle progression in human breast cancer.     

Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.     

Overexpression of Akt1 upregulates glycogen synthase activity and phosphorylation of mTOR in IRS-1 knockdown HepG2 cells

Insulin receptor substrate (IRS) proteins are important docking proteins in mediating the insulin signaling cascade. We have investigated the effect of short interfering RNA (siRNA) mediated knockdown of IRS-1 on insulin signaling cascade in primary human hepatocellular carcinoma HepG2 cell line and HepG2 cells overexpressing Akt1/PKB-alpha (HepG2-CA-Akt/PKB). IRS-1 knockdown in both cell lines resulted in reduction of insulin stimulated Akt1 phosphorylation at Ser 473. In parental HepG2 cells, IRS-1 knockdown resulted in reduction (ca. 50%) in the basal level of phosphorylated mTOR (Ser 2448) irrespective of insulin treatment. In contrast, HepG2-CA-Akt/PKB cells showed an upregulation in the basal level of phosphorylated mTOR (Ser 2448) (ca. 40%). Insulin mediated phosphorylation of mTOR was reduced. IRS-1 knockdown also reduced the cell proliferation of parental HepG2 cells by ca. 30% in the presence/absence of insulin, whereas in HepG2-CA-Akt/PKB the cell proliferation was reduced by 15% and treatment of insulin further reduced it to ca. 50% (vs. control). IRS-1 knockdown also reduced the glycogen synthase (GS) activity in parental HepG2 cells, however, it was upregulated in HepG2-CA-Akt/PKB cells. These results suggest that knockdown of IRS-1 abolished basal as well as insulin mediated phosphorylation/activity of proteins involved in cell proliferation or glycogen metabolism in the parental Hep2 cells. IRS-1 knockdown in cells overexpressing constitutively active Akt1/PKB-alpha either did not change or upregulated the basal levels of phosphorylated/active proteins. However, insulin mediated response was either not altered or downregulated in these cells.     

Further evidence for the involvement of insulin receptor substrates in epidermal growth factor-induced activation of phosphatidylinositol 3-kinase.

In accordance with our recent results obtained with cultured rat hepatocytes [Fujioka, T. & Ui, M. (2001) Eur. J. Biochem. 268, 25-34], epidermal growth factor (EGF) gave rise to transient tyrosine phosphorylation of insulin receptor substrates (IRS-1 and IRS-2), thereby activating the bound phosphatidylinositol 3-kinase in human epidermoid carcinoma A431 cells normally abundant in EGF receptors (EGFR) and Chinese hamster ovary (CHO) cells transfected with full-length EGFR. These actions of EGF, although much smaller in magnitude than those of insulin or IGF-I in the same cells, were accompanied by tyrosine phosphorylation of EGFR rather than insulin or IGF-I receptors, never observed in wild-type CHO cells expressing no EGFR, and totally inhibited by an inhibitor of EGFR kinase, AG1478, that was without effect on insulin or IGF-I actions. Recombinant IRS-1 was phosphorylated on tyrosines upon incubation with purified EGFR from A431 cells and 32P-labeled ATP. When CHO cells were transfected with C-terminal truncated EGFR lacking three NPXY motifs responsible for direct binding to phosphotyrosine-binding domains of IRSs, no effect of EGF could be observed. We suggest that tyrosine phosphorylation of IRS-1 or IRS-2 could mediate EGFR-induced activation of phosphatidylinositol 3-kinase in mammalian cells.