Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M501439200 on April 22, 2005

J. Biol. Chem., Vol. 280, Issue 24, 23173-23183, June 17, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/24/23173    most recent
M501439200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J.-a
Right arrow Articles by Quon, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J.-a
Right arrow Articles by Quon, M. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Phosphorylation of Ser24 in the Pleckstrin Homology Domain of Insulin Receptor Substrate-1 by Mouse Pelle-like Kinase/Interleukin-1 Receptor-associated Kinase

CROSS-TALK BETWEEN INFLAMMATORY SIGNALING AND INSULIN SIGNALING THAT MAY CONTRIBUTE TO INSULIN RESISTANCE*

Jeong-a Kim{ddagger}, Deborah C. Yeh{ddagger}, Marel Ver{ddagger}, Yunhua Li{ddagger}, Andrea Carranza{ddagger}, Thomas P. Conrads§, Timothy D. Veenstra§, Maureen A. Harrington¶, and Michael J. Quon{ddagger}||

From the {ddagger}Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland 20892, §SAIC-Frederick, Inc., Laboratory of Proteomics and Analytical Technologies, Mass Spectrometry Center, NCI, National Institutes of Health, at Frederick, Frederick, Maryland 21702-1201, and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and Walther Cancer Institute, Indianapolis, Indiana 46202

Received for publication, February 7, 2005 , and in revised form, March 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammation contributes to insulin resistance in diabetes and obesity. Mouse Pelle-like kinase (mPLK, homolog of human IL-1 receptor-associated kinase (IRAK)) participates in inflammatory signaling. We evaluated IRS-1 as a novel substrate for mPLK that may contribute to linking inflammation with insulin resistance. Wild-type mPLK, but not a kinase-inactive mutant (mPLK-KD), directly phosphorylated full-length IRS-1 in vitro. This in vitro phosphorylation was increased when mPLK was immunoprecipitated from tumor necrosis factor (TNF)-{alpha}-treated cells. In NIH-3T3IR cells, wild-type mPLK (but not mPLK-KD) co-immunoprecipitated with IRS-1. This association was increased by treatment of cells with TNF-{alpha}. Using mass spectrometry, we identified Ser24 in the pleckstrin homology (PH) domain of IRS-1 as a specific phosphorylation site for mPLK. IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser24) had impaired ability to associate with insulin receptors resulting in diminished tyrosine phosphorylation of IRS-1 and impaired ability of IRS-1 to bind and activate PI-3 kinase in response to insulin. IRS-1-S24D also had an impaired ability to mediate insulin-stimulated translocation of GLUT4 in rat adipose cells. Importantly, endogenous mPLK/IRAK was activated in response to TNF-{alpha} or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser24, we found that interleukin-1 or TNF-{alpha} treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser24. We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-{alpha}-regulated phosphorylation at Ser24 in the pleckstrin homology domain of IRS-1 by mPLK/IRAK represents an additional mechanism for cross-talk between inflammatory signaling and insulin signaling that may contribute to metabolic insulin resistance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biochemical, physiological, and epidemiological studies implicate pro-inflammatory cytokines (e.g. TNF-{alpha},1 IL-1{beta}, and IL-6) in the development of insulin resistance and the pathophysiology of type 2 diabetes and obesity (1–8). These studies suggest an intriguing link between inflammation and metabolic dysregulation. Indeed, I{kappa}B kinase {beta} (IKK{beta}), a critical mediator of inflammatory signaling pathways activating NF-{kappa}B, has been identified as an important inhibitor of metabolic insulin signaling pathways (9–12). Inactivation of IKK{beta} signaling increases insulin sensitivity, whereas overexpression of IKK{beta} or activation of IKK{beta} by pro-inflammatory cytokines (e.g. TNF-{alpha}) leads to insulin resistance (9, 12). Similarly, JNK is another inflammatory signaling molecule that may play a role in the insulin resistance of obesity (13). One potential explanation for these observations is cross-talk between inflammatory signaling and metabolic insulin signaling pathways.

Metabolic actions of insulin such as enhanced glucose uptake into skeletal muscle and adipose tissue are regulated by activation of the insulin receptor tyrosine kinase and subsequent tyrosine phosphorylation of IRS-1. Although other IRS family members including IRS-2, -3, and -4 share similar structures, IRS isoforms have both overlapping and distinct functions (14). For example, IRS-1 knock-out mice have a phenotype of growth retardation and insulin resistance, whereas IRS-2 knock-out mice have a phenotype of frank diabetes due to {beta}-cell failure (15, 16). Thus, IRS-1 seems to be the most important IRS isoform for mediating the metabolic effects of insulin in skeletal muscle and fat. Specific tyrosine-phosphorylated motifs on IRS-1 serve as docking sites for binding and activation of PI-3 kinase. Lipid products of PI 3-kinase stimulate activation of the serine kinase phosphoinositide-dependent protein kinase 1 that then phosphorylates and activates downstream serine kinases including Akt (protein kinase B) and protein kinase C{zeta}. These downstream PI 3-kinase-dependent signaling molecules promote translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface, resulting in increased glucose uptake into the cell (for review of metabolic insulin signaling pathways see Ref. 14). In addition to tyrosine phosphorylation sites on IRS-1 that are necessary for propagation of metabolic insulin signaling, IRS-1 contains numerous serine residues that are phosphorylated in response to treatment of cells with a variety of agents including insulin and TNF-{alpha} (17–22). Increased serine phosphorylation of IRS-1 is generally associated with impaired tyrosine phosphorylation of IRS-1 by the insulin receptor, decreased IRS-1-associated PI 3-kinase binding and activity, and insulin resistance (17, 20, 21, 23, 24). Interestingly, a number of PI 3-kinase-dependent serine kinases including Akt, protein kinase C{zeta}, and glycogen synthase kinase 3 can phosphorylate IRS-1 on serine residues and may participate in feedback regulation of insulin signal transduction (25–28). Moreover, activation of pro-inflammatory kinases IKK{beta} and JNK leads to increased serine phosphorylation of IRS-1 at Ser307 and insulin resistance (29–32). Other serine residues in the C-terminal region of IRS-1 (e.g. Ser612) also undergo phosphorylation in response to pro-inflammatory cytokines (33). The identity of kinases that directly phosphorylate these specific serine residues on IRS-1 in response to activation of IKK{beta} or treatment with pro-inflammatory cytokines remains unclear. In addition, other unidentified serine phosphorylation sites on IRS-1 and additional pro-inflammatory kinases may also participate in cross-talk between inflammatory signaling and insulin signaling. Thus, detailed molecular mechanisms relating inflammation with insulin resistance remain incompletely understood.

Mouse Pelle-like kinase (mPLK) is a Ser/Thr kinase homologous to the Drosophila innate immune kinase Pelle and is the mouse homologue of the human IL-1 receptor-associated kinase-1 (IRAK-1) (34). In response to TNF-{alpha} or IL-1{beta} stimulation, IRAK-1 is recruited to the IL-1 receptor complex through the adaptor MyD88 (35), where IRAK-1 subsequently becomes phosphorylated. IRAK-1 then interacts with TNF receptor-associated factor 6, TGF{beta}-activating kinase, and NF-{kappa}B-inducing kinase (36–38) resulting in activation of IKK{beta} and JNK and finally NF-{kappa}B (39–41). Although IRAK-1 is required for activation of NF-{kappa}B in response to IL-1 (42), the requirement for IRAK-1 kinase activity in NF-{kappa}B activation is controversial (41, 43, 44). mPLK catalytic activity is required for full activation of the TNF-{alpha} pathway, leading to NF-{kappa}B activation. However, physiological substrates for mPLK/IRAK-1 have not been conclusively identified. Because mPLK participates in activation of IKK{beta}, JNK, and NF-{kappa}B, we hypothesized that IRS-1 may be a novel substrate for mPLK, which mediates cross-talk between pro-inflammatory signaling and insulin signaling pathways. In the present study we identified Ser24 in the pleckstrin homology (PH) domain of IRS-1 as a phosphorylation site for mPLK that may contribute to insulin resistance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs
mPLK-WT and mPLK-KD—cDNA for full-length wild type mPLK with a C-terminal Myc-epitope tag was subcloned into pcDNA3.1 as described (45). mPLK-KD is a kinase-inactive point mutant of mPLK-WT (D358N) as described (41).

GST-IRS-1N, GST-IRS-1M, and GST-IRS-1C—GST fusion constructs containing fragments of rat IRS-1 (N, aa 2–516; M, aa 526–859; C, aa 900–1235) were generous gifts from Xiao Jian Sun (46).

IRS-1-WT—cDNA for human IRS-1 with a C-terminal HA-epitope tag was subcloned into pCIS2 mammalian expression vector as described (47).

IRS-1-S24A, IRS-1-S24D, IRS-1-S24E—Point mutants of IRS-1-WT were created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.

GST-IRS-1-(2–336), GST-IRS-1-(2–336)-S24A—Expression vectors for GST fusion proteins containing aa 1–336 of hIRS-1 and the corresponding S24A point mutant were constructed. Human IRS-1 cDNA template was amplified by PCR. Primers 5'-cgc gga tcc GCG AGC CCT CCG GAG AGC-3' and 5'-AT GGT GCC TTC GCC GTC ACT-3' were used for PCR. The PCR product was gel-purified, digested with Bam H1, and ligated into pGex 4T-1 (Amersham Biosciences) that was pre-digested with Bam H1 and SmaI. GST-IRS-1-(1–336)-S24A was constructed from GST-IRS-1-(1–336) using the QuikChange site-directed mutagenesis kit. DNA sequences for both wild type and mutants of GST-IRS-1 constructs were confirmed by direct sequencing.

GLUT4-HA—An expression vector for human GLUT4 with the influenza hemagglutinin epitope (HA1) inserted in the first exofacial loop of GLUT4 was used as described (48).

