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Molecular Endocrinology 18 (2): 350-358
Copyright © 2004 by The Endocrine Society

Negative Regulation of Insulin-Stimulated Mitogen-Activated Protein Kinase Signaling By Grb10

Paul Langlais, Lily Q. Dong, Fresnida J. Ramos, Derong Hu, Yunhua Li, Michael J. Quon and Feng Liu

Department of Biochemistry (P.L., F.L.), Department of Cellular & Structural Biology (L.Q.D.), and Department of Pharmacology (F.J.R., D.H., F.L.), The University of Texas Health Science Center, San Antonio, Texas 78229; and Diabetes Unit, National Center for Complementary and Alternative Medicine (M.J.Q., Y.L.), National Institutes of Health, Bethesda, Maryland 20892-1755

Address all correspondence and requests for reprints to: Feng Liu, Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229. E-mail: liuf{at}uthscsa.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Grb10 is a Pleckstrin homology and Src homology 2 (SH2) domain-containing protein that binds to the tyrosine-phosphorylated insulin receptor in response to insulin stimulation. Loss of Grb10 function in mice results in fetal and placental overgrowth; however, the molecular mechanism remains unknown. In the present study, we show that overexpression of Grb10 in Chinese hamster ovary cells expressing the insulin receptor or in 3T3-L1 adipocytes reduced insulin-stimulated phosphorylation of MAPK. Overexpression of Grb10 in rat primary adipocytes also inhibited insulin-stimulated phosphorylation of the MAPK downstream substrate Elk1. To determine the mechanism by which Grb10 inhibited insulin-stimulated MAPK signaling, we examined whether Grb10 affects the phosphorylation of MAPK upstream signaling components. We found that overexpression of Grb10 inhibited the insulin-stimulated phosphorylation of Shc, a positive regulator of the MAPK signaling pathway. The inhibitory effect was diminished when the SH2 domain of Grb10 was deleted. The negative role of Grb10 in insulin signaling was established by suppression of endogenous Grb10 by RNA interference in HeLa cells overexpressing the insulin receptor, which enhanced insulin-stimulated phosphorylation of MAPK, Shc, and Akt. Taken together, our findings suggest that Grb10 functions as a negative regulator in the insulin-stimulated MAPK signaling pathway. In addition, the inhibitory effect of Grb10 on the MAPK pathway is most likely due to a direct block of insulin-stimulated Shc tyrosine phosphorylation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE BINDING OF insulin to its receptor on the cell membrane results in receptor tyrosine kinase activation, leading to receptor trans-autophosphorylation at several crucial sites required to initiate further downstream signaling events such as activation of the phosphatidylinositol 3'-kinase (PI 3-kinase) pathway, the Cbl-CAP pathway, and the MAPK pathway (1, 2). Autophosphorylation of Tyr960 in the juxtamembrane region of the insulin receptor (IR) creates an NPXpY recognition sequence that functions to recruit phosphotyrosine binding (PTB) domain-containing proteins such as Shc (3). In addition to Tyr960, autophosphorylation also occurs at Tyr1322, creating a pYXXM motif, which has been shown to bind to the Src homology 2 (SH2) domain of Shc (3, 4). Once Shc associates with the IR, it is phosphorylated on tyrosine residues, thus creating a docking site for the SH2 domain of Grb2. Grb2 recruits the guanine nucleotide exchange factor protein, Sos, resulting in a Sos-mediated conversion of the membrane-bound protein Ras from the inactive GDP bound form to the active GTP bound form. Activated Ras then associates with and activates Raf-1, leading to the phosphorylation and activation of MAPK/ERK kinase (MEK) and its downstream substrate MAPK. Activated MAPK phosphorylates a variety of substrates, including Elk-1, and mediates nuclear events that regulate cell growth and differentiation (for a review see Ref. 5).

In addition to Shc, the IR also interacts with several other proteins, in particular, the pleckstrin homology (PH)- and SH2 domain-containing protein Grb10 (6, 7, 8, 9, 10). Through the SH2 domain and a novel domain located between the PH and SH2 domains known as the BPS (between the PH and SH2) domain, Grb10 binds to the autophosphorylated tyrosine residues within the kinase domain of the IR upon insulin stimulation (7, 9, 11, 12, 13, 14, 15).

