SOCS-6 Binds to Insulin Receptor Substrate 4, and Mice Lacking the SOCS-6 Gene Exhibit Mild Growth Retardation

ABSTRACT SOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6. We found that SOCS-6 and SOCS-7 are expressed ubiquitously in murine tissues. Like other SOCS family members, SOCS-6 binds to elongins B and C through its SOCS box, suggesting that it might act as an E3 ubiquitin ligase that targets proteins bound to its SH2 domain for ubiquitination and proteasomal degradation. We investigated the binding specificity of the SOCS-6 and SOCS-7 SH2 domains and found that they preferentially bound to phosphopeptides containing a valine in the phosphotyrosine (pY) +1 position and a hydrophobic residue in the pY +2 and pY +3 positions. In addition, these SH2 domains interacted with a protein complex consisting of insulin receptor substrate 4 (IRS-4), IRS-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase. To investigate the physiological role of SOCS-6, we generated mice lacking the SOCS-6 gene. SOCS-6−/− mice were born in a normal Mendelian ratio, were fertile, developed normally, and did not exhibit defects in hematopoiesis or glucose homeostasis. However, both male and female SOCS-6−/− mice weighed approximately 10% less than wild-type littermates.

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Phosphatidylinositol 3-Kinase and Prostaglandylinositol Cyclic Phosphate (Cyclic PIP), a Mediator of Insulin Action, in the Signal Transduction of Insulin

Biological Chemistry ◽

10.1515/bc.2000.140 ◽

2000 ◽

Vol 381 (11) ◽

pp. 1139-1141 ◽

Cited By ~ 1

Author(s):

A. Gypakis ◽

H.K. Wasner

Keyword(s):

Insulin Receptor ◽

Glucose Transport ◽

Functional Role ◽

Specific Inhibitor ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Downstream Signaling ◽

Cyclic Phosphate ◽

Phosphatidylinositol 3

Abstract It has been suggested that downstream signaling from the insulin receptor to the level of the protein kinases and protein phosphatases is accomplished by prostaglandylinositol cyclic phosphate (cyclic PIP), a proposed second messenger of insulin. However, evidence points also to both phosphatidylinositol 3-kinase, which binds to the tyrosine phosphorylated insulin receptor substrate-1, and the Ras complex in insulin's downstream signaling. We have examined whether a correlation exists between these various observations. It was found that wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, prevented insulin-induced, as well as cyclic PIP-induced activation of glucose transport, indicating that PI 3-kinase action on glucose transport involves downstream signaling of both insulin and cyclic PIP. Wortmannin has no effect on cyclic PIP synthase activity nor on the substrate production for cyclic PIP synthesis either, indicating that the functional role of PI 3-kinase is exclusively downstream of cyclic PIP.

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Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor to insulin receptor substrate 1

Molecular Endocrinology ◽

10.1210/me.9.4.435 ◽

1995 ◽

Vol 9 (4) ◽

pp. 435-442 ◽

Cited By ~ 6

Author(s):

V. Sanchez-Margalet

Keyword(s):

Insulin Receptor ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Receptor Substrate 1 ◽

Adaptor Molecule ◽

Phosphatidylinositol 3

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Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

Molecular and Cellular Biology ◽

10.1128/mcb.16.5.2195 ◽

1996 ◽

Vol 16 (5) ◽

pp. 2195-2203 ◽

Cited By ~ 91

Author(s):

D A Antonetti ◽

P Algenstaedt ◽

C R Kahn

Keyword(s):

Skeletal Muscle ◽

Insulin Receptor ◽

Tissue Distribution ◽

Cardiac Muscle ◽

Splice Variants ◽

Regulatory Subunit ◽

Insulin Receptor Substrate ◽

Insulin Receptor Substrate 1 ◽

Tissue Specific ◽

Sh2 Domains

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.

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SH3 domain of the phosphatidylinositol 3-kinase regulatory subunit is responsible for the formation of a sequestration complex with insulin receptor substrate-1

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2007.10.187 ◽

2008 ◽

Vol 365 (3) ◽

pp. 433-438 ◽

Cited By ~ 2

Author(s):

Yuichi Ikegami ◽

Kouichi Inukai ◽

Takuya Awata ◽

Tomoichiro Asano ◽

Shigehiro Katayama

Keyword(s):

Insulin Receptor ◽

Sh3 Domain ◽

Regulatory Subunit ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Receptor Substrate 1 ◽

Phosphatidylinositol 3

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The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4453 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4453-4465 ◽

Cited By ~ 170

Author(s):

S Pons ◽

T Asano ◽

E Glasheen ◽

M Miralpeix ◽

Y Zhang ◽

...

