RESUMEN
The diverse biological actions of insulin and insulin-like growth factor I (IGF-I) are initiated by binding of the polypeptides to their respective cell surface tyrosine kinase receptors. These activated receptors phosphorylate a series of endogenous substrates on tyrosine, amongst which the insulin receptor substrate (IRS) proteins are the best characterized. Their phosphotyrosine-containing motifs become binding sites for Src homology 2 (SH2) domains on proteins such as SH2 domain-containing protein-tyrosine-phosphatase (SHP)-2/Syp, growth factor receptor bound-2 protein, (Grb-2), and phosphatidyl inositol 3 kinase (PI3 kinase), which participate in activation of specific signaling cascades. However, the IRS molecules are not only platforms for signaling molecules, they also orchestrate the generation of signal specificity, integration of signals induced by several extracellular stimuli, and signal termination and modulation. An extensive review is beyond the scope of the present article, which will be centered on our own contribution and reflect our biases.
Asunto(s)
Insulina/metabolismo , Proteínas de la Leche , Receptor IGF Tipo 1/metabolismo , Proteínas Represoras , Transducción de Señal/fisiología , Factores de Transcripción , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al ADN/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Integrinas/metabolismo , Modelos Biológicos , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas/metabolismo , Factor de Transcripción STAT5 , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/metabolismoRESUMEN
Stat proteins are SH2 domain-containing transcription factors that are activated by various cytokines and growth factors. In a previous work, we have identified Stat 5B as a substrate of the insulin receptor based on yeast two-hybrid and mammalian cell transfection studies. In the present study, we have approached the biological relevance of the interaction between the insulin receptor and the transcription factor Stat 5B. Firstly, we show that both insulin and insulin-like growth factor I lead to tyrosine phosphorylation of Stat 5B, and this promotes binding of the transcription factor to the beta-casein promoter containing a Stat 5 binding site. Further, we demonstrate that insulin stimulates the transcriptional activity of Stat 5B. Activation of Stat 5B by insulin appears to be Jak2-independent, whereas Jak2 is required for GH-induced Stat 5B activation. Hence the pathway by which Stat 5B is activated by insulin is different from that used by GH. In addition, by using Jak1- and Tyk2-deficient cells we exclude the involvement of both Jak1 and Tyk2 in Stat 5B activation by insulin. Taken together, our results strengthen the notion that insulin receptor can directly activate Stat 5B. More importantly, we have identified a Stat 5 binding site in the human hepatic glucokinase promoter, and we show that insulin leads to a Stat 5B-dependent increase in transcription of a reporter gene carrying this promoter. These observations favor the idea that Stat 5B plays a role in mediating the expression of the glucokinase gene induced by insulin. As a whole, our results provide evidence for the occurrence of a newly identified circuit in insulin signaling in which the cell surface receptor is directly linked to nuclear events through a transcription factor. Further, we have revealed an insulin target gene whose expression is, at least in part, dependent on Stat 5B activation and/or binding.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Glucoquinasa/genética , Insulina/farmacología , Proteínas de la Leche , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas , Transactivadores/fisiología , Transcripción Genética , Células 3T3 , Animales , Carcinoma Hepatocelular/metabolismo , ADN/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Janus Quinasa 1 , Janus Quinasa 2 , Hígado/enzimología , Neoplasias Hepáticas/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Fosfotirosina/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción STAT5 , Transfección , Células Tumorales CultivadasRESUMEN