The functional roles of Grb10 in insulin signaling remain controversial. Overexpression of full-length mouse Grb10 has been found to either have no effect (16) or positively regulate insulin-mediated mitogenic and metabolic effects (17, 18). On the other hand, stable expression of the PH domain-truncated human Grb10 isoform in Chinese hamster ovary (CHO)/IR cells reduced insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and inhibited insulin-stimulated activation of PI 3-kinase (6). Overexpression of full-length human Grb10 inhibits insulin-stimulated tyrosine phosphorylation of the 60-kDa GTPase-activating protein-associated protein p62dok (19) and IRS-1 (15) in CHO/IR cells and 3T3-L1 adipocytes. Overexpression of human Grb10 has also been found to decrease insulin-stimulated glycogen synthase activity and glycogen synthesis in primary hepatocytes, albeit without affecting insulin-induced IRS1/2 phosphorylation, PI 3-kinase activation, and ERK1/2 MAPK activity (20). Very recently, it has been shown that disrupting Grb10 function by targeted deletion of the Grb10 gene leads to overgrowth of mice, suggesting that endogenous Grb10 acts as a negative regulator of growth (21).

In this study, we investigated the effect of Grb10 on insulin-stimulated MAPK activation. We found that overexpression of Grb10 in CHO/IR cells, 3T3-L1 adipocytes, and rat primary adipocytes inhibits insulin-stimulated MAPK and Shc phosphorylation. The inhibitory effect of Grb10 was diminished when the SH2 domain of the protein was deleted, suggesting that binding of Grb10 to the IR is necessary for the inhibition. Suppression of Grb10 expression by RNA interference (RNAi) enhanced basal and insulin-stimulated phosphorylation of MAPK and Shc, suggesting that endogenous Grb10 plays a negative role in the MAPK signaling pathway. Because phosphorylation of Shc and MAPK is essential for insulin-stimulated mitogenic events, our results suggest a potential molecular mechanism by which disruption of Grb10 function leads to overgrowth in knockout mice (21).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Grb10 Inhibits MAPK Phosphorylation in CHO/IR Cells and 3T3-L1 Adipocytes
To investigate the potential effect of Grb10 on insulin-stimulated activation of the MAPK signaling pathway, we transiently expressed myc-tagged p42 MAPK in CHO/IR cells either alone or together with human Grb10. Significant phosphorylation of MAPK was observed within 5 min of insulin stimulation (Fig. 1AGo, upper panel, lanes 1 and 2). Overexpression of Grb10 inhibited insulin-stimulated MAPK phosphorylation (Fig. 1AGo, upper panel, lane 2 vs. lane 4). We also tested whether adenoviral-mediated overexpression of Grb10 inhibits insulin-stimulated activation of endogenous MAPK in 3T3-L1 adipocytes, a cell line that is physiologically relevant to insulin signaling. We found that insulin treatment led to a significant phosphorylation of endogenous p44/p42 MAPK within 5 min, which was inhibited by overexpression of Grb10 (Fig. 1BGo, top panel).



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  		Fig. 1. Overexpression of Human Grb10 Inhibits MAPK Signaling

A, CHO/IR cells were transiently transfected with either Myc-tagged p42 MAPK alone or cotransfected with MAPK and Grb10. After 24 h, the cells were washed with 1x PBS, incubated in serum-free medium for 2 h, and subsequently treated with (+) or without (-) insulin (10-8 M) for 5 min. Cell lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blotted with antibody to either the HA tag to Grb10 (bottom panel), or phospho-MAPK (top panel). The phospho-MAPK membrane was stripped and reblotted with Myc antibody to visualize MAPK (middle panel). Data are representative of three independent experiments. B, Fully differentiated 3T3-L1 adipocytes were infected at an infection efficiency of 90% with either green fluorescent protein (GFP) or Grb10 adenovirus. Cells were serum starved overnight, washed with PBS, and then serum starved for 1 additional hour. The cells were subsequently treated with (+) or without (-) 100 nM insulin for 5 min. Cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blotted with an antibody to either the HA tag to Grb10 (bottom panel) or phospho-MAPK (top panel). The whole-cell lysates were reloaded and blotted with an antibody to p44/42 MAPK (middle panel). Data are representative of three independent experiments. C, Rat primary adipocytes were transiently cotransfected with pFA-Elk1, pFR-Luc, and pCIS, PH domain-truncated human Grb10, or full-length human Grb10 by electroporation. After overnight incubation, the cells were treated without or with insulin (100 nM, 5 h). The cells were then lysed and the luciferase activity in each sample was determined. Results shown are the mean ± SEM of six independent experiments performed in triplicate (normalized to the group transfected with pCIS and stimulated with insulin).