Antibodies—Anti-IRS-1, anti-phosphotyrosine (4G10), and the anti-p85 subunit of PI 3-kinase antibodies were purchased from Upstate Biotechnology, Inc. (Charlottesville, VA), anti-IRAK-1 antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and anti-GST antibody was purchased from Amersham Biosciences (Piscataway, NJ). Anti-c-Myc antibody was purchased from Roche Applied Science (Indianapolis, IN). Anti-HA antibody was purchased from Covance (Denver, PA).

Cell Culture and Transfection
NIH-3T3 fibroblasts stably overexpressing human insulin receptors (NIH-3T3IR), Cos-7, HEK293, HepG2, and FaO cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine in a humidified atmosphere with 5% CO2. Bovine aortic endothelial cells (Clonetics Corp., San Diego, CA) in primary culture were grown in EGM-2 MV (Cambrex Biosciences, Walkersville, MD) as described (49) and used at passages 3–4. The evening before transfection cells were seeded in 60-mm dishes at 50% confluence. Polyfect (Qiagen Inc., Valencia, CA) was used to transiently transfect cells according to the manufacturer's instructions. One day after transfection cells were serum-starved overnight and then treated without or with TNF-{alpha} or insulin as described in figure legends. Rat adipose cells in primary culture were prepared and transfected as previously described (50, 51).

Cell Lysis and Immunoblotting
Before cell lysis, cells were briefly washed with ice-cold phosphate-buffered saline. Cells were lysed with lysis buffer containing 50 mM Tris, pH 7.2, 125 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM NaVO3, 20 mM NaF, 1 mM sodium pyrophosphate, and complete protease inhibitor mixture (Roche Applied Science). Cell debris were removed by centrifugation at 17,000 x g for 10 min at 4 °C. Samples were then boiled with Laemmli sample buffer for 5 min, and proteins were resolved by 10% SDS-PAGE. Samples were immunoblotted by standard methods, and blots were quantified by scanning densitometry (Molecular Dynamics).

Purification of GST Fusion Proteins
Bacteria transformed with GST-IRS-1 constructs were grown in LB media containing 50 mg/ml ampicillin. This starter culture was diluted 1/10 in to 100 ml of LB medium containing 50 mg/ml ampicillin and grown at 37 °C for 2 h with vigorous shaking (300 rpm) until the culture reached A 0.6. GST-IRS-1 fusion proteins were induced with 0.1 mM isopropyl 1-thio-{beta}-D-galactopyranoside for 2 h at 37 °C. Bacteria were pelleted by centrifugation at 2236 x g for 10 min. The pellet was resuspended in 4 ml of ice-cold phosphate-buffered saline, and cells were lysed by 20-s/10-s-interval sonication. The lysate was centrifuged at 15,115 x g for 10 min, and the supernatant was incubated with 0.6 ml of 50% glutathione-Sepharose 4B slurry at 4 °C for 30 min. Sepharose beads were washed with ice-cold phosphate-buffered saline 3 times, and GST-IRS-1 fusion proteins were eluted with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0. In some cases proteins were concentrated with a YM-10 column (Millipore Corp., Bedford, MA).

In Vitro Kinase Assays
Cos-7 cells or NIH-3T3IR cells were transiently transfected with empty vector (control), mPLK-WT, or mPLK-KD. In some experiments cells were serum-starved overnight and then treated without or with TNF-{alpha} (10 ng/ml, 5 min). Recombinant mPLK was immunoprecipitated by incubating cell lysates (1 mg of total protein) with 1 µg of anti-Myc antibody for 2 h at 4 °C followed by incubation with protein A/G-agarose beads (Santa Cruz Biotechnology) at 4 °C for 1 h. The immunocomplex was washed once with cell lysis buffer, once with buffer B (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40), and twice with buffer C (20 mM Tris. pH 7.4, 150 mM NaCl). The immunocomplex was then incubated in kinase assay buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM dithiothreitol, 50 µM ATP) with 10 µCi of [{gamma}32P]ATP and either purified full-length IRS-1 (1 µg) (Upstate Biotechnology) or purified GST-IRS-1 fusion proteins (2 µg) for 1 h at 37 °C. The reaction was stopped by adding Laemmli sample buffer and boiling for 5 min. Samples were then subjected to 10% SDS-PAGE, transferred to nitrocellulose membranes, and exposed to x-ray film or phosphor screens for autoradiography or PhosphorImager analysis (Storm 860, Amersham Biosciences Corp.).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1.
mPLK/IRAK is expressed in insulin target cells. A, using an anti-IRAK antibody, we detected expression of endogenous mPLK/IRAK at the expected molecular weight by immunoblotting cell lysates of primary rat adipose cells (lane 1), primary bovine aortic endothelial cells (BAEC) (lane 2), NIH-3T3IR cells (lane 3), and COS-7 cells (lane 4). In each lane, 35 µg of total protein was loaded on the gel. B, using a monoclonal antibody that specifically detects activated IRAK (52), we observed the activation of endogenous IRAK in response to IL-1{beta} (4 ng/ml, 10 min) or TNF-{alpha} treatment (20 ng/ml, 10 min) in primary mouse adipose cells and bovine aortic endothelial cells. Thirty µg of total protein was loaded on each lane of the gel. Activated IRAK was immunoblotted with monoclonal anti-IRAK antibody (BD Bioscience; catalog #610754), and total IRAK was immunoblotted with anti-IRAK antibody (Santa Cruz Biotechnology). Representative blots are shown from experiments that were repeated independently twice. P-, phosphorylated.

 
Assessing IRS-1-associated PI-3 Kinase Binding and Activity
NIH-3T3IR cells transiently transfected with IRS-1-WT, IRS-1-S24A, IRS-1-S24D, or IRS-1-S24E were serum-starved overnight and treated without or with insulin (100 nM, 5 min). Cell lysates were then subjected to immunoprecipitation with anti-HA antibody to recover recombinant IRS-1 proteins. 4/5 of each sample was used for detection of co-immunoprecipitated p85 subunit of PI 3-kinase by immunoblotting with anti-p85 antibody. The remainder of the sample immunoprecipitate was used to detect IRS-1-associated PI 3-kinase activity. For each sample, 10 µg of sonicated phosphatidylinositol substrate (Sigma) was incubated with 50 µl of PI 3-kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.3 mM EGTA, 10 mM MgCl2) and with 10 µCi of [{gamma}32P]ATP for 10 min at 37 °C. The reaction was stopped by adding 100 µl of 0.1 N HCl. Phospholipid was extracted with 200 µl of CHCl3/CH3OH (1:1). The organic phase was collected and applied to silica gel thin layer chromatography plates (Whatman) pre-activated with 1% potassium oxalate. Thin layer chromatography was performed in CHCl3/CH3OH/H2O/NH4OH (60:47:12.3:2). The plates were dried, and results were visualized by autoradiography. Quantification was done by PhosphorImager analysis, and results were normalized for the amount of IRS-1 recovered by immunoprecipitation.

Co-immunoprecipitation
Cell lysates were incubated with primary antibody at 4 °C for 2 h. The immune complex was pulled down with protein A/G beads. The beads were washed three times with cell lysis buffer, and then samples were mixed with SDS-sample buffer and boiled for 5 min. The samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blotted with antibodies as indicated.

GLUT4 Translocation Assay
Rat adipose cells were co-transfected by electroporation with GLUT4-HA and either IRS-1-WT or IRS-1-S24D as described (50, 51). After 20 h cells were processed and treated with insulin at final concentrations of 0, 0.07, or 60 nM for 30 min at 37 °C. Cell surface epitope-tagged GLUT4 was determined using monoclonal anti-HA antibody (HA-11, Covance) in conjunction with 125I-labeled sheep anti-mouse IgG as described (51).

Mass Spectrometry Analysis
One µl of tryptic-digested IRS-1 sample was co-crystallized with 1 µl of {alpha}-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 1% trifluoroacetic acid and spotted directly on a stainless steel matrix-assisted laser desorption ionization (MALDI) plate. Mass spectra were acquired using an Applied Biosystems 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA). For all mass spectra the laser frequency was 200 Hz, and for collision-induced dissociation the collision energy was 1 keV (air was used as the collision gas). MALDI spectra were internally calibrated (<20 ppm) using trypsin autolysis products. Post-acquisition base-line correction and smoothing was carried out using the software provided with the instrument. Spectra were submitted to Mascot (matrixscience.com) for peptide mass fingerprinting.

Phospho-specific Antibody for Ser24 in IRS-1
The phospho-peptide DVRKVGYLRKPKpSMHK (pS, phosphorylated Ser) was synthesized and conjugated to keyhole limpet hemocyanin as an antigen. New Zealand White rabbits were immunized and boosted three times with the conjugate. Anti-serum was further purified by collecting through an affinity column that was coupled with the non-phosphorylated DVRKVGYLRKPKSMHK peptide.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Insulin Target Cells Express mPLK/IRAK—We first examined whether mPLK (or its orthologue such as IRAK) is endogenously expressed in cells from metabolic and vascular insulin target tissues. Endogenous IRAK was detected in lysates of both rat adipose cells and bovine aortic endothelial cells (BAEC) by immunoblotting with anti-IRAK antibody (Fig. 1A). In addition, we also detected endogenous mPLK/IRAK in COS-7 cells and NIH-3T3IR cells. Using an antibody that can detect the activated form of mPLK/IRAK (52), we found that IL-1{beta} or TNF-{alpha} treatment of primary adipose cells or endothelial cells activated endogenous mPLK/IRAK in insulin target cells (Fig. 1B). Interestingly, treatment of Fao hepatoma cells with insulin also activated mPLK/IRAK at levels comparable with those observed in response to IL-1{beta} (data not shown).

