Different Subcellular Localization and Phosphoinositides Binding of Insulin Receptor Substrate Protein Pleckstrin Homology Domains

Giorgia Razzini, Alessandra Ingrosso, Anna Brancaccio, Salvatore Sciacchitano, Diana L. Esposito and Marco Falasca

Unit of Physiopathology of Cell Signaling (G.R., A.I., A.B., M.F.) Laboratory of Cellular and Molecular Endocrinology Department of Cell Biology & Oncology Istituto di Ricerche Farmacologiche "Mario Negri" Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (CH), Italy
Department of Experimental Medicine and Pathology (S.S.) Universita’ "La Sapienza" di Roma 00161 Roma, Italy
Department of Oncology and Neuroscience (D.L.E.) Università G. D’Annunzio Chieti 66100 Chieti, Italy


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3–5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The insulin receptor (IR) is a tyrosine kinase that phosphorylates intermediate adapter proteins on multiple tyrosine residues during its activation. Several insulin receptor substrate (IRS) proteins have been identified, including IRS-1, IRS-2, IRS-3, and IRS-4 (1). These multidocking proteins activate downstream signaling pathways by interacting with SH2-containing proteins, some of which are key players in insulin signal transduction, such as Grb-2, the regulatory subunit of phosphoinositide 3-kinase (PI 3-K) p85, and the phosphotyrosine phosphatase SHP-2 (SH2-containing protein tyrosine phosphatase) (2). Recently, a new IR-docking protein, called Gab-1 (Grb-2-associated binding-1) has been identified (3). The activity of PI 3-K is necessary to elicit many of the effects of insulin on glucose and lipid metabolism, indicating that it is an essential downstream effector of insulin signaling (4). The principal mechanisms through which insulin activates PI 3-K appear to be via IRS proteins. IRS-1 and -2 are widely distributed, whereas IRS-3 expression has been demonstrated in adipose tissue, fibroblasts, and liver cells (5, 6). In adipose cells, the association of the regulatory subunit of PI 3-K p85 is much more rapid for IRS-3 than for IRS-1 (5). In addition, in the same cells, PI 3-K activation by IRS-3 is quantitatively higher than that mediated by IRS-1 or IRS-2. In studies of the specificity of insulin signaling, it is of great interest to determinate the exact contribution of each single IRS protein in the promotion of the downstream effects of insulin. In particular, the formation of complexes catalyzed by IRS proteins in specific compartments appears crucial. All the IRS proteins have similar structures, sharing a highly homologous N terminus pleckstrin homology (PH) domain, and a phosphotyrosine binding (PTB) domain, both of which are important for mediating interactions with the insulin receptor (1). PH domains, which have been found in several signaling proteins, have functional requirements for membrane association (7). The IRS-1 PH domain has been shown to be essential for efficient tyrosine phosphorylation of IRS-1 during insulin stimulation (8, 9). The ligands of IRS PH domains are unknown, although it has been shown that the IRS-1 and -2 PH domains bind to acidic proteins (10). Many PH domains have been found to bind phosphoinositides (11), and this suggests that the PH domains of the IRS proteins might allow the interaction of these with membrane phosphoinositides. Here we have examined the role of the IRS PH domains in membrane targeting and phosphoinositide interactions by using subcellular localization studies in vivo and phosphoinositide-binding assays in vitro. We show that the IRS-1 and IRS-2 PH domains, tagged using green fluorescent protein (GFP), are localized exclusively in the cytoplasm in serum-starved NIH-IR and L6 cells. Stimulation with insulin or insulin-like growth factor I (IGF-I), but not platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), induces a rapid translocation of the GFP-IRS-1 and GFP-IRS-2 PH domains to the plasma membrane. This translocation is blocked by pretreatment with the PI 3-K