 
Human Grb10 Isoforms Inhibit Insulin-Stimulated Elk-1 Activity in Primary Rat Adipocytes
To test whether Grb10 inhibits insulin signaling downstream of MAPK, we examined the effects of overexpressing Grb10 on insulin-stimulated Elk1 phosphorylation in primary rat adipocytes. Elk-1 is a ternary complex factor that is known to be phosphorylated by activated MAPK upon insulin stimulation (22). Plasmids encoding either full-length (11) or the PH domain-truncated (6) human Grb10 isoforms were electroporated into primary rat adipocytes together with a plasmid encoding the GAL4 binding domain fused to the Elk1 activation domain (GAL4/Elk1) and a plasmid encoding a GAL4 binding sequence/luciferase. Phosphorylation of the GAL4/Elk1 fusion protein leads to activation of Elk1 and a subsequent increase in luciferase expression, which can be monitored by luciferase assays. We found that insulin treatment of cells transfected with an empty vector pCIS led to a 2-fold increase in luciferase activity, correlating with an activation of Elk1 (Fig. 1CGo). The insulin-stimulated luciferase activity was significantly decreased (P < 0.006) in cells overexpressing Grb10 (Fig. 1CGo). These data suggest that overexpression of Grb10 is sufficient to inhibit an insulin-stimulated MAPK-dependent mitogenic signaling pathway in adipocytes.

Grb10 Inhibits Insulin-Stimulated Tyrosine Phosphorylation of Shc in CHO/IR Cells and 3T3-L1 Adipocytes
To investigate the mechanism by which Grb10 inhibits insulin-stimulated activation of the MAPK pathway, we tested whether Grb10 inhibits tyrosine phosphorylation of the adapter protein p52 Shc, an adapter protein that functions in close proximity to the IR and mediates insulin-stimulated MAPK phosphorylation. We found that in CHO/IR cells, Shc underwent rapid insulin-stimulated tyrosine phosphorylation (Fig. 2AGo, lane 1 vs. lane 2). Coexpression of Grb10 with Shc resulted in a marked inhibition of insulin-stimulated tyrosine phosphorylation of Shc (Fig. 2AGo, lane 2 vs. lane 4). Shc tyrosine phosphorylation was inhibited by Grb10 in a dose-dependent manner (Fig. 2BGo, lane 2 vs. lanes 3–6). We also tested whether adenoviral-mediated overexpression of Grb10 inhibits insulin-stimulated activation of endogenous Shc in 3T3-L1 adipocytes. We found that insulin treatment led to a notable phosphorylation of endogenous Shc within 5 min, which was inhibited by overexpression of Grb10 (Fig. 2CGo, top panel).



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  		Fig. 2. Grb10 Inhibits Insulin-Stimulated Tyrosine Phosphorylation of Shc

A, Myc-tagged p52 Shc was transiently expressed in CHO/IR cells either alone or with HA-tagged human Grb10. Cells were serum starved for 6 h and then treated with (+) or without (-) 10 nM insulin for 15 min. Cells were lysed and Shc was immunoprecipitated with antibody to the protein. The immunoprecipitates were washed three times in ice-cold buffer B, separated by SDS-PAGE, and transferred onto a nitrocellulose membrane. The tyrosine phosphorylation of Shc was determined by Western blot using an antiphosphotyrosine antibody (top panel). Shc was detected with an antibody to the myc tag (middle panel). The expression of Grb10 was determined by Western blot using an antibody to the HA tag (bottom panel). Data are representative of three independent experiments. B, Myc-tagged Shc was cotransfected with increasing amounts (1, 4, 7, or 10 µg) of HA-tagged hGrb10 into CHO/IR cells. Cells were serum-starved for 4 h, stimulated with (+) or without (-) 10 nM insulin for 5 min, and lysed. Western blot analysis was determined as in panel A. Data are representative of three independent experiments. C, 3T3-L1 adipocytes were infected at an infection efficiency of 90% with either GFP or Grb10 adenovirus. Cells were serum starved overnight, washed with PBS, and then serum starved for 1 additional hour. The cells were subsequently treated with (+) or without (-) 100 nM insulin for 5 min. Cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The tyrosine phosphorylation of Shc was determined by Western blot using an antiphospho Shc antibody (top panel). Shc was detected with an antibody to the protein (middle panel). The expression of Grb10 was determined by Western blot using an antibody to the HA tag (bottom panel). D, Schematic of human Grb10 truncations. E, Myc-tagged Shc was transiently expressed in CHO/IR cells either alone or with HA-tagged truncations of human Grb10. Cells were serum-starved for 2 h and then either left untreated or treated with 10 nM insulin for 5 min. Western blot analysis was determined as in panel A. Data are representative of three independent experiments. F, Myc-tagged p42 MAPK was transiently expressed in CHO/IR cells either alone or with HA-tagged truncations of human Grb10. After 24 h, the cells were washed with 1x PBS, incubated in serum-free medium for 2 h, and subsequently treated with (+) or without (-) insulin (10-8 M) for 5 min. Cell lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blotted with antibody to either the HA tag to Grb10 (bottom panel), phospho-MAPK (top panel), or Myc antibody to visualize MAPK (middle panel). Data are representative of three independent experiments. G, Myc-tagged Shc was transiently expressed in CHO/IR cells alone or with either HA-tagged human Grb10 or human Grb10R520G. Cells were serum starved for 2 h and then left untreated or treated with 10 nM insulin for 5 min. Cells were lysed and Shc was immunoprecipitated using antibody to the Myc tag. Western blot analysis was determined as in panel A. Data are representative of three independent experiments.