IRS-1 Is a Direct Substrate for mPLK in Vitro—We next tested whether mPLK can directly phosphorylate IRS-1 in vitro. An immune-complex kinase assay was performed using full-length purified IRS-1 as substrate and recombinant mPLK-WT or mPLK-KD immunoprecipitated from transiently transfected NIH-3T3IR cells treated without or with TNF-{alpha} (Fig. 2). IRS-1 underwent significant phosphorylation only in the presence of mPLK-WT (Fig. 2, A and C, lanes 3 and 4) but not when incubated with mPLK-KD (A and C, lanes 5 and 6) or control samples (A and C, lanes 1 and 2). Moreover, mPLK-WT immunoprecipitated from cells treated with TNF-{alpha} was able to phosphorylate IRS-1 to a greater extent than mPLK-WT from untreated cells (Fig. 2, A and C, cf. lanes 3 and 4). As expected, mPLK-WT (but not mPLK-KD) underwent autophosphorylation that was increased by TNF-{alpha} treatment (Fig. 2A, lower panel, lanes 3 and 4). Immunoblotting samples with anti-IRS-1 and anti-IRAK antibodies demonstrated comparable amounts of substrate and enzyme in each in vitro kinase assay (Fig. 2B). These results suggest that IRS-1 can function as a direct substrate for mPLK and that TNF-{alpha} treatment increases the kinase activity of mPLK toward IRS-1. This raises the possibility that mPLK may mediate cross-talk between inflammatory signaling and insulin signaling pathways.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 2.
IRS-1 is a direct substrate for mPLK in vitro. Cos-7 cells were transiently transfected with empty vector (Control), Myc-tagged mPLK-WT, or mPLK-KD and then treated without or with TNF-{alpha} (10 ng/ml, 5 min). Recombinant mPLK immunoprecipitated from cell lysates using an anti-myc Ab was then used in an immune-complex kinase assay with purified IRS-1 as the substrate as described under "Materials and Methods." A, autoradiogram from a representative immune-complex kinase assay. B, samples shown in panel A were immunoblotted (IB) with antibodies against IRS-1 and mPLK to demonstrate comparable recovery of enzyme and substrate in each sample. C, quantification of results in panel A, normalized for IRS-1 recovery in panel B. Results shown are the mean ± S.E. for five independent experiments. TNF-{alpha} treatment significantly enhanced the ability of mPLK-WT to phosphorylate IRS-1 (cf. lanes 3 and 4, p < 0.05).

 
mPLK Interacts with IRS-1 in Intact Cells—To test whether IRS-1 can interact with mPLK in intact cells, we transiently co-transfected HEK293 cells with HA-tagged IRS-1 and either mPLK-WT or mPLK-KD. Transfected cells were treated without or with TNF-{alpha}, and then anti-HA immunoprecipitates were immunoblotted with anti-IRAK antibody (Fig. 3). Interestingly, only wild-type mPLK (but not the kinase dead mutant) co-immunoprecipitated with IRS-1 (Fig. 3, top panel). Moreover, the amount of mPLK-WT co-immunoprecipitated with IRS-1 was significantly increased in samples from cells treated with TNF-{alpha} (Fig. 3, cf. lanes 1 and 3). To verify that TNF-{alpha} treatment was effective, we immunoblotted cell lysates with anti-I{kappa}B{alpha} antibody. As expected, TNF-{alpha} treatment resulted in degradation of I{kappa}B{alpha} (Fig. 3, bottom panel). These results suggest that in intact cells, IRS-1 can interact with mPLK in a TNF{alpha}-regulated manner that depends on kinase activity of mPLK.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 3.
TNF-{alpha} regulates interaction of mPLK with IRS-1 in HEK293 cells. HEK293 cells were transiently co-transfected with IRS-1-HA and either mPLK-WT or mPLK-KD. Transfected cells were treated without or with TNF-{alpha} (20 ng/ml) for 15 min. Recombinant HA-tagged IRS-1 was immunoprecipitated (IP) from cell lysates with an anti-HA Ab, and samples were subjected to SDS-PAGE and immunoblotting (IB). mPLK-WT (but not mPLK-KD) co-immunoprecipitated with IRS-1, and this interaction was significantly enhanced after cells were treated with TNF-{alpha}. Comparable recovery of IRS-1 in each sample and comparable expression of recombinant mPLK constructs were demonstrated by immunoblotting (middle panels). Note that mPLK-KD runs on the gel as a more compact band with faster mobility than mPLK-WT presumably because the kinase-dead mutant does not undergo autophosphorylation. Immunoblotting with anti-I{kappa}B{alpha} demonstrates that TNF-{alpha} treatment caused degradation of I{kappa}B{alpha} as expected (lower panel). Representative results are shown from experiments that were repeated independently three times.

 



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 4.
mPLK phosphorylates only N-terminal fragment of GST-IRS-1 (aa 2–516) in vitro. NIH-3T3IR cells were transiently transfected with empty vector (Control), Myc-tagged mPLK-WT, or mPLK-KD. Recombinant mPLK immunoprecipitated from cell lysates using an anti-Myc Ab was then used in an immune-complex kinase assay with purified GST-IRS-1 fragments (N, aa 2–516; M, aa 526–859; C, aa 900–1235) as described under "Materials and Methods." The autoradiogram demonstrates that only the N-terminal fragment of IRS-1 is phosphorylated by mPLK-WT. Comparable recovery of mPLK enzyme and IRS-1 substrate for each of the samples is demonstrated by immunoblotting (IB) with anti-Myc Ab and anti-GST antibody, respectively. Representative results are shown from experiments that were repeated independently five times.

 
Ser24 in the PH Domain of IRS-1 Is the Major mPLK Phosphorylation Site—To begin to identify a specific mPLK phosphorylation site in IRS-1, we used GST-IRS-1 fusion proteins containing N-terminal, C-terminal, or middle fragments of IRS-1 as substrates in our in vitro kinase assays. Middle and C-terminal fragments of IRS-1 were not phosphorylated by mPLK-WT (Fig. 4, lanes 7 and 8). Of note, only the N-terminal fragment of IRS-1, containing amino acids 2–516, was capable of undergoing phosphorylation by mPLK-WT (Fig. 4, lane 6). We next repeated the in vitro kinase assays with the N-terminal fragment of IRS-1 in the presence of unlabeled ATP and analyzed the samples using mass spectrometry. This analysis implicated Ser24 as a potential mPLK phosphorylation site (Fig. 5). Phosphorylation covalently modifies serine, threonine, and tyrosine residues at their hydroxy moieties, resulting in the addition of HPO3 (added mass of 79.9663 Da). The experimental masses from MALDI-TOF mass spectroscopy were compared with a list of theoretical IRS-1 tryptic peptide masses in which the Ser, Thr, and Tyr residues were phosphorylated. A match is considered if the difference between theoretical value and observed value is less than 25 ppm. The observed peptide at m/z 1603.776 (Fig. 5) was identified by this selection criteria and corresponds to VGpYLRKPKpSMHK (pY is phosphorylated Tyr, and pS is phosphorylated Ser). Ser24 is located in an exposed portion of the PH domain of IRS-1 (Fig. 6A). Moreover, this serine residue is conserved in IRS-1 among various species but not in IRS-2 (Fig. 6B). To confirm that Ser24 in IRS-1 is a specific phosphorylation site for mPLK, we repeated the in vitro kinase assay using a GST-IRS-1 fragment (aa 1–336) containing either the wild-type sequence or an S24A point mutation. Importantly, by contrast with the wild-type IRS-1 fragment, the S24A point mutant did not undergo significant phosphorylation by mPLK (Fig. 6C, cf. lanes 2 and 4). These results strongly suggest that Ser24 is a major and specific phosphorylation site on IRS-1 for mPLK.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 5.
Mass spectrometric analysis of IRS-1 before (A) and after (B) in vitro phosphorylation by mPLK. Both IRS-1 samples were digested with trypsin and analyzed by MALDI-TOF mass spectroscopy. Clearly shown is the appearance of the peak at m/z = 1603.776 after phosphorylation in vitro by mPLK that corresponds to the phosphopeptide VGpYLRKPKpSMHK (pY is phosphorylated Tyr, and pS is phosphorylated Ser). Additionally, the observed peak at 1619.784 (B) corresponds to the phosphopeptide oxidized at the methionine residue ({Delta} = 16 Da).

 



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 6.
mPLK specifically phosphorylates IRS-1 at Ser24 in vitro. A, Ser24 is located in an accessible region of the PH domain of IRS-1 (indicated in yellow and by the arrow). This figure was adapted from the Protein Data Bank (www.rcsb.org, ID code 1QQG). B, Ser24 is conserved in IRS-1 across species but not in the homologous region of IRS-2. C, NIH-3T3IR cells were transiently transfected with empty vector (control), Myc-tagged mPLK-WT, or mPLK-KD. Recombinant mPLK immunoprecipitated from cell lysates using an anti-Myc Ab was used in an immune-complex kinase assay with a purified GST-IRS-1 fragment containing aa 1–336 (either WT or S24A) as described under "Materials and Methods." Autoradiograms demonstrate that only the wild-type fragment but not the S24A mutant was phosphorylated by mPLK-WT. The membrane was stained with Ponceau S (lower panel) to demonstrate comparable amounts of GST-IRS-1 in each sample. Representative results are shown from experiments that were repeated independently three times.