inhibitors, wortmannin and LY294002, and it is stimulated by cotransfection of the constitutively active PI 3-K (p110F). In addition, we show that the PH domains of IRS-1 and IRS-2 bind the lipid products of PI 3-K, corroborating the idea that PI 3-K is a determinant for PH domain-mediated anchoring to the plasma membrane. Finally, the PH domain of IRS-3 binds specifically to phosphatidylinositol 3-phosphate (PtdIns-3-P), and is present on the plasma membrane either in serum-starved or insulin-stimulated cells. These results reveal a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and IRS-2 signaling in insulin-stimulated cells, and suggest the differential regulation of IRS-1 and IRS-2 vs. IRS-3.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Specific Binding of the IRS Protein PH Domains
To study the lipid binding properties of the PH domains derived from human (h)IRS-1, murine (m)IRS-2, and mIRS-3, we used a lipid dot-blot assay to analyze the binding of [32P]-labeled glutathione-S-transferase (GST)-PH domain fusions to different phosphoinositides spotted on nitrocellulose, as previously described (12). Figure 1Go shows the results of the dot-blot assay for GST alone (Fig. 1AGo) and GST-PH domain fusions from phospholipase C{delta} (Fig. 1BGo) and protein kinase B (PKB, also known as Akt and RAC; Fig. 1CGo), used as positive controls, and for IRS-1, IRS-2, and IRS-3 (Fig. 1Go, D–F). The control blot using [32P]-labeled GST alone gave no signals above background, whereas the phospholipase C{delta} (PLC{delta}) PH domain bound phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) with high specificity, and the Akt/PKB PH domain bound phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) and phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) as previously shown (13, 14, 15). Analysis of these blots indicated that the IRS-1 PH domain showed a promiscuous pattern with preferential binding to PtdIns-3,4-P2, but also, although weaker, binding to PtdIns-3,4,5-P3 and PtdIns-4,5-P2. Higher specificity was observed in the case of the IRS-2 PH domain, which showed a preferential binding to PtdIns-3,4-P2 (Fig. 1EGo), whereas the IRS-3 PH domain showed specific binding for PtdIns-3-P (Fig. 1FGo). Only small quantities of other phosphoinositides were bound by IRS-1, IRS-2, and -3 PH domains. Other inositol phospholipids tested in the same assay, such as phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, and phosphatidylinositol 3,5-bisphosphate, gave much weaker or no signals (data not shown). Since this assay does not allow a quantitative comparison, we further investigated the phosphoinositide binding to IRS protein PH domains by using a different assay. We incubated a GST-fusion of the IRS PH domains bound to glutathione beads with a mixture of tritiated inositol phosphate standards (see Materials and Methods and Ref. 16). The inositol phosphates present in the mixture (Fig. 2AGo) and those remaining attached to the glutathione beads (Fig. 2Go, B–E) after incubation with the mixture and subsequent washing, were analyzed by HPLC. As shown in Fig. 2BGo, the GRP-1 PH domain, our positive control, bound inositol-1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) with very high specificity, as previously shown (17), while the PLC{delta}1 PH domain bound inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) (not shown) (15). The IRS-1 PH domain showed preferential binding to Ins-1,3,4,5-P4 although substantial binding to inositol-1,3,4-trisphosphate (Ins-1,3,4-P3) and Ins-1,4,5-P3 was also evident (Fig. 2CGo). The binding of the IRS-2 and IRS-3 PH domains for Ins-1,3,4-P3 (Fig. 2DGo) and inositol 3-phosphate (Ins-3-P) (Fig. 2EGo), respectively, was much more specific. In the latter case, we also detected a peak around 71 min that corresponds to inositol-1,3-bisphosphate (Ins-1,3-P2), which is present as a contaminant in the standards pool used in the assay. In addition, it is worth noting that in this pool we used Ins-3-P instead of Ins-1,3-P2.



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Figure 1. Comparison of the Phosphoinositide-Binding Specificities of the GST-IRS PH Domains Using a Dot-Blot Assay

[32P]-labeled GST-PH domain fusion proteins were used to probe nitrocellulose filters onto which specific phosphoinositides had been spotted. The amount of lipid spotted in each row was 0.2, 0.4, 0.6, 0.8, and 1.0 µg.

 


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Figure 2. Inositol Phosphate Binding Specificities of the GST-IRS PH Domains

GST fusion proteins attached to beads were incubated with a mixture of standard [3H]-inositol phosphates. The beads were washed, and then the bound inositol phosphates were eluted with 0.1 N HCl, separated by HPLC, and quantified by an on-line radioactivity flow detector. A, HPLC profile of the mixture of standard [3H]-inositol phosphates (1/10 of mixture incubated with the beads); B, [3H]-inositol phosphates bound to the GST Grp-1 PH domain; C, to the GST IRS-1 PH domain, D, to the GST IRS-2 PH domain, and E, to the GST IRS-3 PH domain. No binding was observed with GST alone.

 
Quantitative analyses of the HPLC patterns, expressing the amount of radioactivity of each compound as a percentage of the total radioactivity present in the mixture, and then, in the case of the IRS-1 PH domain, as a percentage of Ins-1,3,4,5-P4 bound, demonstrate that the IRS-1 PH domain binds most strongly to Ins-1,3,4,5-P4 (100.0 ± 12.0) and, to a lesser extent, to Ins-1,3,4-P3 (38.5 ± 2.5), Inositol 1,4,5-P3 (20.0 ± 5.4), and Ins-3-P (8.5 ± 5.1). The same analyses for the IRS-2 and IRS-3 PH domains show strong preferential binding to Ins-1,3,4-P3 and Ins-3-P, respectively, and weak (<10% of preferential inositol phosphates) or no binding to the other inositol phosphates. Similar data were obtained when the GST fusion of the IRS-1, IRS-2, and IRS-3 PH domains was incubated with deacylated cell-lipid extracts obtained from [3H]-inositol-labeled L6 cells stimulated with insulin. Furthermore, the same relative affinities for binding to standard inositol headgroups were observed when a quantitative analysis was performed using deacylated phosphoinositides (data not shown).

Subcellular Localization of the IRS Protein PH Domains
To explore the possible roles of the PH domains in signal-dependent localization of the IRS proteins, we analyzed the subcellular localization of the IRS PH domains fused to a GFP. We transfected the GFP-IRS PH domains into NIH-IR and L6 cells. In serum-starved NIH-IR cells transfected with the GFP-IRS-1 and GFP-IRS-2 PH domains, all the fluorescence was dispersed in the cytoplasm (Fig. 3Go, A and D). Stimulation of cells with 100 nM insulin induced a translocation of the GFP-IRS-1 and IRS-2 PH domains to the plasma membrane, which was visible after 2–3 min of stimulation, and more evident at longer times (5–10 min; Fig. 3Go, B, C, E, and F). A different behavior was observed when the IRS-3 PH domain was transfected. In fact, as shown in Fig. 3GGo, in serum-starved cells there was a clear plasma membrane localization, and after insulin stimulation there were no qualitative differences, although the localization appeared to be more pronounced (Fig. 3Go, H and I). To obtain a quantitative analysis of these observations, a blind scoring of the intracellular localization was performed and has been shown in Fig. 3JGo; clear quantitative differences exist in the plasma membrane localization of the GFP-IRS-1 and IRS-2 PH domains in unstimulated and insulin-stimulated NIH-IR cells, whereas no difference was found in the case of the GFP-IRS-3 PH domain. The nuclear staining present in both unstimulated and stimulated cells was also found with the empty vector and therefore represents nonspecific staining due to the small size of the fusion protein that permits it to diffuse through the nuclear pores (16).



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Figure 3. Insulin-Induced GFP-IRS PH Domain Membrane Translocation in NIH-IR Cells

Fluorescence micrographs, obtained as described previously (9 ), of NIH-IR cells expressing GFP-IRS-1 (A–C), GFP-IRS-2 (D–F), and GFP-IRS-3 (G–I) PH domains at the indicated times after insulin (100 nM) stimulation. Bar, 10 µm. J, Statistical analysis of the plasma membrane localization in control and insulin-stimulated NIH-IR cells transfected with GFP IRS-1, IRS-2, and IRS-3 PH domains. Means ± SD from three separate experiments, each performed in duplicate, are shown.

 
Effects of Other Growth Factors on the Subcellular Localization of the IRS Protein PH Domains
To determine whether a similar translocation of IRS PH domains takes place in response to stimulation of other receptors that activate PI 3-K, NIH-IR 3T3 and L6 cells were also stimulated with IGF-I, PDGF, and EGF. IGF-I evoked a translocation response of the IRS-1 and -2 PH domains very similar to that observed when insulin was used as the agonist (Fig. 4Go, C and D). Interestingly in this case, under the same conditions, PDGF (Fig. 4Go, E and F) and EGF (Table 1Go) were not able to induce any translocation of the GFP-IRS-1 and GFP-IRS-2 PH domains in L6 and NIH-IR cells after up to 10 min of stimulation, remaining instead like the untreated cells (Fig. 4Go, A and B). In parallel, as a positive control, PDGF was able to induce plasma membrane translocation of GFP-Akt-PH (data not shown). To further analyze this selectivity in IRS-1 PH domain translocation induced by different growth factors, we microinjected the GFP-IRS-1 PH domain DNA into differentiated 3T3 L1 adipocytes, where differences in insulin and PDGF signaling have been well studied. As shown in Fig. 5AGo, in serum-starved adipocytes, no specific localization of the IRS-1 PH domain was observed, while after 10 min of insulin stimulation there is a clear plasma membrane localization (Fig. 5BGo); this was not seen with PDGF stimulation (Fig. 5CGo), confirming the specificity of insulin in inducing plasma membrane localization of the IRS-1 PH domain.