 
The SH2 Domain of Grb10 Is Important in the Inhibition of Insulin-Stimulated Shc Tyrosine Phosphorylation in Cells
Our results suggest that Grb10 inhibits insulin signaling to MAPK through inhibition of insulin-stimulated phosphorylation of the adaptor protein Shc. To understand the mechanism of the inhibition, we expressed Myc-tagged Shc in CHO/IR cells together with either wild-type or various truncation mutants of Grb10 (Fig. 2DGo). Consistent with our previous findings, full-length wild-type Grb10 expression resulted in a considerable decrease in Shc tyrosine phosphorylation (Fig. 2EGo, lane 2 vs. 3). In contrast, coexpression of Grb10 mutants lacking either the SH2 domain alone ({Delta}SH2) or both the BPS and SH2 domains [{Delta}(BPS+SH2)] restored insulin-stimulated tyrosine phosphorylation of Shc (Fig. 2EGo, lane 3 vs. lanes 4 and 5). Deletion of the SH2 domain of Grb10 also restores MAPK phosphorylation in the presence of overexpressed Grb10 (Fig. 2FGo, lane 3 vs. lanes 4 and 5). In addition, the mutant human Grb10R520G, in which the function of the SH2 domain is impaired through mutation of the critical arginine (Arg520) to glycine (15, 23), only partially reversed the ability of Grb10 to inhibit insulin-stimulated Shc tyrosine phosphorylation (Fig. 2GGo, lane 3 vs. 4). Taken together, the data indicate that the SH2 domain of Grb10, which has been shown to mediate the interaction between Grb10 and the IR (7, 11, 12, 13, 15, 23), is critical for the inhibitory effect of Grb10 on insulin-stimulated activation of the MAPK pathway.

Inhibition of Endogenous hGrb10 Expression Results in an Increase of Insulin Signaling to MAPK, Shc, and Akt
Because the above studies were carried out in cells overexpressing Grb10, it is unclear whether the inhibitory effect of Grb10 on MAPK signaling is due to amplifying or blocking the endogenous functions downstream of Grb10. To address this question, we attempted to investigate the functional role of endogenous Grb10 by RNAi. The RNAi target sequence corresponds to nucleotides (nt 498–518) of full-length human Grb10, hGrb10{zeta}, although both the human Grb-IR/hGrb10ß and hGrb10{gamma} isoforms contain the identical sequence in this region (24). We generated a Grb10 RNAi plasmid using the pBS/U6/RNAi plasmid (25) and examined the effect and specificity of the Grb10 RNAi in HeLa/IR cells. As seen in Fig. 3AGo, Western blot analysis of whole-cell lysates showed that transfection of the control plasmid pBS/U6/RNAi in HeLa/IR cells had no effect on the expression levels of Grb10 (lanes 1–3). On the other hand, Grb10 expression was greatly inhibited when cells were cotransfected with the pBS/U6/RNAi/Grb10 plasmid in a dose-dependent manner (lanes 4–6). This inhibition was specific because expression of the Grb10 RNAi plasmid had no effect on the coexpressed p70 S6 kinase (lanes 7–9).