Keyword(s):

Insulin Receptor ◽

Regulatory Subunit ◽

Stable Complex ◽

Phosphatidylinositol 3 Kinase ◽

Sh2 Domains ◽

Cellular Processes ◽

Src Homology 2 ◽

Src Homology ◽

And Function ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling.

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Phosphatidylinositol 3′-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase

Biochemical Journal ◽

10.1042/bj3350397 ◽

1998 ◽

Vol 335 (2) ◽

pp. 397-404 ◽

Cited By ~ 12

Author(s):

Keith A. CENGEL ◽

Rosanne E. KASON ◽

Gregory G. FREUND

Keyword(s):

Insulin Receptor ◽

Immune Complexes ◽

Kinase Activity ◽

Sh2 Domain ◽

Serine Phosphorylation ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Serine Kinase ◽

Insulin Receptor Substrate 1 ◽

Pas Kinase

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3´-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase–IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate.

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Detection of a 60 kDa tyrosine-phosphorylated protein in insulin-stimulated hepatoma cells that associates with the SH2 domain of phosphatidylinositol 3-kinase

Biochemical Journal ◽

10.1042/bj3080579 ◽

1995 ◽

Vol 308 (2) ◽

pp. 579-583 ◽

Cited By ~ 12

Author(s):

K L Milarski ◽

D F Lazar ◽

R J Wiese ◽

A R Saltiel

Keyword(s):

Signal Transduction ◽

Insulin Receptor ◽

Hepatoma Cells ◽

Sh2 Domain ◽

Regulatory Subunit ◽

Dependent Manner ◽

Phosphatidylinositol 3 Kinase ◽

Phosphorylated Protein ◽

Tyrosine Phosphorylated Protein ◽

Phosphatidylinositol 3

Activation of the tyrosine kinase activity of the insulin receptor by autophosphorylation leads to phosphorylation of cellular substrates on tyrosine. Thus far, the best characterized is the insulin receptor substrate (IRS) 1, which has been proposed to serve as a docking protein for other molecules involved in signal transduction. A number of other proteins that become phosphorylated in response to insulin have been identified, some of which are reported to be tissue-specific. A 60 kDa phosphoprotein has been detected in adipocytes after insulin stimulation [Lavan and Lienhard (1993) J. Biol. Chem. 268, 5921-5928]. We have identified a protein of similar molecular mass in rat hepatoma cells transfected with the human insulin receptor. The 60 kDa protein in hepatoma cells is tyrosine-phosphorylated in response to insulin in a dose-dependent manner, with maximal phosphorylation occurring at 50 nM insulin. Although the dose-response of p60 phosphorylation mirrors that of IRS-1, the time course is slightly slower, with maximal phosphorylation observed 5 min after addition of insulin. Like the adipocyte protein, the 60 kDa protein detected in liver cells binds to the SH2 domain of the p85 regulatory subunit of phosphatidylinositol 3-kinase, but not to other SH2 domains. Binding of p60 to p85 is similar to the interaction between p85 and IRS-1 in that a tyrosine-phosphorylated peptide containing the YVXM motif can inhibit the association. The presence of this 60 kDa tyrosine-phosphorylated protein in adipocytes and hepatoma cells suggests that it represents another important intermediate in the insulin-receptor signal-transduction pathway.

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Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7418 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7418-7428 ◽

Cited By ~ 309

Author(s):

X J Sun ◽

D L Crimmins ◽

M G Myers ◽

M Miralpeix ◽

M F White

Keyword(s):

Insulin Receptor ◽

Tyrosine Phosphorylation ◽

Regulatory Elements ◽

Sh2 Domain ◽

Insulin Receptor Substrate ◽

Insulin Stimulation ◽

Intact Cells ◽

Tyrosine Residues ◽

Sh2 Domains ◽

Amino Terminal

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.

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Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7466-7475.1994 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7466-7475

Author(s):

B H Jhun ◽

D W Rose ◽

B L Seely ◽

L Rameh ◽

L Cantley ◽

...

Keyword(s):

Fusion Protein ◽

Dna Synthesis ◽

Insulin Receptor ◽

Signal Transduction Pathway ◽

Sh2 Domain ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Receptor Substrate 1 ◽

Phosphatidylinositol 3

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.

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