The SOCS proteins are induced by several cytokines and are involved in negative feedback loops. Here we demonstrate that in 3T3-L1 adipocytes, insulin, a hormone whose receptor does not belong to the cytokine receptor family, induces SOCS-3 expression but not CIS or SOCS-2. Using transfection of COS-7 cells, we show that insulin induction of SOCS-3 is enhanced upon Stat5B expression. Moreover, Stat5B from insulin-stimulated cells binds directly to a Stat element present in the SOCS-3 promoter. Once induced, SOCS-3 inhibits insulin activation of Stat5B without modifying the insulin receptor tyrosine kinase activity. Two pieces of evidence suggest that this negative regulation likely results from competition between SOCS-3 and Stat5B binding to the same insulin receptor motif. First, using a yeast two-hybrid system, we show that SOCS-3 binds to the insulin receptor at phosphotyrosine 960, which is precisely where Stat5B binds. Second, using confocal microscopy, we show that insulin induces translocation of SOCS-3 from an intracellular compartment to the cell membrane, leading to colocalization of SOCS-3 with the insulin receptor. This colocalization is dependent upon phosphorylation of insulin receptor tyrosine 960. Indeed, in cells expressing an insulin receptor mutant in which tyrosine 960 has been mutated to phenylalanine, insulin does not modify the cellular localization of SOCS-3. We have thus revealed an insulin target gene of which the expression is potentiated upon Stat5B activation. By inhibiting insulin-stimulated Stat5B, SOCS-3 appears to function as a negative regulator of insulin signaling.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Proteínas de la Leche , Proteínas Represoras , Transducción de Señal , Factores de Transcripción , Células 3T3 , Adipocitos/metabolismo , Animales , Células COS , Proteínas de Unión al ADN/metabolismo , Ratones , Microscopía Fluorescente , Mutación , Fosforilación , Unión Proteica , Proteínas , ARN Mensajero/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Factor de Transcripción STAT5 , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/metabolismo , TransfecciónRESUMEN
In L6 muscle cells expressing the Arg1152 --> Gln insulin receptor (Mut), basal tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was increased by 35% compared with wild-type cells (WT). Upon exposure to insulin, IRS-1 phosphorylation increased by 12-fold in both the Mut and WT cells. IRS-2 was constitutively phosphorylated in Mut cells and not further phosphorylated by insulin. The maximal phosphorylation of IRS-2 in basal Mut cells was paralleled by a 4-fold increased binding of the kinase regulatory loop binding domain of IRS-2 to the Arg1152 --> Gln receptor. Grb2 and phosphatidylinositol 3-kinase association to IRS-1 and IRS-2 reflected the phosphorylation levels of the two IRSs. Mitogen-activated protein kinase activation and [3H]thymidine incorporation closely correlated with IRS-1 phosphorylation in Mut and WT cells, while glycogen synthesis and synthase activity correlated with IRS-2 phosphorylation. The Arg1152 --> Gln mutant did not signal Shc phosphorylation or Shc-Grb2 association in intact L6 cells, while binding Shc in a yeast two-hybrid system and phosphorylating Shc in vitro. Thus, IRS-2 appears to mediate insulin regulation of glucose storage in Mut cells, while insulin-stimulated mitogenesis correlates with the activation of the IRS-1/mitogen-activated protein kinase pathway in these cells. IRS-1 and Shc-mediated mitogenesis may be redundant in muscle cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Arginina/metabolismo , Glutamina/metabolismo , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Sustitución de Aminoácidos , Animales , Arginina/genética , Línea Celular , Proteína Adaptadora GRB2 , Glutamina/genética , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas/metabolismo , Ratas , Receptor de Insulina/genética , Relación Estructura-Actividad , Transfección , Dominios Homologos srcRESUMEN
In addition to the pleckstrin homology domain and the phosphotyrosine binding domain in insulin receptor substrate (IRS)-1 and IRS-2, a region between amino acids 591 and 786 in IRS-2 (IRS-2-(591-786)) binds to the insulin receptor. Based on peptide competition studies, this region interacts with the phosphorylated regulatory loop of the insulin receptor; we designate this region the kinase regulatory loop binding (KRLB) domain. Two tyrosine residues in the KRLB domain at positions 624 and 628 are crucial for this interaction. Phosphorylation of tyrosine residues in the KRLB domain by the insulin receptor inhibits the binding to the receptor. These results reveal a novel mechanism regulating the interaction of the insulin receptor and IRS-2 that may distinguish the signal of IRS-2 from IRS-1.