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  		Fig. 3. Suppression of Grb10 Expression by RNAi

A, HeLa/IR cells were transfected with a plasmid encoding HA-tagged Grb10 or HA-tagged p70 S6 kinase (S6K) together with increasing amounts of either the control plasmid pBS/U6/RNAi or the pBS/U6/RNAi/Grb10 plasmid. Cells were lysed 24 h posttransfection, and the expression of HA-tagged proteins was examined by Western blot using an antibody to the HA-tag (upper panel). The Western blot for histone expression is shown to document equal loading of protein (lower panel). B, PCR products amplified from HeLa/IR or HeLa/IR/{Delta}Grb10 cells using Grb10 (left panel) or Grb7 (right panel) specific primers. mRNA abundance was normalized relative to that of GAPDH. C, Cell lysates of either HeLa/IR cells or HeLa/IR/{Delta}Grb10 were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blotted with an antibody to Grb10 to show RNAi-mediated inhibition of Grb10 expression in the HeLa/IR/{Delta}Grb10 cells (upper panel). The Western blot for ß-tubulin expression is shown to document equal loading of protein (lower panel).

 
To investigate the functional role of endogenous Grb10, we generated a HeLa/IR stable cell line in which the expression of endogenous hGrb10 was impaired by RNAi. When probed with antibody to human Grb10, HeLa/IR lysates reveal bands that comigrate only with full-length human Grb10, hGrb10{zeta}, indicating that the experiments conducted would be targeting this human Grb10 isoform. We chose HeLa cells due to the fact that they have been shown to be a successful model system to study RNAi-mediated gene silencing (26). RT-PCR and Western blot analysis revealed that the expression of Grb10 mRNA (Fig. 3BGo) and protein (Fig. 3CGo) was almost completely blocked in Grb10-suppressed HeLa/IR cells. We also examined the effect of Grb10 RNAi on the two other members of the Grb7 family, namely Grb7 and Grb14. RT-PCR analysis revealed that Grb14 mRNA was not expressed in HeLa/IR cells (data not shown). In addition, we found that suppression of Grb10 by RNAi had no effect on Grb7 mRNA expression in the HeLa/IR/{Delta}Grb10 cells (Fig. 3BGo). These findings indicated that suppression of Grb10 expression had no effect on expression levels of other members of the Grb7/10/14 family.

Using the Grb10-deficient cells, we examined the phosphorylation of endogenous MAPK and Shc. We found that both the basal and insulin-stimulated phosphorylation of MAPK (Fig. 4AGo) and Shc (Fig. 4BGo) was notably increased in the HeLa/IR/{Delta}Grb10 cell line as compared with the wild-type HeLa/IR cells. The reduced effect of insulin on MAPK and Shc phosphorylation in HeLa/IR cells compared with the other cell types is most likely due to the difference in the cellular contents of these cell types. Like other cancer cells, HeLa cells have a higher basal phosphorylation and are not very insulin sensitive. However, as demonstrated previously (26) and also confirmed in our studies, HeLa cells provide an excellent model system for RNAi studies. We also examined whether suppression of Grb10 expression had an effect on insulin stimulation of the PI 3-kinase pathway by measuring Akt phosphorylation. Our results showed that insulin-stimulated phosphorylation of Akt at both Thr308 and Ser473 was markedly increased in the HeLa/IR/{Delta}Grb10 cells as compared with the wild-type HeLa/IR cells (Fig. 4CGo). These findings are strong evidence that endogenous Grb10 plays a negative role in insulin signaling.



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  		Fig. 4. Inhibition of Endogenous hGrb10 Expression Results in Increased MAPK, Shc, and Akt Phosphorylation

A, Cell lysates from either HeLa/IR cells or HeLa/IR/{Delta}Grb10 cells treated with (+) or without (-) insulin (100 nM) for 5 min were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blotted with the designated antibodies. Data are representative of three independent experiments. B, HeLa/IR cells or HeLa/IR/{Delta}Grb10 cells were serum starved for 2 h and then left untreated or treated with 100 nM insulin for 5 min. The cells were lysed, and endogenous Shc was immunoprecipitated using antibody to the protein. The tyrosine phosphorylation of Shc was determined by Western blot using an antiphosphotyrosine antibody (upper panel). Shc was detected by Western blot with antibody to the protein (lower panel). Data are representative of three independent experiments. C, HeLa/IR cells or HeLa/IR/{Delta}Grb10 cells were serum starved for 2 h and then left untreated or treated with 100 nM insulin for 5 min. The cells were lysed and endogenous Akt was immunoprecipitated using antibody to the protein. The phosphorylation of Akt was determined by Western blot using either antiphospho-AktT308 (top panel) or antiphospho-AktS473 (middle panel). Akt protein was detected by Western blot with antibody to the protein (bottom panel). Data are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Grb10 is known to associate with the insulin receptor in response to insulin stimulation (6, 7, 8, 9, 12, 27); however, the role of Grb10 in insulin signaling remains controversial. Because most, if not all, of the previous studies were carried out in cells overexpressing Grb10, it is unclear whether the inhibitory or stimulatory roles of Grb10 in insulin signaling were caused by direct inhibition of the signaling pathway or due to sequestration of molecules necessary for signaling.

