chico
The discovery of the first intracellular substrate for insulin, IRS-1, has redirected the field of diabetes research and led to many important
advances in the understanding of insulin action. Detailed analysis of IRS-1 demonstrates structure/function relationships for this modular
docking molecule, including mechanisms of substrate recognition and signal propagation. Recent work has also identified other structurally
similar molecules, including IRS-2, the Drosophila protein DOS, and the Grb2-binding protein Gab1, suggesting that this intracellular
signaling strategy is conserved evolutionarily and is utilized by an expanding number of receptor systems. In fact, IRS-1 itself has been
shown to be important in other growth factor and cytokine signaling systems, including growth hormone and several interleukins. Analysis
of mice lacking IRS-1 confirms an important physiological role for this protein in glucose metabolism and general cell growth in the intact
animal. Disregulation of the signaling pathways integrated by the IRS proteins may contribute to the pathophysiology of
non-insulin-dependent diabetes mellitus or other diseases (Yenush, 1997).
Since the discovery of insulin in 1921, there has been no problem more fundamental to diabetes research than
understanding how insulin works at the cellular level. Insulin binds to the alpha subunit of the insulin receptor, which activates the
tyrosine kinase in the beta subunit, but the molecular events linking the receptor kinase to insulin-sensitive enzymes and transport
processes are unknown. The discovery that insulin stimulates tyrosine phosphorylation of a protein with a relative molecular mass between
165,000 and 185,000 kDa, collectively called pp185, shows that the insulin receptor kinase has specific cellular substrates. The pp185 is
a minor cytoplasmic phosphoprotein found in most cells and tissues; its phosphorylation is decreased in cells expressing mutant
receptors defective in signaling. IRS-1, which encodes a component of the pp185 band, has now been cloned. IRS-1 contains over ten
potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During insulin stimulation, the IRS-1 protein
undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-kinase, suggesting that IRS-1 acts as a multisite 'docking'
protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains. Thus IRS-1 may link the
insulin receptor kinase and enzymes regulating cellular growth and metabolism (Sun, 1991).
Insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate of the insulin and insulin-like growth factor (IGF)-1 receptors.
Transgenic mice lacking IRS-1 are resistant to insulin and IGF-1, but exhibit significant residual insulin action that corresponds to
the presence of an alternative high molecular weight substrate in liver and muscle. 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, has
significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice,
immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays were performed using specific antibodies to 4PS.
Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle; it binds to the p85 subunit of PI 3-kinase, and
activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient
mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression.
Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in
IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal
transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal
transduction to these two major pathways of insulin signaling (Patti, 1995).
Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct
set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic
signal transduction. By using the technique of isothermal titration calorimetry, the thermodynamics of binding of
the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding
sites were determined. The results show that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific
phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor,
ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. In contrast, the recognition of the
phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process.
Mutagenesis and amino acid substitution experiments show that in addition to the tyrosine-phosphorylated NPXY motif, the PTB
domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where
pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB
domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms
to recognize tyrosine-phosphorylated receptors (Farooq, 1999).
Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue
in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth
factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2
domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data reveal new potential Grb2 binding sites
at Tyr-1114 [epidermal growth factor receptor (EGFR) C-tail]; Tyr-743 [platelet-derived growth factor receptor (PDGFR) insert
region], Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None
of the phosphopeptides from the juxtamembrane or C-tail regions of IR bind Grb2 significantly, and only one phosphopeptide from
the basic fibroblast growth factor receptor (Tyr-556) binds Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of
EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the
Grb2 SH2 domain. With regard to Shc binding, the data reveal new potential binding sites at Tyr-703 and Tyr-789 from the catalytic
domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. The data also identify new potential Shc binding sites at Tyr-764,
in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be
required for Shc phosphorylation in response to insulin. The study confirms the previous identification of Tyr-992 and Tyr-1173 in
the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None
of the 34 phosphopeptides from IRS-1 bind Shc strongly, although Tyr-690 shows medium strength binding. The specificity
characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly
scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling
pathways (Ward, 1996).