Asunto(s)
Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosforilación , Tirosina/metabolismoRESUMEN
We have screened a human placenta library using the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Stat 5B and the receptor requires a functional insulin-receptor kinase, Tyr960 of insulin receptor is implicated in the interaction with Stat 5B, whereas asparagine and proline forming the NPEY960-motif are not, and Stat 5B mutated at Thr684, a potential phosphorylation site of mitogen-activated protein kinase, loses its ability to interact with the insulin receptor. Further, we found that insulin promotes rapid tyrosine phosphorylation of endogenous Stat 5B in 293 EBNA cells overexpressing insulin receptor and in NHIR cells. Taken together, our findings suggest that Stat 5B corresponds to a substrate for the insulin-receptor kinase, and this widens the repertoire of insulin-signaling pathways.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de la Leche , Receptor de Insulina/metabolismo , Transactivadores/metabolismo , Células Cultivadas , Humanos , Fosforilación , Factor de Transcripción STAT5 , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
Activated insulin and insulin-like growth factor-I receptors transmit downstream signals via the insulin receptor substrate (IRS-1 and IRS-2) and a series of proteins containing Src homology-2 (SH2) domains, including SH2-containing protein tyrosine phosphatase 2 (SHP-2). In the present study, we analyzed in the yeast two-hybrid system the interaction between both receptors and SHP-2. We found that a catalytically inactive SHP-2 is able to bind to tyrosine-phosphorylated IR beta-subunit and IGF-I R beta-subunit. However, with wild-type SHP-2, we were unable to detect an interaction with these receptors, which is likely to be due to dephosphorylation of the receptors by the phosphatase. Further, our results demonstrate that tyrosine 1322 of the IR, and the corresponding tyrosine 1316 of the IGF-I R are implicated in the interaction with the SHP-2 SH2 domain. At the level of SHP-2, it would appear that both SH2 domains of SHP-2 are necessary for optimal association with either receptor. Finally, using several insulin and IGF-I receptor mutants, we found that the kinase regulatory autophosphorylation sites play an important role in the interaction of these receptors with the SHP-2 SH2 domain. These sites are also necessary for the interaction with full-length IRS-1. We conclude that 1) the IR and IGF-I R directly interact with SHP-2; 2) the C-terminus autophosphorylation of these receptors sites are involved in this process; and 3) the receptors' kinase autophosphorylation sites are necessary for the interaction with SHP-2 and also with IRS-1.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Dominios Homologos src , Clonación Molecular , ADN Complementario , Genes Reporteros , Humanos , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Sustancias Macromoleculares , Mutagénesis Sitio-Dirigida , Fosfoproteínas/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Multimerización de Proteína , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system. Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively. The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1. Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1. This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation.
Asunto(s)
Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN , Técnicas In Vitro , Proteínas Sustrato del Receptor de Insulina , Ratones , Datos de Secuencia Molecular , Fosforilación , Fosfotirosina/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Activation of the phosphatidyl inositol 3-kinase (PI 3-kinase) is one of the immediate events in signaling by the insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF-IR). The IR and IGF-IR are two closely related tyrosine kinases, which are activated on binding of their respective ligands. Previous studies have proposed that the two receptors interact directly with the SH2 domains of the Mr 85K regulatory subunit (p85) of PI 3- kinase via phosphorylated Y1322THM and Y1316AHM sequences located in the carboxyl-terminal domain of the IR and the IGF-IR, respectively. We In this study we have used the yeast two-hybrid system to compare the interaction of the cytoplasmic domains of the IR and the IGF-IR with p85. We found that the IR is more efficient in interacting with the p85 than is the IGF-IR. For both receptors, deletion of the region containing the Y1322THM sequence in the IR and the Y1316AHM-similar sequence in the IGF-IR decreases their ability to interact with p85. However, these mutated receptors still interacted with the full-length p85, suggesting that other regions in the receptors might be involved in the interaction. Furthermore, mutations of the three major autophosphorylation sites indicate that interactions with p85 are dependent on the receptor tyrosine kinase activity. Finally, we asked whether the two SH2 domains of p85 (n-SH2 and c-SH2) are involved in the same fashion in their association with the two receptors. Interestingly, we observed that the carboxyl- terminal domain of the IGF-IR associates only with the p85 c-SH2 domain, whereas the corresponding domain of the IR interacts with both the n-SH2 and the c-SH2 domains. In combination, both SH2 domains (n/c-SH2) contribute to the maximal interaction observed with the full-length p85. Although the precise impact on signaling resulting from these differences in the interaction of p85 with the IR vs. the IGF-IR remains to be determined, it is tempting to propose that they contribute, at least in part, to the specificity of the biological responses induced by insulin vs. IGF-1.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Using the yeast two-hybrid system, a genetic assay for studying protein-protein interactions, we have examined and compared the interaction of the insulin-like growth factor-I receptor (IGF-IR) and the insulin receptor (IR) with their two known substrates p52Shc and the insulin receptor substrate-1 (IRS-1). We also mapped the specific domains of the IGF-IR and p52Shc participating in these interactions. Our findings can be summarized as follows: (i) the tyrosine kinase activity of the IGF-IR is essential for the interaction with p52Shc and IRS-1, (ii) p52Shc and IRS-1 bind to the IGF-IR in the NPEY-juxtamembrane motif, (iii) contrary to p52Shc, IRS-1 binds also to the major autophosphorylation sites (Tyr-1131, -1135, and -1136) of the IGF-IR, and (iv) the amino-terminal domain of p52Shc is required for its association with the IR and the IGF-IR. We propose that (i) the IGF-IR and the IR share at least in part the same molecular mechanism underlying their interplay with their two substrates, p52Shc and IRS-1, and (ii) IRS-1 interacts with the IGF-IR in a fashion that is different from that used by p52Shc. Finally, our data highlight the crucial role of the juxtamembrane domain in signaling by both the IR and the IGF-IR.