In the present study, we show that overexpression of Grb10 inhibits insulin-stimulated MAPK signaling in a variety of cells. In addition, we showed that suppression of Grb10 expression by RNAi led to enhanced insulin-stimulated Akt, Shc, and MAPK phosphorylation (Fig. 4Go). This is the first evidence suggesting that endogenous Grb10 plays a negative role in insulin signaling. As for other members of the Grb7/10/14 family (24, 28), suppression of Grb10 by RNAi did not affect the expression of Grb7. Thus, the inhibitory effect of Grb10 is specific and is not caused by altering the expression levels of other members of the Grb7/10/14 family, which may potentially up-regulate insulin signaling. Therefore, the most likely explanation for our results is that endogenous Grb10 normally functions to negatively regulate insulin signaling and that removal of this suppression up-regulates insulin signaling. Our results are consistent with a recent finding showing that disruption of Grb10 function by gene targeting leads to mouse overgrowth, suggesting that endogenous Grb10 is a potent growth inhibitor in vivo (21).

Recently, we have shown that human Grb10 inhibits insulin-stimulated activation of the PI-3 kinase pathway by blocking the insulin-stimulated association between the IR and IRS, resulting in the inhibition of IRS tyrosine phosphorylation (15). Results from the present study suggest that Grb10 may use a similar mechanism to negatively regulate the MAPK pathway. Consistent with this, we found that overexpression of Grb10 inhibits insulin-stimulated tyrosine phosphorylation of the adapter protein Shc, which has been shown to mediate insulin-stimulated MAPK phosphorylation. This result is in agreement with a recent finding that Grb10 inhibits Shc tyrosine phosphorylation by the IR in vitro (14). The idea that Grb10 reduces MAPK phosphorylation by inhibiting Shc tyrosine phosphorylation is consistent with the finding that a mutation in the SH2 domain, which inhibits the interaction between Grb10 and the tyrosine-phosphorylated IR (7, 11, 12, 13), reduces the inhibitory effect of Grb10 on insulin-stimulated Shc tyrosine phosphorylation (Fig. 2Go, E and G). However, because we (11) and others (7, 12, 13) have previously found that Grb10 binds to the kinase domain of the IR whereas Shc binds to autophosphorylated tyrosines in either the juxtamembrane region (3) or at the C terminus of the IR (3, 4), it is likely that the inhibition of Shc association with the IR is caused by either a Grb10-induced conformational change of the IR or a physical hindrance by Grb10, which prevents the IR from phosphorylating Shc, rather than by competitive binding of Grb10 and Shc for the same site on the IR. We attempted to verify, by coimmunoprecipitation studies, whether Grb10 inhibits Shc binding to the IR; however, we were unable to detect an interaction between the IR and Shc in our cell systems (data not shown), perhaps due to the transient nature of the association.

In summary, we show that overexpression of Grb10 inhibits insulin signaling to the MAPK pathway in CHO/IR cells as well as in cells physiologically relevant to insulin action such as 3T3-L1 adipocytes and rat primary adipocytes. In addition, we show that suppression of endogenous Grb10 expression enhanced insulin-stimulated phosphorylation of MAPK, Shc, and Akt. These results strongly suggest that Grb10 normally functions as a negative regulator of insulin-mediated metabolic and mitogenic events. Our results elucidate a molecular mechanism for the recent observation that disruption of Grb10 expression leads to mouse overgrowth (21).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
The CHO cell line overexpressing the IR (CHO/IR) was described previously (6, 8, 23). 3T3-L1 cells were from ATCC (Manassas, VA). The HeLa/IR cell line, a human IR-overexpressing cell line, was created by cotransfecting HeLa cells with cDNA encoding the human IR (29) and the puromycin resistance vector pSV2 using the LipofectAMINE reagent according to the manufacturer’s protocol (Life Technologies, Gaithersburg, MD). Transfectants were selected with puromycin, and colonies were screened by Western blot analysis with an anti-IRß antibody. Monoclonal antibody to the hemagglutinin (HA) tag was purchased from COVANCE (Berkeley, CA). Anti-Myc antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-specific antibodies to MAPK, Akt, and ShcY317 and antibodies to Akt and hGrb10 were obtained from Cell Signaling Technology (Beverly, MA). The polyclonal phosphotyrosine (RC20) antibody was purchased from Transduction Laboratories, Inc. (Lexington, KY). Monoclonal and polyclonal Shc antibodies were purchased from Santa Cruz Biotechnology, Inc. and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively. Secondary antibodies conjugated to alkaline phosphatase and horseradish peroxidase were purchased from Promega Corp. (Madison, WI). Protein G- and protein A-conjugated Sepharose beads were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL).

Construction of Plasmids and Adenoviruses
Plasmids encoding HA-tagged PH domain-truncated human Grb10, full-length human Grb10, and the SH2 domain point mutant human Grb10R520G were described previously (6, 8, 23). The Myc-tagged Shc expression vector, pRK5/myc p52 Shc, was a generous gift from Dr. Ben Margolis (University of Michigan, Ann Arbor, MI). Myc-tagged p42 MAPK was a gift from Dr. Kun-Liang Guan (University of Michigan). Adenoviruses encoding human Grb10 were described previously (15).

Generation of Grb10 Truncation Mutants
A cDNA encoding the SH2 domain-deleted mutant of Grb10 (amino acid residues 1–492; Grb10{Delta}SH2) was generated by PCR using full-length hGrb10 cDNA (8, 11) as a template. The PCR primers used were forward (FW): 5'-TGCTCTAGACAGTGCTACAGAGCCAAC-3'; and reverse (RV): 5'-GCGAATTCGTGCTGTGTCCTGTGAATC-3'. Using the same FW primer and a RV primer (5'-GCG AAT TCC TTC CTC TGC TGA GGG ATT C-3'), we generated another truncation mutant of Grb10 [amino acids 1–414; Grb10 {Delta}(BPS+SH2)]. After restriction digestion, the cDNA fragments were subcloned into the mammalian expression plasmid pBEX, in frame at their C termini with a sequence encoding the HA tag.

Cell Culture
CHO/IR cells were maintained in Ham’s F-12 medium (Life Technologies) supplemented with 10% newborn calf serum and 1% penicillin/streptomycin. HeLa/IR and HeLa/IR/{Delta}Grb10 cells were grown in DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin. 3T3-L1 cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were grown to confluency and then differentiated by addition of the same medium containing isobutylmethylxanthine (500 µM), dexamethasone (25 µM), and insulin (4 µg/ml) for 3 d and then by addition of the medium containing insulin for 3 additional days. The medium was then changed every 3 d until the cells were fully differentiated, which typically occurred on the 10th day.

Immunoprecipitation and Western Blot
Transfections of CHO/IR cells were performed in either 60-mm or 100-mm plates with 5 or 10 µg total recombinant plasmid, respectively, using the LipofectAMINE reagent according to the manufacturer’s protocol (Life Technologies). Twenty-four hours after transfection, cells were treated accordingly and then lysed in 300 µl of buffer A (50 mM HEPES, pH 7.6; 150 mM NaCl; 1% Triton X-100; 10 mM NaF; 20 mM sodium pyrophosphate; 20 mM ß-glycerol phosphate; 1 mM sodium orthovanadate; 10 µg/ml leupeptin; 10 µg/ml aprotinin; 1 mM phenylmethylsulfonyl fluoride). The lysates were centrifuged (14,000 x g, 4 C, 10 min), and the supernatants were incubated with specific antibodies preadsorbed to protein G beads (Amersham Pharmacia Biotech) overnight at 4 C with gentle rotation. The immunoprecipitates were then washed three times with ice-cold buffer B (50 mM HEPES, pH 7.6; 150 mM NaCl; and 0.1% Triton X-100). Proteins bound to the beads were eluted by heating at 95 C for 4 min in SDS-PAGE sample loading buffer. The eluted proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and detected with specific antibodies.

Transfection of Rat Adipose Cells and Elk-1 Phosphorylation Assay
Rat adipose cells in primary culture were prepared from epididymal fat pads and transfected by electroporation as described (30). Each experimental group was transfected with a total of 5 µg DNA/cuvette. After transfected cells were processed and cultured (31), the Path-Detect system (Stratagene, La Jolla, CA) was used to assess the effects of Grb10 constructs on the phosphorylation of an Elk-1 reporter as described previously (32). The adipocytes were subject to serum starvation overnight (as described in the legend to Fig. 1CGo), treated with or without insulin (100 nM, 5 h), and cell lysates were assayed for luciferase activity. Paired t tests were used to compare results from the Elk-1 phosphorylation experiments. Values of P < 0.05 were considered to represent statistical significance.

Generation of the pBS/U6/Grb10 RNAi Construct and the Grb10-Suppressed HeLa/IR Cell Line
To generate the pBS/U6/Grb10 RNAi construct, a 22-nt oligo corresponding to nt 498–518 of full-length human Grb10 (8, 11) (GGTTCTTTACCTCCGAGCCAG) was first inserted into the ApaI (blunted) and XhoI sites of pBS/U6/RNAi construct [a generous gift of Dr. Y. Shi, Harvard Medical School, Boston, MA (25)]. The selected coding sequence has no significant sequence homology to genes other than Grb10 isoforms as determined by BLAST (basic local alignment search tool) search. The second step involved subcloning the inverted sequence that contains a 6-nt spacer and five Ts into the HindIII and EcoRI sites of the intermediate plasmid. Oligo 1 is 5'-GGTTCTTTACCTCCGAGCCAGA-3' (FW) and 5'-AGCTTCTGGCTCGGAGGTAAAGAACC-3' (RV). Oligo 2 is 5'-AGCTTCTGGCTCGGAGGTAAAGAACCCTTTTTG-3' (FW) and 5'-AATTCAAAAAGGGTTCTTTACCTCCGAGCCAG-3' (RV). The added restriction sites are underlined. The successful generation of the pBS/U6/Grb10 RNAi construct was confirmed by DNA sequencing. The HeLa/IR/{Delta}Grb10 stable cell line was created by cotransfecting the pBS/U6/Grb10 RNAi plasmid with the puromycin resistance vector pSV2, using the LipofectAMINE reagent according to the manufacturer’s protocol (Life Technologies). Transfectants were selected with puromycin, and colonies were screened by immunoblot analysis with an anti-hGrb10 antibody (Cell Signaling Technology).

RT-PCR Experiments
Total RNA was isolated from either wild-type HeLa/IR or HeLa/IR/{Delta}Grb10 cells using Trizol (Life Technologies, Inc.). RT-PCR was performed using the SuperScript First-Strand Synthesis System RT-PCR kit from Invitrogen (San Diego, CA) according to the manufacturer’s instructions. cDNA was synthesized from total RNA (5 µg) using oligo(deoxythymidine) primers and Superscript II RT (Invitrogen). Of the resulting single-strand cDNA reaction (20 µl), 2 µl were used as templates for RT-PCR. FW and RV primers used to amplify human Grb10, Grb7, Grb14, and glyceraldehyde phosphate dehydrogenase (GAPDH) were as follows: Grb10-FW (5'-GACCTGGAAGCCCTGGTG-3'), Grb10-RV (5'-CGTGAGCACAGGGGGGCT3'); Grb7-FW (5-TGTGAAATGCTGGTGCAGCGAGC-3), Grb7-RV (5'-ATCTGAGGCACTTCTCAAGGGTGG-3'); Grb14-FW (5'-ACCAGCAGGGCTTTAGATGTACC-3'), Grb14-RV (5'-GAACTGCAGCCACTTCTACCTTG-3'); GAPDH-FW (5'-ACCACAGTCCATGCCATCAC-3'), GAPDH-RV (5'-TCCACCACCCTGTTGCTGTA-3'). Expected sizes for the Grb10 fragment, Grb7 fragment, and the GAPDH fragment are 0.3 kb, 0.7 kb, and 0.4 kb, respectively.


   ACKNOWLEDGMENTS
 
We thank Drs. Ben Margolis, Kun-Liang Guan, and Y. Shi for providing plasmid constructs used in this study. We also thank Drs. KeriLyn Wick, Mike Wick, and Mei Ann Lim for their insightful comments.


   FOOTNOTES
 
This work was supported by NIH R01 Grant DK52933.

Abbreviations: BPS, Between the PH and SH2; CHO, Chinese hamster ovary; FW, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; IR, insulin receptor; IRS, IR substrate; nt, nucleotide; PH, pleckstrin homology; PI 3-kinase, phosphatidylinositol 3'-kinase; RNAi, RNA interference; RV, reverse; SH2, Src-homology 2.

Received for publication April 3, 2003. Accepted for publication November 5, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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