Interaction domains located in the NH2 terminus of IRS-1 mediate its recognition by the insulin receptor. Alignment of IRS-1 and IRS-2 reveals two homology regions: the IH1(PH) contains a pleckstrin homology (PH) domain, and the IH2(PTB) contains a phosphotyrosine binding (PTB) domain. A third region in IRS-1 called SAIN has been proposed to contain another functional PTB domain. Peptide competition experiments demonstrate that the IH2(PTB) in IRS-2, like the corresponding domain in IRS-1, binds directly to peptides containing NPXY motifs. In contrast, these peptides do not bind to IH1(PH) or the SAIN regions. In 32D cells the IH1(PH) is essential for insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin-stimulated phosphatidylinositol 3-kinase activity and p70(s6k: see Drosophila RPS6-p70-protein kinase) phosphorylation. In contrast, the IH2(PTB) and the SAIN regions are not required for these insulin actions; however, the IH2(PTB) improves the coupling between IRS-1 and the insulin receptor. Overexpression of the insulin receptor in 32DIR cells increases IRS-1 tyrosine phosphorylation and mediates insulin-stimulated DNA synthesis. The sensitivity of these responses is partially reduced by deletion of either the IH1(PH) or the IH2(PTB) and significantly reduced when both regions are deleted. Thus, the PH and PTB domains equally couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB domain is inefficient and the PH domain is essential for a productive interaction (Yenush, 1996b).
Adipocytes contain three major substrate proteins of the insulin receptor, termed IRS-1, IRS-2, and IRS-3. IRS-1 and IRS-2 are located mainly in the low density microsome (LDM) fraction and are tyrosine phosphorylated in response to
insulin stimulation, leading to phosphatidylinositol (PI) 3-kinase activation. In contrast, IRS-3 is located mainly in the plasma
membrane (PM) fraction and contributes to PI 3-kinase activation in the PM fraction. The different cellular localizations of IRS
proteins may account for the mechanism of insulin resistance induced by a high fat diet, considering that PI 3-kinase activation in
the LDM fraction is reportedly essential for the translocation of GLUT4 in adipocytes. High fat feeding in rats increases both
protein and mRNA levels of IRS-3 but decreases those of IRS-1 and IRS-2 in epididymal adipocytes. As a result, selective
impairment of insulin-induced PI 3-kinase activation is observed in the LDM fraction, whereas PI 3-kinase activation is
conserved in the PM fraction. This is the first report showing that different IRS proteins function in different subcellular
compartments, which may contribute to determining the insulin sensitivity in adipocytes (Anai, 1998).
Insulin stimulation of 3T3-L1 adipocytes results in rapid activation of the insulin receptor tyrosine kinase followed by
autophosphorylation of the receptor and phosphorylation of insulin receptor substrate 1 (IRS-1), its major substrate. The insulin
receptor resides mostly at the cell surface of 3T3-L1 adipocytes under basal conditions, while about two-thirds of IRS-1 fractionates
with intracellular membranes and one-third fractionates with cytosol. To test whether insulin receptor internalization is required for
optimal tyrosine phosphorylation of IRS-1, 3T3-L1 adipocytes and CHO-T cells were incubated at 4 degrees C: this inhibits receptor
endocytosis but not its tyrosine kinase activity. Under these conditions, tyrosine phosphorylation of IRS-1 in the low density
microsome fraction in response to insulin is as intense as that observed at 37 degrees C, indicating that endocytosis of insulin
receptors is not necessary for tyrosine phosphorylation of IRS-1 to occur. Surprisingly, at 37 degrees C, insulin action on 3T3-L1
adipocytes progressively decreases the amount of IRS-1 protein associated with the low density microsome fraction and increases that
in the cytosol. This redistribution of IRS-1 from the low density microsome fraction to the cytosol in response to insulin is
accompanied by decreased electrophoretic mobility of IRS-1 on SDS-polyacrylamide gel electrophoresis. Incubation of adipocytes at 4
degrees C blocks the appearance of tyrosine-phosphorylated IRS-1 in the cytosol. Taken together, these data indicate that insulin
receptors phosphorylate IRS-1 at the cell surface, perhaps in coated pits that are included in the low density microsome fraction. The
results also suggest a desensitization mechanism in which the tyrosine-phosphorylated membrane-bound IRS-1, associated with
signaling molecules such as phosphatidylinositol 3-kinase, is released into the cytoplasm in concert with its serine/threonine
phosphorylation (Heller-Harrison, 1995).