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Figure 4. IGF-I, but Not PDGF, Induces IRS-1 and IRS-2 PH Domain Membrane Translocation in NIH-IR Cells

Cells were transiently transfected with GFP-IRS-1 and GFP-IRS-2 PH. After 24 h of serum starvation, the cells were stimulated with IGF-I (10 ng/ml, panel B), or PDGF (25 ng/ml; panel C). Bar, 10 µm.

 

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Table 1. Binding Specificities and Conditions Affecting Plasma Membrane Localization of the IRS PH Domains

 


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Figure 5. Insulin, but Not PDGF, Induces IRS-1 PH Domain Membrane Translocation in 3T3 L1 Adipocytes

3T3 L1 adipocytes were serum-starved for 12 h and then microinjected with GFP-IRS-1. After 4 h, cells remained unstimulated (A), were stimulated with insulin (100 nM, panel B), or PDGF (25 ng/ml, panel C).

 
Effect of PI 3-K Inhibitors on the IRS Protein PH Domain Localization
To test whether this event was PI 3-kinase-dependent, as our binding data suggested, we preincubated NIH-IR and L6 cells with the PI 3-K inhibitors wortmannin (Fig. 6Go, B and D) and LY294002 (Table 1Go) before insulin stimulation. This pretreatment completely abolished insulin-dependent translocation to the plasma membrane of the GFP-IRS-1 and –2 PH domains in NIH-IR (Fig. 6Go, B and D) and L6 cells (Table 1Go). In addition, similar data were obtained by coexpression with a dominant negative p85 (the regulatory subunit of PI 3-K) mutant (Table 1Go). When the effect of the inhibitors of PI 3-K on IRS-3 PH domain localization was examined, we found that wortmannin inhibited GFP IRS-3 PH domain plasma membrane targeting in both insulin-stimulated (Fig. 6Go, E and F) and serum-starved NIH-IR (Fig. 6Go, G and H) and L6 cells (Table 1Go), whereas coexpression with a dominant negative p85 had no effect (Table 1Go). Next, we examined the effect of expression of a constitutively activated PI 3-K (p110F) on the cellular localization of the GFP-IRS-1 and -2 PH domains. In L6 cells, membrane localization of the IRS-1 and -2 PH domains was not seen in cells that express a membrane-targeted kinase-inactive form of PI 3-K (p110KD) (Fig. 6Go, I and J). In contrast, in L6 cells that overexpress the membrane-targeted PI 3-K, the PH domains of IRS-1 and IRS-2 were targeted to the plasma membrane even in serum-starved cells (Fig. 6Go, K and L). These data suggest that the IRS-1 and -2 PH domain binding to the PI 3-K products plays a role in their membrane translocation. In addition, the membrane targeting of the IRS-3 PH domain seems not to be dependent on p110 activity, but should be dependent on the level of PtdIns-3-P itself.



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Figure 6. Effect of PI 3-Kinase Inhibition or Activation on Membrane Localization of the GFP-IRS PH Domains

A–D, Fluorescence micrographs of serum-starved NIH-IR cells expressing the GFP-IRS-1 and IRS-2 PH fusion proteins, stimulated with 100 nM insulin for 10 min, with 100 nM wortmannin pretreatment for 10 min. E–H, Fluorescence micrographs of serum-starved NIH-IR cells expressing the GFP-IRS-3 PH fusion protein, stimulated (E and F) or unstimulated (G and H) with 100 nM insulin for 10 min, with 100 nM wortmannin pretreatment for 10 min (F and H). I–L, Fluorescence micrographs of serum-starved L6 cells that had been cotransfected with a vector expressing the GFP-IRS-1 and GFP-IRS-2 PH fusion proteins and inactivated (p110KD) p110ß (PI 3-K) and membrane-targeted activated (p110F). All p110 variants have been described and characterized previously (37 ). In all transfection experiments the total amount of DNA in each transfection was held constant by the addition of empty vector. Bar, 10 µm.