 



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 7.
Endogenous IRS-1 is phosphorylated at Ser24 in response to IL-1{beta} or TNF-{alpha} treatment in intact cells. A, specificity of phospho-specific IRS-1 Ser24 antibody. HEK293 cells were transiently transfected with empty vector (control), Myc-tagged mPLK-WT, or mPLK-KD. Recombinant mPLK immunoprecipitated from cell lysates using an anti-Myc antibody was used in an immune-complex kinase assay with a purified GST-IRS-1 fragment containing aa 1–336 (either WT or S24A) as described under "Materials and Methods" (without labeled ATP). Samples were subjected to immunoblotting (IB) with a phospho-specific antibody that detects IRS-1 phosphorylated at Ser24 (upper panel). Only the wild-type fragment but not the S24A mutant was recognized by the phospho-specific antibody. The membrane was stained with Ponceau S (lower panel) to demonstrate comparable amounts of GST-IRS-1 in each sample (middle panel). Immunoblotting with anti-IRAK antibody demonstrated comparable amounts of mPLK in each sample (lower panel). B, Fao cells were treated with IL-1{beta} (4 ng/ml, 10 min) or TNF-{alpha} (20 ng/ml, 10 min), and then cell lysates containing 50 µg of total protein were subjected to SDS-PAGE and immunoblotted with anti-phospho-IRS-1 Ser24 antibody (upper panel). Another gel was run in parallel and immunoblotted for total IRS-1 (lower panel). Representative blots are shown from experiments that were repeated independently three times.

 
Phosphorylation of Endogenous IRS-1 at Ser24 in Intact Cells—To demonstrate that endogenous IRS-1 can undergo phosphorylation at Ser24 in an intact insulin target cell in response to manipulation of innate immune signaling pathways, we developed a phospho-specific antibody that can specifically detect IRS-1 when it is phosphorylated at Ser24. The specificity of this antibody was demonstrated by its ability to detect phosphorylation of Ser24 in a GST-IRS-1 fragment containing the wild-type sequence but not the S24A mutant after in vitro phosphorylation by wild-type mPLK (but not kinase-inactive mPLK) (Fig. 7A). Using this phospho-specific antibody, we found that IL-1{beta} or TNF-{alpha} treatment of Fao cells (a rat hepatoma cell line) causes increased phosphorylation of endogenous IRS-1 at Ser24 (Fig. 7B). We observed similar results in HepG2 cells (another liver cell line), and insulin treatment also caused increased phosphorylation of IRS-1 at Ser24 (data not shown). Thus, Ser24 is a bona fide serine phosphorylation site on IRS-1 whose phosphorylation is increased upon activation of the innate immune system in an insulin target cell.

Functional Role of Ser24—The PH domain of IRS-1 plays a crucial role in membrane localization of IRS-1 and the ability of IRS-1 to undergo tyrosine phosphorylation by the insulin receptor (53). Thus, it is possible that phosphorylation at Ser24 may alter PH domain function and result in impaired insulin signaling. We explored the functional role of Ser24 in IRS-1 by using point mutants with substitutions of alanine, aspartate, or glutamate for serine at position 24. The alanine substitution should prevent phosphorylation at Ser24, whereas substitutions of aspartate or glutamate were intended to mimic phosphorylation at Ser24. NIH-3T3IR cells transiently transfected with HA-tagged IRS-1 constructs were treated without or with insulin. Recombinant IRS-1 was immunoprecipitated from cell lysates, and samples were immunoblotted with anti-phosphotyrosine antibodies to evaluate tyrosine phosphorylation of IRS-1 and co-immunoprecipitated insulin receptor (Fig. 8). As expected, wild-type IRS-1 underwent robust tyrosine phosphorylation in response to insulin stimulation, and co-immunoprecipitation with the tyrosine-phosphorylated insulin receptor was also easily detectable (Fig. 8, lanes 1 and 2). Results with the S24A mutant of IRS-1 were similar to those obtained with wild-type IRS-1. By contrast, S24D and S24E mutants had an impaired ability to undergo tyrosine phosphorylation in response to insulin and a diminished capacity to co-immunoprecipitate with the tyrosine-phosphorylated IRS-1 (Fig. 8, lanes 5–8). These results suggest that phosphorylation of IRS-1 at Ser24 may impair the ability of IRS-1 to interact with the activated insulin receptor, resulting in diminished tyrosine phosphorylation of IRS-1 in response to insulin stimulation.

Tyrosine-phosphorylated IRS-1 propagates insulin signaling by binding and activating a variety of downstream signaling molecules including PI 3-kinase and Grb2. Therefore, we examined the ability of Ser24 point mutants of IRS-1 to bind the p85 regulatory subunit of PI 3-kinase and activate PI 3-kinase in response to insulin stimulation. As expected, insulin stimulated a significant increase in p85 binding and PI 3-kinase activity associated with wild-type IRS-1 (Fig. 9, lanes 1 and 2). Results with the S24A mutant of IRS-1 were comparable with those obtained with wild-type IRS-1 (Fig. 9, lanes 3 and 4). However, IRS-1-S24D and -S24E mutants had a significantly impaired ability to bind and activate PI 3-kinase in response to insulin stimulation (~50% decrease compared with control; Fig. 9, lanes 5–8). Quantification of the results from multiple independent experiments for p85 binding and PI 3-kinase activity associated with IRS-1 are shown in Figs. 9, B and C, respectively. Thus, phosphorylation of IRS-1 at Ser24 may also impair its ability to bind and activate PI 3-kinase and lead to insulin resistance. Similar results were observed with the PI 3-kinase-dependent serine kinase Akt. That is, phosphorylation of HA-tagged Akt at Ser473 in response to insulin was impaired in cells co-transfected with IRS-1-S24D and -S24E mutants (when compared with wild-type IRS-1) (data not shown). In addition, IRS-1-S24D and -S24E mutants also had and impaired ability to bind Grb2 in response to insulin (data not shown). Thus, multiple signaling pathways downstream from IRS-1 may be impaired by phosphorylation at Ser24.

To determine whether mPLK is capable of impairing IRS-1 function in intact cells, we co-expressed HA-tagged IRS-1 with vector control, mPLK-WT, or mPLK-KD and examined p85 binding to IRS-1 in response to insulin by co-immunoprecipitation (Fig. 10). As expected, insulin stimulated a significant increase in p85 binding to IRS-1 in control cells (Fig. 10A, lanes 1 and 4). Importantly, overexpression of mPLK-WT significantly decreased the amount of p85 co-immunoprecipitated with IRS-1 in response to insulin by ~40% (when compared with vector control) (Fig. 10A, lanes 4 and 5). The amount of p85 associated with IRS-1 in insulin-treated cells overexpressing mPLK-WT was also significantly less than that observed in cells expressing mPLK-KD (Fig. 10A, lanes 5 and 6). These data indicate that mPLK kinase activity can result in impaired function of IRS-1 similar in magnitude to that observed in IRS-1 mutants (S24D and S24E), which mimics phosphorylation at Ser24. Taken together, our data suggest that phosphorylation of IRS-1 by mPLK may result in impaired insulin signaling and insulin resistance.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 8.
Phosphorylation of IRS-1 at Ser24 may impair the ability of IRS-1 to interact with insulin receptor and undergo tyrosine phosphorylation. NIH-3T3IR cells transiently transfected with HA-tagged IRS-1-WT, IRS-1-S24A, S24D, or S24E mutants were treated without or with insulin (100 nM, 5 min). A, recombinant IRS-1 was immunoprecipitated from cell lysates with anti-HA antibody. Immunoprecipitated (IP) samples were immunoblotted (IB) with anti-phosphotyrosine ({alpha}-pY) antibody. Tyrosine-phosphorylated IRS-1 is shown in the top panel. Tyrosine-phosphorylated insulin receptor {beta} subunit (IR{beta}) is shown in the middle panel. Immunoblotting control for IRS-1 recovery is shown in the lower panel. Representative results are shown from experiments that were repeated independently three times. B, densitometric quantification of tyrosine-phosphorylated IRS-1. C, densitometric quantification of tyrosine-phosphorylated insulin receptor associated with IRS-1. Results in panels B and C are the mean ± S.E. of three independent experiments.

 
Insulin-stimulated translocation of GLUT4 in adipose cells is a PI 3-kinase- and Akt-dependent function (51, 54). Therefore, we evaluated the ability of Ser24 point mutants of IRS-1 to mediate translocation of GLUT4 in transfected rat adipose cells in primary culture. Cells co-transfected with GLUT4-HA and vector-only, wild-type IRS-1, or IRS-1-S24D were stimulated without or with increasing concentrations of insulin. The amount of GLUT4-HA recruited to the cell surface in response to insulin was measured using a double antibody assay. In control cells, insulin stimulated a dose-dependent increase in cell surface GLUT4-HA (Fig. 11). As reported previously (47), overexpression of IRS-1-WT caused a significant increase in basal cell surface GLUT4 (in the absence of insulin), whereas maximal insulin-stimulated translocation of GLUT4 was similar to results from control cells. Interestingly, cells overexpressing IRS-1-S24D had an impaired ability to recruit GLUT4 to the cell surface under both basal and insulin-stimulated conditions (when compared with cells overexpressing IRS-1-WT). Thus, the impaired tyrosine phosphorylation of IRS-1-S24D that leads to decreased binding and activation of PI 3-kinase and Akt has a functionally important metabolic consequence. These results suggests that phosphorylation at Ser24 by mPLK may impair critical metabolic actions of insulin including GLUT4 translocation.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 9.
Phosphorylation of IRS-1 at Ser24 may impair the ability of IRS-1 to bind and activate PI 3-kinase in response to insulin. NIH-3T3IR cells transiently transfected with HA-tagged IRS-1-WT, IRS-1-S24A, S24D, or S24E mutants were treated without or with insulin (100 nM, 5 min). Recombinant IRS-1 was immunoprecipitated (IP) from cell lysates with anti-HA antibody. A, co-immunoprecipitation of the p85 regulatory subunit of PI 3-kinase (top panel) and PI 3-kinase activity (middle panel) were detected by immunoblotting (IB) with anti-p85 antibody or a lipid kinase assay, respectively, as described under "Materials and Methods." Comparable recovery of IRS-1 in each sample was demonstrated by immunoblotting samples with anti-IRS-1 antibody (lower panel). B, densitometric quantification of p85 subunit binding to IRS-1. Insulin-stimulated association of p85 with IRS-1-WT is not significantly different from that with IRS-1-S24A (p > 0.6) but is significantly greater than that with IRS-1-S24D (p < 0.03) or IRS-1-S24E (p < 0.01). C, PhosphorImager quantification of the [32P]PIP3 product from lipid kinase assay. Insulin-stimulated association of PI 3-kinase activity with IRS-1-WT is not significantly different from that with IRS-1-S24A (p > 0.4) but is significantly greater than that with IRS-1-S24D (p < 0.0001) or IRS-1-S24E (p < 0.0002). Results in panels B and C are mean ± S.E. of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
mPLK/IRAK is a serine/threonine kinase involved in both TNF-{alpha} and IL-1{beta} signaling (39, 41, 55, 56). That is, mPLK/IRAK is responsive to stimulation by both IL-1{beta} and TNF-{alpha}. In IL-1{beta} signaling, mPLK/IRAK has been linked to pathways that activate both IKK{beta} and JNK (39, 55). Even though mPLK undergoes autophosphorylation and can interact with proteins such as NF-{kappa}B-inducing kinase and IKK{beta} (40, 41), direct substrates for mPLK (other than itself) have not yet been conclusively identified. Indeed, in the IL-1{beta} signaling pathway, some functions of mPLK such as activation of NF-{kappa}B may not require intact mPLK kinase activity (43, 44). In embryonic fibroblasts and macrophages isolated from mPLK knock-out mice, activation of NF-{kappa}B, p38, and JNK is impaired in response to IL-1{beta} (56, 57), and NF-{kappa}B activation is impaired in response to TNF-{alpha} (56). IL-6 and TNF-{alpha} production in response to IL-1{beta} is also significantly reduced in skin fibroblasts or macrophages from mPLK-null mice (56, 57). As a result of this impaired inflammatory response, IRAK-deficient mice are more resistant to lethal effects of endotoxin and lipopolysaccharide (58, 59). Although an immunological phenotype for IRAK knock-out mice has been defined, a metabolic phenotype for these mice has not yet been evaluated or reported. Our observation that mPLK/IRAK is endogenously expressed in metabolic and vascular insulin targets such as adipose tissue and endothelium raises the possibility that mPLK may mediate cross-talk between inflammatory signaling and insulin signaling that leads to insulin resistance.