CSF-1 is equipotent with insulin in its ability to stimulate 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes expressing the colony
stimulating factor-1 receptor/insulin receptor chimera (CSF1R/IR). However, CSF-1-stimulated glucose uptake and glycogen
synthesis is reduced by 50% in comparison to insulin in 3T3-L1 cells expressing a CSF1R/IR mutated at Tyr960 (CSF1R/IRA960).
CSF-1-treated adipocytes expressing the CSF1R/IRA960 are impaired in their ability to phosphorylate insulin receptor substrate 1
(IRS-1) but not in their ability to phosphorylate IRS-2. Immunoprecipitation of IRS proteins followed by Western blotting reveals
that the intact CSF1R/IR co-precipitates with IRS-2 from CSF-1-treated cells. In contrast, the CSF1R/IRA960 co-precipitates poorly
with IRS-2. These observations suggest that Tyr960 is important for interaction of the insulin receptor cytoplasmic domain with
IRS-2, but it is not essential to the ability of the insulin receptor tyrosine kinase to use IRS-2 as a substrate. These observations also
suggest that in 3T3-L1 adipocytes, tyrosine phosphorylation of IRS-2 by the insulin receptor tyrosine kinase is not sufficient for
maximal stimulation of receptor-regulated glucose transport or glycogen synthesis (Chaika, 1999).
In L6 muscle cells expressing the Arg1152 --> Gln insulin receptor (Mut), basal tyrosine phosphorylation of insulin receptor substrate
(IRS)-1 is increased by 35% compared with wild-type cells (WT). Upon exposure to insulin, IRS-1 phosphorylation increases by
12-fold in both Mut and WT cells. IRS-2 is constitutively phosphorylated in Mut cells and not further phosphorylated by insulin.
The maximal phosphorylation of IRS-2 in basal Mut cells is 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
reflects the phosphorylation levels of the two IRSs. Mitogen-activated protein kinase activation and [3H]thymidine incorporation closely
correlates with IRS-1 phosphorylation in Mut and WT cells, while glycogen synthesis and synthase activity correlates with IRS-2
phosphorylation. The Arg1152 --> Gln mutant does 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 (Miele, 1999).
Immortalized fetal brown adipocyte cell lines have been generated from insulin receptor substrate 1 (IRS-1) knockout mice and an impairment in insulin-induced lipid synthesis has been demonstrated. The consequences of IRS-1 deficiency on mitogenesis in response to insulin have been investigated. The lack of IRS-1 results in the
inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the
cell cycle. These cells show a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44
mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lack tyrosine phosphorylation of SHC and show no
SHC-Grb-2 association in response to insulin. The mitogenic response to insulin can be partially restored by enhancing IRS-2 tyrosine phosphorylation and
its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown
adipocytes with wild-type IRS-1 restores insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2
associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1
mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restores SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2
association impairs MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1
and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes (Valverde, 2001).
GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homolog of the Caenorhabditis
elegans protein, sem-5. Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras
signaling by receptor tyrosine kinases. In response to insulin, GRB2 forms a stable complex with two
tyrosine-phosphorylated proteins. One protein is the major insulin receptor substrate IRS-1 and the second is the SH2
domain-containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the
insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc.
Although GRB2 associates with IRS-1 and Shc, it is not tyrosine-phosphorylated after insulin stimulation, implying that GRB2 is
not a substrate for the insulin receptor. Furthermore, a short sequence motif (YV/IN) present in IRS-1, EGFR
and Shc, has been identified that specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and
phosphatidylinositol-3 (PI-3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule,
suggesting a mechanism whereby IRS-1 could provide the core for a large signaling complex. A model is proposed whereby
insulin stimulation leads to formation of multiple protein-protein interactions between GRB2 and the two targets IRS-1 and Shc.
These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules (Skolnik, 1993).
The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), only Ser-318, but not Ser-307 phosphorylation was detected, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. Since the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, the role of Shp2 in this context was examined. Phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depends on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 are blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1 (Mussig, 2005).
continues...see chico: Evolutionary homologs part 2/3
| part 3/3
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
chico:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.