Correspondence to Lewis C. Cantley: lewis_cantley{at}hms.harvard.edu
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Abbreviations used in this paper: IGF, insulin-like growth factor; IRS, insulin receptor substrate; MEF, mouse embryonic fibroblasts; PH, pleckstrin homology; PI, phosphoinositide; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PI-3,4-P2, phosphatidylinositol-3,4-bisphosphate; SH, Src homology; TIRFM, total internal reflection fluorescence microscopy.
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Introduction |
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Class IA PI 3-kinase is a heterodimer consisting of a regulatory subunit (p85) and a catalytic subunit (p110). The p85 regulatory subunit stabilizes the p110 catalytic subunit and holds it in a low activity state (Yu et al., 1998b). The p85 regulatory subunit contains two Src homology (SH) 2 domains that recognize phosphotyrosine residues in the context pYxxM (pY, phosphotyrosine; x, any amino acid) on activated receptors or their adaptor molecules (Songyang et al., 1993). The binding of p85 to tyrosine-phosphorylated proteins serves both to relieve inhibition on the p110 catalytic subunit as well as to recruit PI 3-kinase from the cytosol to the plasma membrane, where its substrate, PI-4,5-P2, resides (Rordorf-Nikolic et al., 1995). Mammals contain three different genes for the p85 regulatory subunit (p85, p85ß, and p55
) and three different genes for the class IA catalytic subunit (p110
, p110ß, and p110
). The major p85 isoform p85
also exists as two shorter splice variants (p55
and p50
) that lack the NH2-terminal SH3 and RhoGAP homology domains of p85
(Fruman et al., 1998).
Both the insulin receptor and the highly homologous insulin-like growth factor (IGF) 1 receptor activate class IA PI 3-kinase indirectly through phosphorylation of the insulin receptor substrate (IRS) family of adaptor molecules. These receptors phosphorylate tyrosine residues on IRS to create p85-binding sites (for review see Butler et al., 1998; Virkamaki et al., 1999). PI 3-kinase is a key mediator of metabolic signaling downstream of the insulin receptor, and it also mediates cell differentiation, survival, and proliferation downstream of both insulin and IGF-1 receptors (Dufourny et al., 1997; Kulik et al., 1997; Saltiel and Kahn, 2001; Tureckova et al., 2001). Despite the key role PI 3-kinase plays in mediating glucose uptake downstream of the insulin receptor, mice lacking various isoforms of the p85 or p85ß subunit of PI 3-kinase demonstrated the paradoxical phenotype of increased insulin sensitivity, which was caused by improved PI 3-kinase signaling downstream of IRS proteins (Terauchi et al., 1999; Fruman et al., 2000; Mauvais-Jarvis et al., 2002; Ueki et al., 2002b; Chen et al., 2004). It was subsequently shown that the molecular balance between monomeric p85 and the p85p110 dimer can influence the extent of PI 3-kinase signaling downstream of the insulin receptor, as monomeric p85 can negatively regulate PI 3-kinase signaling by competing with the p85p110 dimer for IRS binding (Ueki et al., 2002a, 2003). Recently, it was also reported that the protein SOCS-6 can selectively bind to monomeric p85 and that overexpression of SOCS-6 improves insulin signaling in vivo (Li et al., 2004). Furthermore, the expression of the human placental hormone in transgenic mice results in the up-regulation of p85 and subsequent insulin resistance in muscle (Barbour et al., 2004). These lines of evidence suggest that monomeric p85 acts as a negative regulator of insulin signaling in vivo by controlling the extent of PI 3-kinase activation downstream of the receptor.
In this study, we have investigated the spatial translocation of the p85 regulatory subunit of PI 3-kinase in response to IGF-1 receptor activation. We find that 510 min after IGF-1 stimulation, p85 and IRS-1 assemble into large complexes (foci) in the cytosol. This complex formation appears to be driven preferentially by monomeric p85 rather than by p85p110, and these complexes do not appear to be sites of PI-P3 production. These results indicate a novel mechanism for limiting IRS-1/PI 3-kinase signaling that could explain why insulin/IGF-1 signaling is acutely sensitive to p85 levels.