IRS-1 Is a Novel Substrate for mPLK—In the present study we identified IRS-1 as a novel direct substrate for mPLK. Because we used an immune-complex kinase assay, it is possible that another kinase co-immunoprecipitating with mPLK is responsible for phosphorylation of IRS-1 in vitro. However, this seems highly unlikely because under similar conditions, the kinase-dead mutant of mPLK was unable to phosphorylate IRS-1. That is, phosphorylation of IRS-1 in our in vitro assay required intact kinase activity of mPLK. Moreover, the interaction of mPLK with IRS-1 in cells also depended on the presence of kinase activity in mPLK. IRS-1 represents the first direct substrate for mPLK (other than itself) that has been reported.

Phosphorylation of Endogenous IRS-1 at Ser24 in Intact Cells—Using a phospho-specific antibody that can detect phosphorylation of IRS-1 at Ser24, we demonstrated that TNF-{alpha} or IL-1{beta} treatment of Fao and HepG2 cells caused increased phosphorylation of endogenous IRS-1 at Ser24. These are liver cell lines. Thus, manipulation of the innate immune signaling pathway in an intact insulin target cell results in increased phosphorylation of endogenous IRS-1 at Ser24. This phospho-specific antibody may be a useful tool to study in vivo phosphorylation of IRS-1 at Ser24 in animals and humans in future studies.

Role of Ser24 Phosphorylation in IRS-1 Function—We identified the N-terminal portion of IRS-1 as the only fragment that undergoes phosphorylation by mPLK in vitro. Mass spectrometry of this fragment suggested that Ser24 in the PH domain of IRS-1 was a specific phosphorylation site for mPLK. We confirmed that Ser24 was indeed the major specific phosphorylation site for mPLK since the S24A point mutant did not undergo significant phosphorylation by mPLK in vitro. Ser24 is the first putative phosphorylation site identified within the PH domain of IRS-1. It is notable that Ser24 is located in an exposed region of the PH domain where it is accessible to kinases such as mPLK. Indeed, IRS-1 and mPLK interact in intact cells (as assessed by co-immunoprecipitation), and this interaction is enhanced by TNF-{alpha}. In addition, Ser24 is conserved across species, but it is not present in a homologous region of IRS-2. Thus, phosphorylation of IRS-1 at Ser24 by mPLK and other kinases may contribute to specificity of IRS-1 signaling.

The N-terminal region of IRS-1 contains both a PH domain and a phosphotyrosine binding (PTB) domain that cooperate in targeting IRS-1 to the insulin receptor upon stimulation by insulin. The PTB domain binds to the NPEY motif in the juxtamembrane region of the insulin receptor (60). By contrast, the PH domain may not directly interact with the insulin receptor but, rather, seems to be involved with localizing IRS-1 to phospholipids in the plasma membrane (53, 61). Indeed, there is evidence that the PH domain of IRS-1 is critical for coupling signaling from the insulin receptor through IRS-1 to PI 3-kinase (62–64). In addition, the PH domain of IRS-1 seems to be specific for this function since the substitution of heterologous PH domains from unrelated proteins into IRS-1 prevents tyrosine phosphorylation of IRS-1 by the insulin receptor (65). The role of the PH and PTB domains in IRS-1 may be more critical than in IRS-2 since deletion of the PH and PTB domains in IRS-2 does not impair the ability of IRS-2 to mediate cell proliferation in myeloid cells, whereas similar deletions in IRS-1 do impair IRS-1 function (66). The presence of a putative regulatory phosphorylation site in the PH domain of IRS-1 at Ser24 and the absence of such a phosphorylation site in a homologous region of IRS-2 are consistent with these recent observations. Interestingly, Arg28 in the PH domain of IRS-1 is crucial for the ability of IRS-1 to interact with phospholipids and the plasma membrane. Point mutants of IRS-1 with substitutions at Arg28 have an impaired ability to bind and activate PI 3-kinase in response to insulin (67). Interaction of acidic molecules with the PH domain of IRS-1 may depend in part on the basic residue Arg28 (68). Because Ser24 is in close proximity to Arg28, it is possible that phosphorylation at Ser24 may interfere with the PH domain function of IRS-1 and contribute to impaired insulin signaling and insulin resistance. PH domains of various signaling molecules share structural similarities that are important for membrane localization and interactions with phospholipids in the plasma membrane (69–73). It is possible that this regulatory mechanism is also operative in other signaling molecules that contain PH domains. Indeed, serine phosphorylation in the PH domain of diacylglycerol kinase {delta}1 at Ser22 and Ser26 inhibits its function and plasma membrane localization (74).



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 10.
Overexpression of mPLK-WT impairs insulin-stimulated association of p85 with IRS-1. NIH-3T3IR cells transiently co-transfected with HA-tagged IRS-1-WT and vector control (pCIS2), mPLK-WT, or mPLK-KD were treated without or with insulin (100 nM, 5 min). Recombinant IRS-1 was immunoprecipitated (IP) from cell lysates with anti-HA antibody. A, co-immunoprecipitation of the p85 regulatory subunit of PI 3-kinase (top panel) was detected by immunoblotting (IB) with anti-p85 antibody. Recovery of IRS-1 in each sample was detected by immunoblotting with anti-IRS-1 antibody (lower panel). B, densitometric quantification of p85 subunit binding to IRS-1 normalized for IRS-1 recovery from three independent experiments (mean ± S.E.). Insulin-stimulated association of p85 with IRS-1 in cells overexpressing mPLK-WT was significantly lower than that in the control group (lanes 4 and 5, p < 0.02) or in the cells expressing mPLK-KD (lanes 5 and 6, p < 0.04). There was also a modest decrease in insulin-stimulated p85 binding to IRS-1 in cells expressing mPLK-KD when compared with the vector control (lanes 4 and 6, p < 0.03).

 



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 11.
IRS-1-S24D mutant has an impaired ability to mediate translocation of GLUT4 in rat adipose cells. Rat adipose cells in primary culture were transiently co-transfected with GLUT4-HA and IRS-1-WT or IRS-1-S24D. Cells were then treated with insulin (0, 0.07 nM, 60 nM) for 30 min, and cell-surface GLUT4 was quantified as described under "Materials and Methods." Results shown are the mean ± S.E. of six independent experiments. IRS-1-S24D had an impaired ability to mediate translocation of GLUT4 under both basal and insulin-stimulated conditions when compared with wild-type IRS-1 (p < 0.02 by MANOVA).

 
We used point mutants S24D and S24E to mimic phosphorylation of IRS-1 at Ser24 in order to explore the functional importance of this mPLK phosphorylation site. Interestingly, S24D and S24E mutants of IRS-1 had an impaired ability to interact with the insulin receptor and consequently exhibited decreased tyrosine phosphorylation in response to insulin stimulation when compared with wild-type IRS-1. These results are consistent with an impairment of PH domain function caused by phosphorylation at Ser24. PI 3-kinase binding and activity associated with IRS-1 and subsequent activation of Akt were also impaired in the IRS-1 mutants that mimicked phosphorylation at Ser24. These defects in insulin signaling were manifested as a decreased ability to mediate translocation of GLUT4 in primary adipose cells. Thus, phosphorylation of IRS-1 at Ser24 in the PH domain of IRS-1 may have important consequences for impairing metabolic functions of insulin such as glucose uptake. The S24A mutant of IRS-1 behaved similarly to wild-type IRS-1 with respect to association with the insulin receptor and PI 3-kinase. If there was significant basal phosphorylation of IRS-1 at Ser24, one might expect that the S24A mutant would have enhanced binding and activation of PI 3-kinase when compared with wild-type IRS-1. Thus, our results suggest that basal phosphorylation of IRS-1 at Ser24 is not sufficient to affect function.