 
Phosphoinositide Specificity and Subcellular Localization of the IRS-1 PH Domain Mutated in the Variable Loop
Our localization studies indicated that the IRS-1 PH domain translocation is PI 3-K dependent, while our binding data indicated that the IRS-1 PH domain binds weakly to PI 3-K lipid products. To further investigate whether the IRS-1 PH domain is able to bind to membrane PtdIns-3,4,5-P3 in intact cells, we examined the binding of an IRS-1 PH domain mutant. In PH domains of proteins known to bind products of PI 3-K, such as Bruton tyrosine kinase, Grp-1, and Akt, it has been shown that an arginine located in the ß2-strand (Fig. 7aGo) is critical for lipid binding as well as plasma membrane localization. Therefore, we mutated this residue (R28C) and tested whether the PH domain was still able to localize to the plasma membrane, and whether the lipid binding was affected. As shown in Fig. 7Go, B and C, the IRS-1 mutant neither localized to the plasma membrane upon insulin stimulation, nor bound the phosphoinositides in our binding assays (Table 1Go), indicating that phosphoinositide binding of the IRS-1 PH domain is essential for its recruitment to the plasma membrane.



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Figure 7. Insulin-Induced Changes in the Localization of the Wild-Type and R28C Mutant GFP IRS-1 PH Domain in Transfected NIH-IR Cells

a, Schematic representation of the IRS-1 structure and alignment of the ß2-strand of the IRS-1 PH domain with other PH domains of proteins known to bind the products of PI 3-K. b, Fluorescence micrographs of NIH-IR cells expressing GFP-IRS-1 (A and B) and GFP-IRS-1 R28C (C). Bar, 10 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we have examined the subcellular localization of the PH domains derived from IRS-1, IRS-2, and IRS-3, and their binding to inositol phospholipids and phosphates. In summary, our results clearly show that the IRS-1 PH domain has a weaker preferential affinity for PtdIns-3,4-P2 and PtdIns-3,4,5-P3, the IRS-2 PH domain binds to PtdIns-3,4-P2, and the IRS-3 PH domain binds to PtdIns-3-P. In addition, insulin and IGF-I stimulate the translocation of the IRS-1 and IRS-2 PH domains from the cytosol to the plasma membrane in NIH-IR and L6 cells, and this translocation is dependent upon insulin-stimulated PI 3-K activity; the IRS-3 PH domain did not show this insulin-dependent translocation.

These observations enlarge on the concept that several PH domains play a determinant role in the translocation of cytosolic proteins to the plasma membrane, where the lipid products of PI 3-K are formed. The observed PI 3-K-dependent localization of the IRS-1 and IRS-2 PH domains to the plasma membrane supports the intriguing idea that PI 3-K is both required for, and a consequence of, IRS-1 signaling. PI 3-K is an essential enzyme in the insulin-signaling cascades that mediate both metabolic and mitogenic cellular responses (4). Insulin can activate both IRS-associated and non-IRS-associated PI 3-K activities (4, 18). It is of interest to determine the exact way through which insulin activates PI 3-K-mediated recruitment of the IRS-1 and IRS-2 PH domains. It is thus possible that the direct activation of PI 3-K by insulin or IGF-I receptors (19, 20), or the PI 3-K activity recruited by the other IR docking proteins, could be involved in IRS-1 and IRS-2 membrane targeting. One possible explanation for these results is that IRS-3 is responsible for PI 3-K activation after insulin stimulation, and thereby promotes IRS-1 and IRS-2 translocation. Interestingly, PDGF, a potent stimulator of PI 3-K, whose activity in terms of lipid production was confirmed in the cell types used in the present study (data not shown), does not induce membrane translocation of the IRS-1 and IRS-2 PH domains. This result suggests that the PI 3-K-induced translocation of certain PH domains is specific for certain growth factors. This specificity could be explained as previously proposed (21, 22, 23), as a consequence of different quantitative or spatio-temporal accumulation of PI 3-K products, possibly due to differential localization and regulation of PI 3-K by insulin vs. PDGF. It has been shown that in 3T3-L1 adipocytes, insulin and PDGF activate PI 3-K rapidly, although the time courses of activation differ, since insulin activation of PI 3-K is more persistent compared with the transient activity induced by PDGF (24). Alternatively, and as recent results suggest (10), it could be that PI 3-K is necessary, but not sufficient, to induce this translocation, and that other factors, such as specific proteins, are involved. Our binding data establish clearly that the PH domains of IRS-2 and IRS-3 preferentially bind PtdIns-3,4-P2 and PtdIns-3-P, respectively, whereas the binding specificity of the IRS-1 PH domain appears less clear. In fact, the dot-blot analysis seems to indicate a higher affinity of the IRS-1 PH domain for PtdIns-3,4-P2, whereas the binding on glutathione beads suggests a preferential binding to PtdIns-3,4,5-P3. This discrepancy can be explained by the fact that PtdIns-3,4,5-P3 is more water soluble and hence can be washed out of the filter, thus giving an underestimation of its binding to the IRS-1 PH domain, as already discussed (16). At the same time, and taking into consideration that there is preferential binding to PtdIns-3,4,5-P3, we also observed substantial binding to PtdIns-4,5-P2; the latter is much more abundant than the 3-phosphorylated phosphoinositides (25), even in activated cells, and may suggest that in the cell the PH domain would interact with PtdIns-4,5-P2. In addition, as shown in Fig. 2Go and according to previous data (14), Grp-1 PH domain binding to PtdIns-3,4,5-P3 is much stronger compared with that observed with the IRS-1 PH domain. This evidence may indicate the absence of any physiological role for this interaction. However, our experiments demonstrating the importance of PI 3-K in IRS-1 PH domain translocation imply that this event is most likely regulated by PI 3-K products. In addition, the evidence that the IRS-1 PH domain does not localize in serum-starved cells indicates that the potential interaction with PtdIns-4,5-P2 is not relevant. In other words, we can hypothesize that binding of IRS-1 to PtdIns-3,4,5-P3 occurs only where there is a substantial and localized accumulation of PtdIns-3,4,5-P3. Consistent with this hypothesis is the evidence that constitutively active PI 3-K alone was sufficient to cause the translocation of the IRS-1 PH domain. A possible explanation for these data, other than cooperation between the phosphoinositides and further proteins, as already proposed for the ß-adrenergic receptor kinase PH domain (26), could be that the IRS-1 PH domain requires oligomerization for high-affinity phosphoinositide binding, as already shown for PH domains of dynamin isoforms (27). Our binding data cannot rule out a role for protein binding to IRS PH domains, as recent data have suggested for the IRS-1 and IRS-2 PH domains (10). In fact, this interaction could give further specificity, together with lipid binding, or may be involved in a different step of IRS activation. Previous studies using cell fractionation have shown that IRS-1 and -2 are mainly located in the low density microsome fraction, showing a different pattern of localization compared with IRS-3, which is located mainly in the plasma membrane fraction. IRS-1 and -2 localization to the intracellular membranes seems not to involve the PH domains. The mechanism by which the binding of PI 3-K lipid products to the isolated PH domains of the IRS proteins play a role in the regulation of the intact proteins in the cells remains unknown, but is at present under investigation. In the case of IRS-1 and IRS-2, this interaction could play a role in the correct orientation of the docking protein for efficient tyrosine phosphorylation by the receptor. A similar model for IRS-1 has been recently proposed on the basis of structural and functional data (28). Therefore, in the case of IRS-1 and IRS-2, although the PH domain localization to the plasma membrane is physiologically relevant, it is not sufficient to localize the entire protein. This explains why its localization has not been found previously on the plasma membrane, although it is fundamental for its correct orientation and targeting to the plasma membrane. In fact, as depicted in our model (Fig. 8Go), we hypothesize that the IRS-1 and IRS-2 proteins are targeted to intracellular membranes, and upon insulin stimulation the protein could, through its PH domain, interact with the plasma membrane; this could potentially occur in specific areas, such as with the cytoskeleton. This could thus be an intriguing explanation of the specificity of insulin vs. PDGF signaling and also underlines the importance of the localization of IRS-1 and IRS-2 to specific intracellular compartments.



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Figure 8. Model Outlining the Role of Phosphoinositide-IRS-1 and IRS-2 PH Domain Interactions in Insulin Signaling

LDM, Low-density microsome.

 
Alternatively, a different mechanism could be hypothesized. It has recently been shown that a truncated IRS-1 protein that retains only the PH and the PTB domains inhibits apoptosis and replication of 32DIR cells during insulin stimulation, and that although PI 3-K was not activated, its lipid products were required since LY294002 inhibited these responses (29). These results indicate that novel phosphotyrosine-independent signaling pathways may be regulated by the PH and PTB domains of IRS proteins, suggesting a possible functional role of 3-phosphoinositide interactions with IRS-PH domains.

Recent results confirm that the IRS-1 and -2 PH domains are functionally similar and are required for interaction with 3-phosphorylated phosphoinositides to facilitate interactions between IRS proteins and the insulin receptor (30). In fact, the IRS-2 domain can substitute for the IRS-1 domain of a chimeric IRS protein, whereas heterologous PH domains from the ß-adrenergic receptor kinase, phospholipase C{gamma} (the putative split PH domain not the N-terminal) or spectrin, which did not show binding preference for 3-phosphorylated phosphoinositides in our assays (data not shown and Ref. 16), fail to mediate tyrosine phosphorylation of chimeric IRS-1 proteins (30). The preferential binding of the IRS-2 PH domain to PtdIns-3,4-P2 suggests an important role for this lipid in insulin signaling by the addition of a downstream target together with Akt/PKB. In addition, these data underscore the potential implication of a PtdIns-3,4,5-P3 5'-phosphatase, the enzyme that has PtdIns-3,4,5-P3 as substrate, and produces PtdIns-3,4-P2. In fact, it has been previously shown that insulin activates the formation of complexes containing PtdIns-3,4,5-P3 5'-phosphatase (31). Thus, it is possible that the insulin-sensitive PtdIns-3,4,5-P3 5'-phosphatase has a positive signaling function.

The finding that the IRS-3 PH domain binds to PtdIns-3-P introduces the novelty that a PH domain binds specifically to PtdIns-3-P, and that this interaction occurs at the plasma membrane. Previous data have shown that a protein domain called FYVE, from the initials of the proteins where it was first characterized, binds specifically to PtdIns-3-P and localizes the protein to the Golgi compartment (32, 33, 34). Interestingly, the FYVE domain of EEA1 also binds to Rab5 (35), suggesting that the binding specificity is given not only by PtdIns-3-P, but that it also requires a protein interaction. The localization of the IRS-3 PH domain to the plasma membrane, even in serum-starved cells, and its displacement by pretreatment with wortmannin (known to reduce the level of PtdIns-3-P) suggest not only that a PH domain may bind specifically to this lipid, but also that a portion of the PtdIns-3-P is constitutively present on the plasma membrane.

When our results are combined with previous reports (36), it appears that the regulation of IRS-1 and -2 is different from that of IRS-3. Further studies will be required to clarify which IRS protein transduces the signal inducing the individual insulin-induced cell activities; perhaps the use of single PH domains as dominant negatives will help in the comprehension of signals transduced by each of the IRS proteins.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PDGF-BB and IGF-I were purchased from PeproTech Ltd. (London, UK). DMEM, calf serum (CS), and FCS were obtained from Life Technologies, Inc. (Gaithersburg, MD). All other biochemicals were of the highest possible purities and were obtained from Sigma (St. Louis, MO).

Expression of GST-PH Fusion Proteins
The cDNA fragments corresponding to the PH domains from human IRS-1 (residues 1–134), mouse IRS-2 (residues 1–144), and mouse IRS-3 (20–145) were amplified using PCR. The PCR products were digested with BamHI plus EcoRI and were ligated into the appropriately digested pGEX-2TK (Amersham Pharmacia Biotech, Arlington Heights, IL) bacterial expression vector to direct expression of the GST-PH domain fusion proteins. The DNA sequence of each PH domain insert was verified by dideoxynucleotide sequencing. The GSTPH domain fusions were expressed and purified using glutathione-agarose, as described previously (16). The point mutation in IRS-1 was generated by PCR mutagenesis using the Quick Change Mutagenesis System (Stratagene, La Jolla, CA).

Dot-Blot Assay
Phosphoinositides (Matreya, Inc., Pleasant Gap, PA; and Echelon, Salt Lake City, UT) in 1:1 chloroform-methanol containing 0.1% HCl (12 N) were spotted onto nitrocellulose filters, as shown. After drying, the nitrocellulose filters were blocked for 6–8 h at 4 C in Tris-buffered saline (TBS) containing 1% BSA, without detergent. The [32P]-labeled GST-PH domain fusion proteins, labeled as previously described (12), were then added to TBS/1% BSA to a final concentration of 0.5 µg/ml, and this solution was used to probe the phosphoinositide-containing nitrocellulose for 30 min at room temperature. Filters were washed five times with TBS and dried, and the bound radioactivity was visualized and quantified using a PhosphorImager (Molecular Dynamics, Inc.).

Analysis of [3H]-Inositol Phosphate and [3H]-Glycerophosphoinositide Binding to PH Domains
Mixtures of standard [3H]-inositol phosphates [NEN Life Science Products (Boston, MA), 21 Ci/mmol; ARC, Inc. (St. Louis, MO)], used at the same concentrations, were added to the GST-PH fusion proteins (50 µg) on glutathione-agarose beads. After incubation at 25 C for 10 min, the beads were washed three times with 10 mM Tris, pH 7.4, and the bound counts eluted with 0.1 N HCl. The eluted material was analyzed by strong anion exchange (SAX) HPLC as described previously (16), using a Partisil 10 SAX analytical column, eluting with a shallow gradient from 0.0 to 1.0 M (NH4)2HPO4 (pH 3.80). Radioactivity in the eluate was monitored with an on-line radioactivity flow detector (FLO ONE A-525, Packard Instruments, Meriden, CT). Quantitative analysis was obtained by calculating the amount of radioactivity under the peaks and using the same protein concentrations and specific activities of the different [3H]-inositol phosphates. This assay was initially tested by using several PH domains, and other putative lipid-binding domains, derived from different proteins, and gave very reproducible results, consistent with those obtained with other binding assays. In addition, with several PH domains, such as those from pleckstrin and dynamin, we did not find any binding at all (as for GST alone), indicating that only specific binding was detected.

Subcellular Localization Studies
cDNA encoding the different PH domains was subcloned (in frame with GFP) into the GFP fusion protein expression vector pEGFP-C1 (CLONTECH Laboratories, Inc., Palo Alto, CA), using the BglII and EcoRI sites for expression of an EGFP-PH domain fusion protein in mammalian cells. NIH-IR cells, kindly provided by Professor E. Skolnik (New York University, New York, NY) and L6 cells were maintained in DMEM supplemented with 10% CS and FCS, respectively. Cells were seeded onto 12-mm circular glass coverslips in wells of a six-well plate and transfected with 1 µg of EGFP fusion protein. LipofectAMINE (Life Technologies, Inc.) was used for the transfections according to the manufacturer’s suggestions. Twenty-four hours after transfection, the NIH-IR and L6 cells were serum-starved overnight in 0.5% CS and 0% FCS, respectively. After stimulation with growth factors, cells were washed in PBS and fixed in 4% paraformaldehyde/PBS and mounted for fluorescence microscopy. Microscopy was performed using a LSM 510 laser confocal microscope system connected to an Axiovert 100M (Carl Zeiss, Oberkochen, Germany) and using a 63x objective. Statistical analysis of the plasma membrane localization (no score, 0–25% membrane localization; score 1, >25% membrane localization) was performed by blind scoring of at least 100 cells from three separate experiments, each performed in duplicate.

Microinjection
3T3 L1 adipocytes, differentiated as described previously (24), were plated at a density of 3.5 x 104 onto 13-mm glass coverslips. The following day, the cDNA of PH-IRS1 GFP in PBS was microinjected in cells in HEPES-buffered culture medium, using an Axiovert IM 35 microscope (Carl Zeiss), a micromanipulator 5171 (Eppendorf, Hamburg, Germany), and microinjector 5246. At 4 h after microinjection, the cells were serum starved in 0.5% for 12 h in DMEM. Cells were then stimulated, as described above.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. G. Rodrigues and Dr. C. P. Berrie for constructive comments on the manuscript, Dr. M. A. Lemmon for many valuable discussions, and A. Marchegiani and C. Iurisci for excellent technical assistance.


    FOOTNOTES
 
Address requests for reprints to: M. Falasca, Unit of Physiopathology of Cell Signaling, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (CH) Italy.

This work was supported by Telethon Italy (Grant 328/bi to M.F. and Grant E.606 to D.L.E.) and in part by the Italian National Research Council (Convenzione C.N.R., Consorzio Mario Negri Sud).

Received for publication November 15, 1999. Revision received March 15, 2000. Accepted for publication March 22, 2000.


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