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Results |
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EGFPp85 foci are complexes of p85 and tyrosine-phosphorylated IRS-1
Like the insulin receptor, the IGF-1 receptor activates PI 3-kinase through tyrosine phosphorylation of the IRS family of adaptor molecules, with IRS-1 being the best-characterized member (White, 1998). The pYxxM motifs on IRS-1 directly bind to the SH2 domains of p85 to recruit the p85p110 complex (Backer et al., 1992; for review see Butler et al., 1998). Therefore, we tested whether the translocation of EGFPp85 to foci that are downstream of the IGF-1 receptor requires its interaction with IRS-1. An arginine residue in the conserved FLVRD/E motif of the p85 SH2 domains is critical for coordinating the phosphate moiety of phosphotyrosine (Nolte et al., 1996). Mutations of this arginine to alanine in each of the p85 SH2 domains have been shown to abolish binding to phosphotyrosine without affecting their overall folding (Yu et al., 1998a). Thus, we introduced the same mutations (R358A and R649A in the FLVRD/E motif of each of the SH2 domains of p85
) to generate the EGFPp85
RARA mutant. As expected, although this mutant bound the PI 3-kinase p110 catalytic subunit normally, it could no longer bind tyrosine-phosphorylated IRS-1 after IGF-1 stimulation (Fig. 1 B). Furthermore, the EGFPp85
RARA mutant was much less effective at inhibiting Akt activation when overexpressed in cells (Fig. 1 C), indicating that functional SH2 domains are required for the inhibition of PI 3-kinase signaling by p85. When we coexpressed the CFP-tagged mutant ECFP-p85 RARA with the YFP-tagged wild-type EYFP-p85 in the same cell, only the wild-type EYFP-p85 translocated to foci in response to IGF-1, whereas the SH2 domain mutant ECFP-p85 RARA did not (Fig. 3 A). This indicates that the SH2 domains of p85 are essential in mediating such translocation.
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To rule out the possibility that only overexpressed p85 that is additional to endogenous p85 proteins can form such foci with IRS-1, we expressed EGFPp85
and p85
-FLAG in mouse embryonic fibroblasts (MEFs) that were null for both the p85
and p85ß genes (Brachmann et al., 2005b). We observed IGF-1induced p85
foci formation in p85
/p85ß/ MEFs expressing even the lowest detectable levels of p85
proteins. Furthermore, the introduction of FLAG-tagged p85
into p85
/p85ß/ MEFs by retrovirus infection (using conditions that we have previously shown to result in exogenous p85
levels similar to or lower than endogenous p85
of wild-type MEFs; Brachmann et al., 2005b) led to p85
foci in response to IGF-1 (Fig. 3 D). Thus, p85
IRS-1 foci appear in response to IGF-1 in cells in which total p85
is at or below endogenous levels.
Interestingly, we found that the shorter isoform p55, which lacks the NH2-terminal SH3 and RhoGAP homology domains of p85
, did not translocate to foci in response to IGF-1 (Fig. 3 E). A similar result was obtained with the other shorter isoform p50
(unpublished data). This suggests that SH3 and/or RhoGAP homology domains of p85
play a role in its localization to these foci.
EGFPp85IRS-1 complexes are sites of IRS-1 sequestration
We next investigated whether these EGFPp85IRS-1 foci are sites of PIP3 production. We first used AktPH domainEGFP fusion protein as a reporter to detect PIP3 production in the cell (Gray et al., 1999). We were able to improve the sensitivity of this reporter by trimerizing the PH domain (EGFP-[3]AktPH). As expected, EGFP-(3)AktPH translocated to the plasma membrane in response to stimulation by growth factors, whereas an analogous construct bearing an R25C mutation in each of the PH domains that abolishes binding to the 3' phosphate of PIP3 (Varnai et al., 1999) failed to do so (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200503088/DC1). In addition, the membrane translocation of EGFP-(3)AktPH was also blocked by the PI 3-kinase inhibitor wortmannin (unpublished data). After IGF-1 stimulation, EGFP-(3)AktPH consistently translocated to plasma membrane patches and ruffles within 12 min, which is a time course that is more rapid than that of the EGFPp85
translocation to foci. The pattern of Akt PH domain localization was much more diffused than that of the EGFPp85
foci (Fig. S3). When EYFP-p85
and ECFP-(3)AktPH were coexpressed in the same cell and imaged simultaneously, the translocation of the ECFP-(3)AktPH domain to the plasma membrane was often found to be suppressed (unpublished data), again indicating the down-regulation of PI 3-kinase signaling by p85. In cells in which ECFP-(3)AktPH did show detectable membrane translocation, ECFP-(3)AktPH patches did not colocalize with the EYFP-p85
foci (Fig. 4, A and B), suggesting that these EYFP-p85
foci are not the sites of PIP3 production. This conclusion is further supported by the observation that IRS-1 phosphorylation, PI 3-kinase activation, and Akt activation precedes the appearance of EGFPp85
foci in CHO-K1 cells. As shown in Fig. 4 C,
90% of maximum tyrosine phosphorylation on IRS-1 occurred within the first minute after IGF-1 stimulation. The recruitment of PI 3-kinase to IRS-1 was equally rapid with a nearly identical time course. It took
2 min, however, for the majority of Akt to become activated; this short delay presumably reflects the time required for PI 3-kinase to generate PIP3 at the plasma membrane. Furthermore, there was an
5-min delay before most cells were observed to harbor EGFPp85
foci. The activation of Akt, therefore, precedes the formation of EGFPp85
foci.