There are several other specific serine phosphorylation sites on IRS-1 that have previously been implicated in regulation of IRS-1 function. These include Ser302 (75), Ser307 (29, 31, 32, 75–78), Ser612/632/662/731 (23, 79, 80), Ser636/639 (81, 82), and Ser789 (24). These serine residues are close to the PTB domain or near tyrosine phosphorylation motifs that interact with SH2 domains of downstream signaling molecules. Thus, the mechanisms by which phosphorylation at these sites regulates insulin signaling may differ from a phosphorylation site in the PH domain of IRS-1.

Role of mPLK Kinase Activity—The importance of mPLK kinase activity in the function of mPLK is somewhat controversial. mPLK/IRAK is necessary for activation of NF-{kappa}B by IL-1{beta}, but its kinase activity does not appear to be required for this function (41, 43, 44). However, mPLK kinase activity is important for full activation of TNF-{alpha}-stimulated NF-{kappa}B activity (41). A definitive role for mPLK kinase activity has not been identified in part because direct substrates for mPLK were unknown. We demonstrated that the kinase activity of mPLK against IRS-1, mPLK autophosphorylation, and interactions of mPLK with IRS-1 in intact cells were all enhanced by treatment of cells with TNF-{alpha}. Importantly, overexpression of mPLK-WT caused a significant ~40% decrease in the amount of p85 associated with IRS-1 in response to insulin that was similar to the impairment observed with our IRS-1 mutants mimicking phosphorylation at Ser24. This impairment was also significant when compared with the effects of expressing mPLK-KD. Given that mPLK can phosphorylate IRS-1 at Ser24 in vitro, these data link kinase activity of mPLK with impairment in insulin signaling through IRS-1. Interestingly, when compared with control cells, there was a modest effect of mPLK-KD to decrease p85 binding to IRS-1 that may be the result of non-kinase-dependent actions of mPLK on activation of IKK-{beta} and JNK (39, 55). Elevated levels of TNF-{alpha} and IL-1{beta} are present in insulin-resistant subjects (83), and the ability of these two cytokines to impair insulin signaling pathways may involve activation of IKK{beta} (12) by distinct mechanisms via specific TNF receptor-associated factors for each cytokine (40, 84, 85). In addition to activating IKK{beta} and JNK, an additional mechanism by which TNF-{alpha} treatment enhances serine phosphorylation of IRS-1 may involve mPLK activation. Indeed, a novel function for the kinase activity of mPLK may be to mediate cross-talk between inflammatory signaling and insulin signaling pathways via serine phosphorylation of IRS-1.

Role of mPLK in Cross-talk between Inflammatory Signaling and Insulin Signaling—There are several potential mechanisms for mPLK to participate in cross-talk between inflammatory signaling and insulin signaling that lead to insulin resistance. First, mPLK is known to participate in activation of NF-{kappa}B and inflammatory signaling pathways involving IKK{beta} and JNK (39, 56), which in turn have been implicated in increased serine phosphorylation of IRS-1 and insulin resistance (9, 12, 13, 29, 31, 32). The role of mPLK in these pathways may be independent of mPLK kinase activity. In the present study we have now identified an additional mechanism whereby mPLK directly phosphorylates Ser24 in the PH domain of IRS-1. This leads to impaired PI 3-kinase-dependent insulin signaling and defects in metabolic actions of insulin. TNF-{alpha} treatment enhances the interaction of mPLK with IRS-1 and increases the activity of mPLK to phosphorylate IRS-1. Thus, pro-inflammatory states with elevated TNF-{alpha} levels that are associated with obesity and increased circulating levels of free fatty acids may mediate insulin resistance in part through serine phosphorylation of IRS-1 at Ser24 by mPLK/IRAK. Of interest, we also identified cross-talk in the other direction since insulin stimulation led to activation of mPLK/IRAK and increased phosphorylation of IRS-1 at Ser24. In addition to mPLK/IRAK, it is also possible that there are other serine kinases in the insulin signaling pathway that may phosphorylate IRS-1 at Ser24 in a negative feedback loop similar to other feedback pathways previously identified (25–28). In future studies it will be of interest to determine the phosphorylation status of Ser24 in relevant tissues from animals or humans with insulin resistance (e.g. in diabetes or obesity) and to follow changes in the phosphorylation status of Ser24 after therapeutic interventions.


   FOOTNOTES
 
* This work was supported by a Research Award from the American Diabetes Association (to M. J. Q.). The mass spectrometry was funded in part with Federal funds from NCI, National Institutes of Health, under Contract NO1-CO-12400. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Diabetes Unit, NC-CAM, National Institutes of Health, Bldg. 10, Rm. 6C-205, 10 Center Dr. MSC 1632, Bethesda, MD 20892-1632. Tel.: 301-496-6269; Fax: 301-402-1679; E-mail: quonm{at}nih.gov.

1 The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; IKK{beta}, I{kappa}B kinase {beta}; PI, phosphatidylinositol; PH, pleckstrin homology; mPLK, Mouse Pelle-like kinase; IRAK, IL-1 receptor-associated kinase; JNK, c-Jun N-terminal kinase; KD, kinase dead; WT, wild type; GST, glutathione S-transferase; aa, amino acids; HA, hemagglutinin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PTB, phosphotyrosine binding; Ab, antibody; Tx, transfection. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Xu, H., Barnes, G. T., Yang, Q., Tan, G., Yang, D., Chou, C. J., Sole, J., Nichols, A., Ross, J. S., Tartaglia, L. A., and Chen, H. (2003) J. Clin. Investig. 112, 1821-1830[CrossRef][Medline] [Order article via Infotrieve]
  2. Weisberg, S. P., McCann, D., Desai, M., Rosenbaum, M., Leibel, R. L., and Ferrante, A. W., Jr. (2003) J. Clin. Investig. 112, 1796-1808[CrossRef][Medline] [Order article via Infotrieve]
  3. Hotamisligil, G. S., Shargill, N. S., and Spiegelman, B. M. (1993) Science 259, 87-91[Abstract/Free Full Text]
  4. Hotamisligil, G. S., and Spiegelman, B. M. (1994) Diabetes 43, 1271-1278[Abstract]
  5. Uysal, K. T., Wiesbrock, S. M., Marino, M. W., and Hotamisligil, G. S. (1997) Nature 389, 610-614[CrossRef][Medline] [Order article via Infotrieve]
  6. Ventre, J., Doebber, T., Wu, M., MacNaul, K., Stevens, K., Pasparakis, M., Kollias, G., and Moller, D. E. (1997) Diabetes 46, 1526-1531[Abstract]
  7. Pickup, J. C., Mattock, M. B., Chusney, G. D., and Burt, D. (1997) Diabetologia 40, 1286-1292[CrossRef][Medline] [Order article via Infotrieve]
  8. Festa, A., D'Agostino, R., Jr., Howard, G., Mykkanen, L., Tracy, R. P., and Haffner, S. M. (2000) Circulation 102, 42-47[Abstract/Free Full Text]
  9. Yuan, M., Konstantopoulos, N., Lee, J., Hansen, L., Li, Z. W., Karin, M., and Shoelson, S. E. (2001) Science 293, 1673-1677[Abstract/Free Full Text]
  10. Shoelson, S. E., Lee, J., and Yuan, M. (2003) Int. J. Obes. Relat. Metab. Disord. 27, Suppl. 3, 49-52
  11. Hundal, R. S., Petersen, K. F., Mayerson, A. B., Randhawa, P. S., Inzucchi, S., Shoelson, S. E., and Shulman, G. I. (2002) J. Clin. Investig. 109, 1321-1326[CrossRef][Medline] [Order article via Infotrieve]
  12. Kim, J. K., Kim, Y. J., Fillmore, J. J., Chen, Y., Moore, I., Lee, J., Yuan, M., Li, Z. W., Karin, M., Perret, P., Shoelson, S. E., and Shulman, G. I. (2001) J. Clin. Investig. 108, 437-446[CrossRef][Medline] [Order article via Infotrieve]
  13. Hirosumi, J., Tuncman, G., Chang, L., Gorgun, C. Z., Uysal, K. T., Maeda, K., Karin, M., and Hotamisligil, G. S. (2002) Nature 420, 333-336[CrossRef][Medline] [Order article via Infotrieve]
  14. Nystrom, F. H., and Quon, M. J. (1999) Cell. Signal. 11, 563-574[CrossRef][Medline] [Order article via Infotrieve]
  15. Araki, E., Lipes, M. A., Patti, M. E., Bruning, J. C., Haag, B., III, Johnson, R. S., and Kahn, C. R. (1994) Nature 372, 186-190[CrossRef][Medline] [Order article via Infotrieve]
  16. Withers, D. J., Gutierrez, J. S., Towery, H., Burks, D. J., Ren, J. M., Previs, S., Zhang, Y., Bernal, D., Pons, S., Shulman, G. I., Bonner-Weir, S., and White, M. F. (1998) Nature 391, 900-904[CrossRef][Medline] [Order article via Infotrieve]
  17. Tanti, J. F., Gremeaux, T., van Obberghen, E., and Le Marchand-Brustel, Y. (1994) J. Biol. Chem. 269, 6051-6057[Abstract/Free Full Text]
  18. Ricort, J. M., Tanti, J. F., Van Obberghen, E., and Le Marchand-Brustel, Y. (1995) Diabetologia 38, 1148-1156[Medline] [Order article via Infotrieve]
  19. Kroder, G., Bossenmaier, B., Kellerer, M., Capp, E., Stoyanov, B., Muhlhofer, A., Berti, L., Horikoshi, H., Ullrich, A., and Haring, H. (1996) J. Clin. Investig. 97, 1471-1477[Medline] [Order article via Infotrieve]
  20. Kanety, H., Feinstein, R., Papa, M. Z., Hemi, R., and Karasik, A. (1995) J. Biol. Chem. 270, 23780-23784[Abstract/Free Full Text]
  21. Hotamisligil, G. S., Peraldi, P., Budavari, A., Ellis, R., White, M. F., and Spiegelman, B. M. (1996) Science 271, 665-668[Abstract]
  22. Paz, K., Hemi, R., LeRoith, D., Karasik, A., Elhanany, E., Kanety, H., and Zick, Y. (1997) J. Biol. Chem. 272, 29911-29918[Abstract/Free Full Text]
  23. Mothe, I., and Van Obberghen, E. (1996) J. Biol. Chem. 271, 11222-11227[Abstract/Free Full Text]
  24. Qiao, L. Y., Zhande, R., Jetton, T. L., Zhou, G., and Sun, X. J. (2002) J. Biol. Chem. 277, 26530-26539[Abstract/Free Full Text]
  25. Paz, K., Liu, Y. F., Shorer, H., Hemi, R., LeRoith, D., Quon, M. J., Kanety, H., Seger, R., and Zick, Y. (1999) J. Biol. Chem. 274, 28816-28822[Abstract/Free Full Text]
  26. Li, J., DeFea, K., and Roth, R. A. (1999) J. Biol. Chem. 274, 9351-9356[Abstract/Free Full Text]
  27. Ravichandran, L. V., Esposito, D. L., Chen, J., and Quon, M. J. (2001) J. Biol. Chem. 276, 3543-3549[Abstract/Free Full Text]
  28. Eldar-Finkelman, H., and Krebs, E. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9660-9664[Abstract/Free Full Text]
  29. Aguirre, V., Uchida, T., Yenush, L., Davis, R., and White, M. F. (2000) J. Biol. Chem. 275, 9047-9054[Abstract/Free Full Text]
  30. Rui, L., Aguirre, V., Kim, J. K., Shulman, G. I., Lee, A., Corbould, A., Dunaif, A., and White, M. F. (2001) J. Clin. Investig. 107, 181-189[CrossRef][Medline] [Order article via Infotrieve]
  31. Gao, Z., Hwang, D., Bataille, F., Lefevre, M., York, D., Quon, M. J., and Ye, J. (2002) J. Biol. Chem. 277, 48115-48121[Abstract/Free Full Text]
  32. Aguirre, V., Werner, E. D., Giraud, J., Lee, Y. H., Shoelson, S. E., and White, M. F. (2002) J. Biol. Chem. 277, 1531-1537[Abstract/Free Full Text]
  33. Rotter, V., Nagaev, I., and Smith, U. (2003) J. Biol. Chem. 278, 45777-45784[Abstract/Free Full Text]
  34. Vigon, I., Mornon, J. P., Cocault, L., Mitjavila, M. T., Tambourin, P., Gisselbrecht, S., and Souyri, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5640-5644[Abstract/Free Full Text]
  35. Wesche, H., Henzel, W. J., Shillinglaw, W., Li, S., and Cao, Z. (1997) Immunity 7, 837-847[CrossRef][Medline] [Order article via Infotrieve]
  36. Jiang, Z., Ninomiya-Tsuji, J., Qian, Y., Matsumoto, K., and Li, X. (2002) Mol. Cell. Biol. 22, 7158-7167[Abstract/Free Full Text]
  37. Takaesu, G., Ninomiya-Tsuji, J., Kishida, S., Li, X., Stark, G. R., and Matsumoto, K. (2001) Mol. Cell. Biol. 21, 2475-2484[Abstract/Free Full Text]
  38. Wang, Q., Dziarski, R., Kirschning, C. J., Muzio, M., and Gupta, D. (2001) Infect. Immun. 69, 2270-2276[Abstract/Free Full Text]
  39. Li, X., Commane, M., Jiang, Z., and Stark, G. R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4461-4465[Abstract/Free Full Text]
  40. Vig, E., Green, M., Liu, Y., Yu, K. Y., Kwon, H. J., Tian, J., Goebl, M. G., and Harrington, M. A. (2001) J. Biol. Chem. 276, 7859-7866[Abstract/Free Full Text]
  41. Vig, E., Green, M., Liu, Y., Donner, D. B., Mukaida, N., Goebl, M. G., and Harrington, M. A. (1999) J. Biol. Chem. 274, 13077-13084[Abstract/Free Full Text]
  42. Croston, G. E., Cao, Z., and Goeddel, D. V. (1995) J. Biol. Chem. 270, 16514-16517[Abstract/Free Full Text]
  43. Maschera, B., Ray, K., Burns, K., and Volpe, F. (1999) Biochem. J. 339, 227-231[CrossRef][Medline] [Order article via Infotrieve]
  44. Knop, J., and Martin, M. U. (1999) FEBS Lett. 448, 81-85[CrossRef][Medline] [Order article via Infotrieve]
  45. Trofimova, M., Sprenkle, A. B., Green, M., Sturgill, T. W., Goebl, M. G., and Harrington, M. A. (1996) J. Biol. Chem. 271, 17609-17612[Abstract/Free Full Text]
  46. Zhande, R., Mitchell, J. J., Wu, J., and Sun, X. J. (2002) Mol. Cell. Biol. 22, 1016-1026[Abstract/Free Full Text]
  47. Quon, M. J., Butte, A. J., Zarnowski, M. J., Sesti, G., Cushman, S. W., and Taylor, S. I. (1994) J. Biol. Chem. 269, 27920-27924[Abstract/Free Full Text]
  48. Quon, M. J., Guerre-Millo, M., Zarnowski, M. J., Butte, A. J., Em, M., Cushman, S. W., and Taylor, S. I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5587-5591[Abstract/Free Full Text]
  49. Zeng, G., and Quon, M. J. (1996) J. Clin. Investig. 98, 894-898[Medline] [Order article via Infotrieve]
  50. Quon, M. J., Zarnowski, M. J., Guerre-Millo, M., de la Luz Sierra, M., Taylor, S. I., and Cushman, S. W. (1993) Biochem. Biophys. Res. Commun. 194, 338-346[CrossRef][Medline] [Order article via Infotrieve]
  51. Quon, M. J., Chen, H., Ing, B. L., Liu, M. L., Zarnowski, M. J., Yonezawa, K., Kasuga, M., Cushman, S. W., and Taylor, S. I. (1995) Mol. Cell. Biol. 15, 5403-5411[Abstract]
  52. Mamidipudi, V., Lin, C., Seibenhener, M. L., and Wooten, M. W. (2004) J. Biol. Chem. 279, 4161-4165[Abstract/Free Full Text]
  53. Voliovitch, H., Schindler, D. G., Hadari, Y. R., Taylor, S. I., Accili, D., and Zick, Y. (1995) J. Biol. Chem. 270, 18083-18087[Abstract/Free Full Text]
  54. Cong, L. N., Chen, H., Li, Y., Zhou, L., McGibbon, M. A., Taylor, S. I., and Quon, M. J. (1997) Mol. Endocrinol. 11, 1881-1890[Abstract/Free Full Text]
  55. Li, X., Commane, M., Burns, C., Vithalani, K., Cao, Z., and Stark, G. R. (1999) Mol. Cell. Biol. 19, 4643-4652[Abstract/Free Full Text]
  56. Kanakaraj, P., Ngo, K., Wu, Y., Angulo, A., Ghazal, P., Harris, C. A., Siekierka, J. J., Peterson, P. A., and Fung-Leung, W. P. (1999) J. Exp. Med. 189, 1129-1138[Abstract/Free Full Text]
  57. Thomas, J. A., Allen, J. L., Tsen, M., Dubnicoff, T., Danao, J., Liao, X. C., Cao, Z., and Wasserman, S. A. (1999) J. Immunol. 163, 978-984[Abstract/Free Full Text]
  58. Swantek, J. L., Tsen, M. F., Cobb, M. H., and Thomas, J. A. (2000) J. Immunol. 164, 4301-4306[Abstract/Free Full Text]
  59. Thomas, J. A., Haudek, S. B., Koroglu, T., Tsen, M. F., Bryant, D. D., White, J., Kusewitt, D. F., Horton, J. W., and Giroir, B. P. (2003) Am. J. Physiol. Heart Circ. Physiol. 285, 597-606
  60. Eck, M. J., Dhe-Paganon, S., Trub, T., Nolte, R. T., and Shoelson, S. E. (1996) Cell 85, 695-705[CrossRef][Medline] [Order article via Infotrieve]
  61. Dhe-Paganon, S., Ottinger, E. A., Nolte, R. T., Eck, M. J., and Shoelson, S. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8378-8383[Abstract/Free Full Text]
  62. Myers, M. G., Jr., Grammer, T. C., Brooks, J., Glasheen, E. M., Wang, L. M., Sun, X. J., Blenis, J., Pierce, J. H., and White, M. F. (1995) J. Biol. Chem. 270, 11715-11718[Abstract/Free Full Text]
  63. Yenush, L., Makati, K. J., Smith-Hall, J., Ishibashi, O., Myers, M. G., Jr., and White, M. F. (1996) J. Biol. Chem. 271, 24300-24306[Abstract/Free Full Text]
  64. Vainshtein, I., Kovacina, K. S., and Roth, R. A. (2001) J. Biol. Chem. 276, 8073-8078[Abstract/Free Full Text]
  65. Burks, D. J., Pons, S., Towery, H., Smith-Hall, J., Myers, M. G., Jr., Yenush, L., and White, M. F. (1997) J. Biol. Chem. 272, 27716-27721[Abstract/Free Full Text]
  66. Sun, H., and Baserga, R. (2004) 145, 5332-5343
  67. Jacobs, A. R., LeRoith, D., and Taylor, S. I. (2001) J. Biol. Chem. 276, 40795-40802[Abstract/Free Full Text]
  68. Burks, D. J., Wang, J., Towery, H., Ishibashi, O., Lowe, D., Riedel, H., and White, M. F. (1998) J. Biol. Chem. 273, 31061-31067[Abstract/Free Full Text]
  69. Maffucci, T., Razzini, G., Ingrosso, A., Chen, H., Iacobelli, S., Sciacchitano, S., Quon, M. J., and Falasca, M. (2003) Mol. Endocrinol. 17, 1568-1579[Abstract/Free Full Text]
  70. Milburn, C. C., Deak, M., Kelly, S. M., and Price, N. C. (2003) Biochem. J. 375, 531-538[CrossRef][Medline] [Order article via Infotrieve]
  71. Cozier, G. E., Bouyoucef, D., and Cullen, P. J. (2003) J. Biol. Chem. 278, 39489-39496[Abstract/Free Full Text]
  72. Tuzi, S., Uekama, N., Okada, M., Yamaguchi, S., Saito, H., and Yagisawa, H. (2003) J. Biol. Chem. 278, 28019-28025[Abstract/Free Full Text]
  73. Chen, R. H., Corbalan-Garcia, S., and Bar-Sagi, D. (1997) EMBO J. 16, 1351-1359[CrossRef][Medline] [Order article via Infotrieve]
  74. Imai, S. I., Kai, M., Yamada, K., Kanoh, H., and Sakane, F. (2004) Biochem. J. 382, 957-966[CrossRef][Medline] [Order article via Infotrieve]
  75. Werner, E. D., Lee, J., Hansen, L., Yuan, M., and Shoelson, S. E. (2004) J. Biol. Chem. 279, 35298-35305[Abstract/Free Full Text]
  76. Yu, C., Chen, Y., Cline, G. W., Zhang, D., Zong, H., Wang, Y., Bergeron, R., Kim, J. K., Cushman, S. W., Cooney, G. J., Atcheson, B., White, M. F., Kraegen, E. W., and Shulman, G. I. (2002) J. Biol. Chem. 277, 50230-50236[Abstract/Free Full Text]
  77. Lee, Y. H., Giraud, J., Davis, R. J., and White, M. F. (2003) J. Biol. Chem. 278, 2896-2902[Abstract/Free Full Text]
  78. Jiang, G., Dallas-Yang, Q., Liu, F., Moller, D. E., and Zhang, B. B. (2003) J. Biol. Chem. 278, 180-186[Abstract/Free Full Text]
  79. De Fea, K., and Roth, R. A. (1997) Biochemistry 36, 12939-12947[CrossRef][Medline] [Order article via Infotrieve]
  80. Delahaye, L., Mothe-Satney, I., Myers, M. G., White, M. F., and Van Obberghen, E. (1998) Endocrinology 139, 4911-4919[Abstract/Free Full Text]
  81. Bouzakri, K., Roques, M., Gual, P., Espinosa, S., Guebre-Egziabher, F., Riou, J. P., Laville, M., Le Marchand-Brustel, Y., Tanti, J. F., and Vidal, H. (2003) Diabetes 52, 1319-1325[Abstract/Free Full Text]
  82. Ozes, O. N., Akca, H., Mayo, L. D., Gustin, J. A., Maehama, T., Dixon, J. E., and Donner, D. B. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4640-4645[Abstract/Free Full Text]
  83. Hotamisligil, G. S., Arner, P., Caro, J. F., Atkinson, R. L., and Spiegelman, B. M. (1995) J. Clin. Investig. 95, 2409-2415[Medline] [Order article via Infotrieve]
  84. Gao, Z., Zuberi, A., Quon, M. J., Dong, Z., and Ye, J. (2003) J. Biol. Chem. 278, 24944-24950[Abstract/Free Full Text]
  85. Qian, Y., Commane, M., Ninomiya-Tsuji, J., Matsumoto, K., and Li, X. (2001) J. Biol. Chem. 276, 41661-41667[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Circ. Res.Home page
K. Tanigaki, C. Mineo, I. S. Yuhanna, K. L. Chambliss, M. J. Quon, E. Bonvini, and P. W. Shaul
C-Reactive Protein Inhibits Insulin Activation of Endothelial Nitric Oxide Synthase via the Immunoreceptor Tyrosine-Based Inhibition Motif of Fc{gamma}RIIB and SHIP-1
Circ. Res., June 5, 2009; 104(11): 1275 - 1282.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
U. Maitra, S. Davis, C. M. Reilly, and L. Li
Differential Regulation of Foxp3 and IL-17 Expression in CD4 T Helper Cells by IRAK-1
J. Immunol., May 1, 2009; 182(9): 5763 - 5769.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
S. Boura-Halfon and Y. Zick
Phosphorylation of IRS proteins, insulin action, and insulin resistance
Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E581 - E591.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Zhang, Z. Gao, J. Yin, M. J. Quon, and J. Ye
S6K Directly Phosphorylates IRS-1 on Ser-270 to Promote Insulin Resistance in Response to TNF-{alpha} Signaling through IKK2
J. Biol. Chem., December 19, 2008; 283(51): 35375 - 35382.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
C. Weigert, M. Kron, H. Kalbacher, A. K. Pohl, H. Runge, H.-U. Haring, E. Schleicher, and R. Lehmann
Interplay and Effects of Temporal Changes in the Phosphorylation State of Serine-302, -307, and -318 of Insulin Receptor Substrate-1 on Insulin Action in Skeletal Muscle Cells
Mol. Endocrinol., December 1, 2008; 22(12): 2729 - 2740.
[Abstract] [Full Text] [PDF]