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EGFPp85IRS-1 complexes are cytosolic protein complexes
Previous studies in 3T3-L1 cells have suggested that IRS-1 and p85 are selectively enriched in intracellular membrane fractions after insulin stimulation (Nave et al., 1996; Inoue et al., 1998). To determine the subcellular localization and structural nature of EGFPp85 foci, we first assessed their membrane proximity with total internal reflection fluorescence microscopy (TIRFM) to visualize EGFPp85
that was within
100 nm of the plasma membrane (Toomre and Manstein, 2001). Interestingly, as with epifluorescence microscopy, we did not observe prominent global translocation of EGFPp85
to the plasma membrane upon IGF-1 stimulation. TIRFM revealed that many of the EGFPp85
foci formed near the plasma membrane (Fig. 5 A and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200503088/DC1). Once formed, however, many foci internalized away from the plasma membrane as they became invisible under TIRFM, whereas they were still visible under epifluorescence microscopy (Fig. 5 B).
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Discussion |
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In the current study, by using EGFPp85 as a reporter for the localization of p85 in live cells, we found that IGF-1 receptor activation induces the translocation of EGFPp85
to distinct, foci-like complexes that contain p85 bound to tyrosine-phosphorylated IRS-1 (Figs. 2 and 3). The interaction between p85 and IRS-1 is critical for the dominant negative effect of monomeric p85, as the SH2 domainmutated p85 that fails to bind IRS-1 and form foci is far less effective at inhibiting PI 3-kinase signaling (Fig. 1, B and C). Surprisingly, these complexes contain primarily monomeric p85 with less of the p85p110 dimer and appear to be protein-only complexes that are devoid of membrane components. Because these complexes are not on the membrane, the enzyme would not have access to its lipid substrate even if some p85p110 dimer was present in the complex (Figs. 4 and 5). These observations suggest a novel mechanism to explain how monomeric p85 inhibits PI 3-kinase signaling downstream of IRS-1 (Fig. 5 F). In this modified model, in addition to being a competitive inhibitor for p85p110 binding to IRS-1, monomeric p85 sequesters IRS-1 in cytosolic protein complexes and renders it incapable of stimulating PIP3 production. The failure to form these sequestration complexes in mice lacking all isoforms of p85
(Fruman et al., 2000) would explain why insulin signaling in these animals is more efficient even though less PI 3-kinase is present. As the absence of monomeric p85 would eliminate the sequestration of IRS-1, this allows the small amount of p85p110 dimers that form complexes with IRS-1 to efficiently signal for a longer period of time.
The two mechanisms of negative regulation by monomeric p85 (competitive binding vs. sequestration) are likely to operate in concert to limit both the amplitude and duration of PI 3-kinase signaling downstream of IRS-1. The competitive binding of monomeric p85 for IRS-1 modulates the amplitude of PI 3-kinase signaling at early time points (e.g., 12 min) after IGF-1 stimulation, whereas the sequestration of IRS-1 by p85 in a nonsignaling complex at later time points (1020 min) serves to limit the duration (and possibly the overall amplitude) of PI 3-kinase signaling. In comparison to the competitive binding model, the sequestration model displays two additional properties that make it an attractive mechanism for signal down-regulation. First, the formation of the p85IRS-1 complex occurs with a 510-min time delay (Fig. S1 A), thus allowing IRS-1 a short window of time to recruit and activate p85p110 PI 3-kinase (which occurs within 1 min of receptor ligation; Fig. 4 C); thereafter, the sequestration of IRS-1 would rapidly attenuate the signal. Furthermore, as the p85IRS-1 complexes persist for an extended period of time after IGF-1 withdrawal (30 min1 h; Fig. S1 C), they could also serve as a mechanism of recalcitrance to subsequent IGF-1 or insulin stimulation. Second, the sequestration model provides a mechanism by which a small amount of monomeric p85 could have a profound impact on PI 3-kinase signaling, as a relatively small change in the monomeric p85 level could lead to a relatively large change in the amount of tyrosine-phosphorylated IRS-1 that is available to recruit the p85p110 dimer.
Existing antibodies to p85 localize to foci-like structures in a number of cell types. However, these antibodies also detect foci-like structures in p852/2p85ß2/2 fibroblasts, hence precluding their specificity towards p85 in immunofluorescence staining. Although we were unable to identify a p85 antibody that was suitable for the localization of endogenous p85 by immunofluorescence, it is likely that endogenous, monomeric p85 also forms sequestration complexes with IRS-1 in response to IGF-1 or insulin signaling. Low levels of p85
expression alone in p85
2/2p85ß2/2 MEFs that completely lacked endogenous p85 proteins were sufficient for IGF-1induced p85
IRS-1 foci (Fig. 3 D), suggesting that such complex formation can occur at p85 levels close to that of endogenous p85 proteins. Our biochemical analysis of the levels of IRS-1associated endogenous p85 and p110 reveals that there is far less p110 associated with IRS-1 at 20 min post-stimulation compared with that at 2 min, whereas the amount of p85 that is associated with IRS-1 remains essentially unchanged at these time points (Fig. 4 F). The IRS-1associated p85/p110 ratio, therefore, shifts toward p85 at a later time (20 min postIGF-1 stimulation) and in temporal correlation with the appearance of p85IRS-1 complexes. Consistent with this model, the elevation of in vivo p85 levels (with no change in p110 levels) occurs in response to placental growth hormone, which has been shown to correlate with pregnancy-induced insulin resistance (Barbour et al., 2004; Kirwan et al., 2004). Thus, the changes in insulin or IGF-1 sensitivity that we observed upon manipulation of the levels of p85 isoforms in mice or in cell lines are in agreement with pathophysiological insulin resistance that occurs in correlation with the elevation of p85 levels in vivo.
Finally, it is of interest to note that only the full-length isoforms of p85 (p85 and p85ß), but not the shorter isoforms (p55
and p50
), could form complexes with IRS-1 (Fig. 3 D), albeit p55
and p50
possess the same SH2 domains as p85
. This suggests that in addition to the SH2 domains, the NH2-terminal half of p85 is also necessary for the formation of the p85IRS-1 complex. It has been shown that p85 can homodimerize through its NH2-terminal SH3 and RhoGAP homology domains (Harpur et al., 1999). This observation provides an intriguing mechanism of p85IRS-1 complex formation. A p85 homodimer would have four SH2 domains available (two on each p85 monomer) that could, in theory, bind to various combinations of at least six phosphotyrosine motifs on IRS-1. Thus, a given p85 homodimer could bind to two or more distinct IRS-1 molecules and vice versa. This would allow the multimerization of p85 and IRS-1 that leads to the formation of a large globular structure, which is consistent with the EM image of the p85IRS-1 complex (Fig. 5 E). Alternatively, other molecules that interact with the NH2-terminal half of p85 may be required for the formation of the p85IRS-1 sequestration complex.
In conclusion, we demonstrate in this study that the p85 regulatory subunit of PI 3-kinase, when in excess over the p110 catalytic subunit, can drive the formation of large complexes that contain tyrosine-phosphorylated IRS-1 after IGF-1 receptor ligation. The p85IRS-1 complex localizes to the cytosol and, thus, is not a site for PI-3,4,5-P3 production. We propose that the formation of this complex provides a mechanism for limiting insulin and IGF-1 signaling in vivo. It also offers an explanation for why the elevation of p85 under pathophysiological conditions correlates with insulin resistance, whereas the deletion of p85 genes results in increased insulin sensitivity even under conditions in which total PI 3-kinase activity is reduced.
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Materials and methods |
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CHO-K1 cells were maintained in DME-F12 and 10% FCS with 10% CO2, whereas p852/2p85ß2/2 MEFs were maintained in DME and 15% FCS with 10% CO2. Transient transfection was performed using LipofectAMINE Plus (Invitrogen). Stable CHO-K1 cells expressing EGFPp85
under an ecdysone-inducible promoter were established by serial transfection of the cells first with the plasmids pEcR, pRXR, and pTK-hyg, and then with the plasmids pSV40/Zeo2 and pFR-MCS-EGFPp85 (RHeoGene). Stable clones were screened for the inducible expression of EGFPp85
in response to the ecdysone analogue GS-E and were maintained in DME-F12 and 10% FCS supplemented with 350 µg/ml hygromycin (GIBCO BRL) and 400 µg/ml Zeocin (Invitrogen). The expression of EGFPp85
was induced with 5 µM GS-E for 2448 h.
Anti-p85 and anti-pTyr antibodies have been described previously (Fruman et al., 2000). AntiIRS-1 and anti-p110 antibodies were obtained from Upstate Biotechnology; anti-EEA1 and anticaveolin-1 antibodies were from Transduction Laboratories; antiphospho-Akt serine-473, antiphospho-Akt threonine-308, and anti-Akt antibodies were from Cell Signaling Technology; anti-HA antibody was from Sigma-Aldrich; anti-EGFP antibody was from CLONTECH Laboratories, Inc.; Texas redconjugated secondary antibodies were from Jackson ImmunoResearch Laboratories; and colloidal gold-conjugated secondary antibody was from Utrecht University Medical Center. IGF-1 and PDGF were from Austral Biologicals.
Protein immunoblotting, immunoprecipitation, and immunostaining
Cells were lysed in lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM EDTA, 1 mM DTT, 20 mM NaF, 10 mM sodium pyrophosphate, 50 mM ß-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, and 4 µg/ml each of leupeptin, aproteinin, and pepstatin). Clarified lysate was subjected to immunoprecipitation overnight at 4°C, and the immunoprecipitate was washed three times with wash buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 10 mM sodium pyrophosphate, and 50 mM ß-glycerophosphate). Proteins were separated on SDS-PAGE gels and subjected to Western blotting. Quantitation of the Western signal was performed using an imaging system (Odyssey; LI-COR Biosciences). Immunofluorescence staining was essentially the same as described previously (Kanai et al., 2001), as was immunogold staining for EM (Griffiths, 1993).
Microscopy
For live cell imaging experiments, cells were maintained in phenol redfree DME-F12 in an environment chamber on a 37°C heated microscope stage. A 10% CO2 environment was also applied for imaging experiments exceeding 1 h. IGF-1 or PDGF was directly added to the environment chamber to achieve the desired final concentrations. Ligand was removed with acid wash in some experiments (Jones and Kazlauskas, 2001). Live cell epifluorescence video microscopy was performed on a microscope (Diaphot 300; Nikon) with filter sets for EGFP, EYFP, and ECFP fluorescence using a 60x oil immersion objective or a 20x objective, and images were captured with ImagePro 4 software (Media Cybernetics). Live cell TIRFM was performed on a modified microscope (model IX70; Olympus) with a single green laser using a 60x oil immersion objective, and images were captured with Metamorph 5 software (Universal Imaging Corp.). Immunofluorescence imaging of fixed cells was performed on a confocal microscope (model E800; Nikon/Bio-Rad Laboratories) with a dual green/red laser using a 100x oil immersion objective, and images were captured with LaserSharp 2000 software (Bio-Rad Laboratories). Immunogold transmission EM was performed on an electron microscope (model 1200EX; JEOL). Raw images were further processed by using Adobe Photoshop 7 for presentation, and, in some cases, brightness and contrast were adjusted linearly and consistently for all images within the same experiment. Videos were compiled and annotated by using QuickTime Pro software (Apple Computer, Inc.).
Online supplemental material
Fig. S1 shows the dynamics of EGFPp85 foci in CHO-K1 cells after IGF-1 stimulation and the subsequent removal of IGF-1. Fig. S2 shows EGFPp85
foci formation in NIH3T3 cells and EYFP-p85ß foci formation in CHO-K1 cells after IGF-1 stimulation. Fig. S3 shows translocation of the EGFP-(3)AktPH reporter construct in response to IGF-1 stimulation in CHO-K1 cells. Videos 13 are time-lapse videos of CHO-K1 cells expressing EGFPp85
that are stimulated with either IGF-1 or PDGF. Selected frames of these videos are shown in Figs. 2 (A and B) and 5 A, respectively.
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Acknowledgments |
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This work was supported by a Howard Hughes Medical Institute (HHMI) Predoctoral Fellowship to J. Luo, by an HHMI Physician Postdoctoral Fellowship and an NIH grant (K08 DK065108-01) to S.J. Field, and by NIH grants (GM41890 and CA089021) to L.C. Cantley.
Submitted: 16 March 2005
Accepted: 20 June 2005
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