Home page
 JPEN J Parenter Enteral NutrHome page
A. Shah, N. Mehta, and M. P. Reilly
Adipose Inflammation, Insulin Resistance, and Cardiovascular Disease
JPEN J Parenter Enteral Nutr, November 1, 2008; 32(6): 638 - 644.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Lockett, M. G. Goebl, and M. A. Harrington
Transient membrane recruitment of IRAK-1 in response to LPS and IL-1{beta} requires TNF R1
Am J Physiol Cell Physiol, August 1, 2008; 295(2): C313 - C323.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
Z. Liberman, B. Plotkin, T. Tennenbaum, and H. Eldar-Finkelman
Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase C{beta}II in the diabetic fat tissue
Am J Physiol Endocrinol Metab, June 1, 2008; 294(6): E1169 - E1177.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Lee, E. G. Lynn, J.-a Kim, and M. J. Quon
Protein Kinase C-{zeta} Phosphorylates Insulin Receptor Substrate-1, -3, and -4 But Not -2: Isoform Specific Determinants of Specificity in Insulin Signaling
Endocrinology, May 1, 2008; 149(5): 2451 - 2458.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
D. Pflieger, M. A. Junger, M. Muller, O. Rinner, H. Lee, P. M. Gehrig, M. Gstaiger, and R. Aebersold
Quantitative Proteomic Analysis of Protein Complexes: Concurrent Identification of Interactors and Their State of Phosphorylation
Mol. Cell. Proteomics, February 1, 2008; 7(2): 326 - 346.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
H. Sharfi and H. Eldar-Finkelman
Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling
Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E307 - E315.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Luo, P. Langlais, Z. Yi, N. Lefort, E. A. De Filippis, H. Hwang, C. Y. Christ-Roberts, and L. J. Mandarino
Phosphorylation of Human Insulin Receptor Substrate-1 at Serine 629 Plays a Positive Role in Insulin Signaling
Endocrinology, October 1, 2007; 148(10): 4895 - 4905.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. A. Cooper, A. Whaley-Connell, J. Habibi, Y. Wei, G. Lastra, C. Manrique, S. Stas, and J. R. Sowers
Renin-angiotensin-aldosterone system and oxidative stress in cardiovascular insulin resistance
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2009 - H2023.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
R. Muniyappa, M. Montagnani, K. K. Koh, and M. J. Quon
Cardiovascular Actions of Insulin
Endocr. Rev., August 1, 2007; 28(5): 463 - 491.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Herschkovitz, Y.-F. Liu, E. Ilan, D. Ronen, S. Boura-Halfon, and Y. Zick
Common Inhibitory Serine Sites Phosphorylated by IRS-1 Kinases, Triggered by Insulin and Inducers of Insulin Resistance
J. Biol. Chem., June 22, 2007; 282(25): 18018 - 18027.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-a Kim, G. Formoso, Y. Li, M. A. Potenza, F. L. Marasciulo, M. Montagnani, and M. J. Quon
Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn
J. Biol. Chem., May 4, 2007; 282(18): 13736 - 13745.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
Y. Luo, H.-J. Kwon, S. Montano, M. Georgiadis, M. G. Goebl, and M. A. Harrington
Phosphorylation of SIMPL modulates RelA-associated NF-{kappa}B-dependent transcription
Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1013 - C1023.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Sanchez-Alavez, I. Klein, S. E. Brownell, I. V. Tabarean, C. N. Davis, B. Conti, and T. Bartfai
Night eating and obesity in the EP3R-deficient mouse
PNAS, February 20, 2007; 104(8): 3009 - 3014.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
K. Morino, K. F. Petersen, and G. I. Shulman
Molecular Mechanisms of Insulin Resistance in Humans and Their Potential Links With Mitochondrial Dysfunction
Diabetes, December 1, 2006; 55(Supplement_2): S9 - S15.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
R. Nawaratne, A. Gray, C. H. Jorgensen, C. P. Downes, K. Siddle, and J. K. Sethi
Regulation of Insulin Receptor Substrate 1 Pleckstrin Homology Domain by Protein Kinase C: Role of Serine 24 Phosphorylation
Mol. Endocrinol., August 1, 2006; 20(8): 1838 - 1852.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
J.-a Kim, M. Montagnani, K. K. Koh, and M. J. Quon
Reciprocal Relationships Between Insulin Resistance and Endothelial Dysfunction: Molecular and Pathophysiological Mechanisms
Circulation, April 18, 2006; 113(15): 1888 - 1904.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/24/23173    most recent
M501439200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J.-a
Right arrow Articles by Quon, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J.-a
Right arrow Articles by Quon, M. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement