Role of Pleckstrin Homology Domain in Regulating Membrane Targeting and Metabolic Function of Insulin Receptor Substrate 3

Tania Maffucci, Giorgia Razzini, Alessandra Ingrosso, Hui Chen, Stefano Iacobelli, Salvatore Sciacchitano, Michael J. Quon and Marco Falasca

The Sackler Institute for Muscular Skeletal Research (T.M., G.R., A.I., M.F.), Department of Medicine, University College London, London WC1E 6JJ, United Kingdom; Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland 20893-1632; Department of Oncology and Neurosciences (S.I.), Section of Medical Oncology, Universita’ "G. D’Annunzio," 66100 Chieti, Italy; and Chair of Endocrinology (S.S.), 2nd Faculty of Medicine, Universita’ "La Sapienza" di Roma, Centro Ricerca Ospedale S. Pietro Fatebenefratelli, Associazione Fatebenefratelli per la Ricerca, 00189 Rome, Italy

Address all correspondence and requests for reprints to: Marco Falasca, The Sackler Institute, University College London, Rayne Building, 5 University Street, London WC1E 6JJ, United Kingdom. E-mail: m.falasca{at}ucl.ac.uk.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The insulin receptor phosphorylates insulin receptor substrate (IRS) proteins on multiple tyrosine residues that act as docking sites to recruit a number of downstream signaling molecules. Here we show that IRS3 is localized both at the plasma membrane and in the nucleus. Interestingly, the nuclear localization of the protein is restricted to specific regions involved in mRNA processing and known as speckles. By using different truncated versions of the protein, we demonstrate that the pleckstrin homology (PH) domain is involved in IRS3 localization at the level of both plasma membrane and nucleus. To our knowledge this is the first report of a PH domain responsible for a nuclear targeting of the host protein. By site-directed mutagenesis, we identify residues within the PH domain critical for proper localization of IRS3. Mutations within the PH domain preventing IRS3 intracellular localization result in an inhibition of IRS3-induced glucose uptake. We conclude that the PH domain is required for IRS3 intracellular localization and, furthermore, that it has a key role in metabolic functions of IRS3. In particular, our data suggest that IRS3 intracellular localization at the plasma membrane and in the nucleus is the result of two different cooperative mechanisms both involving the PH domain.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INSULIN RECEPTOR SUBSTRATE (IRS) proteins play a crucial role in insulin signaling in many tissues as the major intracellular substrates for the insulin receptor. Thus far, at least four IRS proteins (IRS1, -2, -3, and -4) have been identified in various mammalian species sharing a similar overall structure (1). All isoforms contain a pleckstrin homology (PH) domain and a phosphotyrosine-binding (PTB) domain at the N terminus that are both required for efficient phosphorylation by the insulin receptor (2, 3). By contrast, the C-terminal regions have less sequence homology, although they all contain many conserved tyrosine phosphorylation motifs responsible for interaction of IRS proteins with downstream SH2 domain-containing signaling molecules such as phosphatidylinositol 3- kinase (PI 3-K) (4). IRS isoforms are likely to be functionally redundant since they can activate similar signaling pathways. However, the existence of some unique structural features suggests that each IRS isoform may have specific functions in different tissues.

One example is the phosphorylation domain of IRS3 that is far smaller than that of other IRS proteins (5, 6). This observation suggests that IRS3 may lack residues required for binding to specific downstream molecules, thus resulting in different effects compared with other IRS isoforms. Indeed it has been recently demonstrated that IRS1 associates with Bcl-2 via its C-terminal domain and synergistically up-regulates Bcl-2 antiapoptotic function (7). IRS3, which lacks the required binding region, abrogates the survival effects of IRS1 in a dominant-negative manner (7).

IRS3 represents the principal substrate for the insulin receptor tyrosine kinase in adipocytes from IRS1 knockout mice (8). Surprisingly, the phenotype of IRS3 null mice is quite normal with respect to both growth and glucose homeostasis (8), and the absence of IRS3 does not lead to detectable increases in tyrosine phosphorylation of IRS1–2 or in their association with PI 3-K. Nevertheless, overexpression of wild-type IRS3 in rat adipose cells causes significant translocation of glucose transporter 4 in the absence of insulin, and expression of a mutant IRS3 unable to bind PI 3-K is sufficient to block insulin-stimulated translocation of glucose transporter 4 (9). Thus, the precise role of IRS3 in insulin signaling is still unknown.

The observation that IRS3 phosphorylation domain contains several motifs not present in other isoforms further suggests that IRS3 may engage distinct downstream molecules and activate specific functional pathways yet to be elucidated. Indeed it has been reported that a green fluorescent protein (GFP) fusion protein of IRS3 is localized at the plasma membrane and in the nucleus of COS-7 cells and that the C-terminal region of IRS3 possesses transcription-regulating activity (10). In addition, it has been recently shown that the mIRS3 promoter is regulated by p53 at the transcriptional level, thus suggesting that IRS3 could participate to p53-regulated cell growth and differentiation (11).

Taken together these data lead to the conclusion that the physiological roles of IRS3 are not completely understood.

We have recently reported that the isolated PH domain from IRS3 has a different intracellular localization when compared with the PH domains from both IRS1 and IRS2 (12). In an effort to understand whether this difference may reflect a different intracellular localization of the full-length protein that, in turn, might be at least partially responsible for IRS3 peculiar functions, we analyzed the intracellular localization of wild-type IRS3 and found that IRS3 is localized at the level of both plasma membrane and in the nucleus. More interestingly, we observed that IRS3 colocalizes with component of the mRNA-processing machinery in the nucleus, indicating that its localization is restricted to nuclear speckles. Our data suggest that two different mechanisms, both involving the PH domain, are responsible for plasma membrane and nuclear localization of the protein, respectively. Mutations within the PH domain preventing IRS3 intracellular localization impair IRS3-induced glucose uptake. Thus our study defines the crucial role of the PH domain in IRS3 intracellular localization and then in metabolic functions of IRS3.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Intracellular Localization of IRS3
We have previously reported that the isolated PH domain from IRS3 is constitutively associated with the plasma membrane (Ref. 12 and Fig. 1Go) whereas PH domains from both IRS1 and IRS2 are localized exclusively in the cytoplasm (12). To study the intracellular localization of the full-length IRS3 and compare it to the PH domain localization, we transfected L6 myoblasts with cDNAs encoding the IRS3 open reading frame [amino acids (a.a.) 1–495] or the isolated PH domain (a.a. 20–107) fused to the GFP. Control cells were transfected with cDNA encoding the GFP alone.



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Fig. 1. Intracellular Localization of IRS3 PH Domain

L6 cells were transfected with a cDNA encoding GFP-PH IRS3. After 24 h, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.25% Triton X-100, and then incubated with Rhodamine Reda-X1,2-dihexadecanoyl-sns-glycero-3-phosphoethanolamine or Vybrant DiI for 10 min and analyzed by confocal microscopy. Arrows indicate the plasma membrane localization. Bar, 10 µm.

 
Confocal microscopy analysis revealed that both GFP-IRS3 (Fig. 2AGo) and GFP-PH IRS3 (Fig. 2BGo) were localized to the plasma membrane and into the nucleus. By contrast, GFP was essentially dispersed in the cytoplasm (Fig. 2CGo). Interestingly, the nuclear localization of the two proteins appeared quite different, with the GFP-IRS3 showing a punctuate staining through the whole nucleus except for the nucleoli (Fig. 2AGo). By contrast, GFP-PH IRS3 appeared more diffuse into the nucleus (Fig. 2BGo). Similar results were obtained in NIH-IR fibroblasts (data not shown).



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Fig. 2. Intracellular Localization of Full-Length IRS3 and Isolated PH Domain

A–C, L6 cells were transfected with cDNAs encoding either the full-length IRS3 (A) or the isolated PH domain from IRS3 (B) fused to the GFP or the GFP alone (C). After 24 h, cells were fixed and analyzed by confocal microscopy. Arrows indicate the plasma membrane localization. Bar, 10 µm. D, L6 cells were transfected with either GFP (lanes 1, 4, and 7) or GFP-PH IRS3 (lanes 2, 5, and 8) or GFP-IRS3 (lanes 3, 6, and 9). After 24 h, nuclear (N, lanes 1–3), membrane (M, lanes 4–6), and cytosolic (C, lanes 7–9) fractions were prepared as described in Materials and Methods. Proteins were then separated by SDS-PAGE, and Western blotting analysis was performed by using an anti-GFP antibody (CLONTECH). Arrowheads mark the position of the fusion proteins. Purity and equivalent loading of separate fractions were monitored by using anti-c-Jun, antiflotillin, and anti-GAPDH antibodies.

 
Subcellular fractionations from L6-transfected cells confirmed that GFP-IRS3 and GFP-PH IRS3 were present both in the nuclear and in the plasma membrane fractions, whereas GFP was more abundant in the cytosolic fraction (Fig. 2DGo). Purity of the different fractions was monitored by using antibodies anti c-Jun (nuclear fractions), flotillin (membrane fractions), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, cytosolic fractions).

To rule out the possibility of a mistargeting induced by GFP, L6 cells were transfected with a cDNA encoding a myc-tagged full-length IRS3, and the intracellular localization of the fusion protein was assessed by confocal microscopy. Different sections of the same myc-IRS3-expressing cell are shown in Fig. 3Go, revealing both a punctuate nuclear distribution (panels A and B) and a plasma membrane localization of the protein (panels C and D). Similar results were obtained in 3T3-L1 adipocytes microinjected with myc-IRS3 (data not shown).



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Fig. 3. Intracellular Localization of myc-Tagged IRS3

L6 cells were transfected with a cDNA encoding a myc-tagged version of the full-length IRS3. After 24 h, cells were briefly extracted with 0.2% Triton X-100 and fixed with 4% paraformaldehyde. The localization of the fusion protein was assessed by using an anti-myc mouse monoclonal antibody and Alexa 488 secondary antibody. Panels represent four thin optical sections of the same cell obtained by confocal laser scanning microscopy. Bar, 10 µm.

 
These data indicate that both the full-length IRS3 and the isolated PH domain are localized to the plasma membrane and into the nucleus and that the IRS3 nuclear localization is more concentrated in discrete punctuate structures.

IRS3 Association with Nuclear Speckles
Punctuate nuclear staining of both GFP-IRS3 and myc-IRS3 suggested a possible localization of IRS3 at the level of speckles, nuclear structures the function of which is likely to be as temporary storage sites or recycling centers for multiple factors required for mRNA biogenesis (13). To test this hypothesis, GFP-IRS3-transfected L6 cells were incubated with a monoclonal antibody (anti-Sm, Y12 antibody) that stains the small nuclear ribonucleoproteins core proteins, thereby visualizing nuclear speckles. Confocal analysis showed a significant colocalization of IRS3 with nuclear speckles (Fig. 4Go, A–C). To further confirm this result, GFP-IRS3-transfected L6 cells were treated with the RNA polymerase II inhibitor {alpha}-amanitin that has been demonstrated to cause a reorganization of nuclear speckles into fewer and larger structures (14, 15). As shown in Fig. 4Go (D–F), {alpha}-amanitin induced the same nuclear redistribution of both GFP-IRS3 and Sm, thus confirming a physical and dynamic association of IRS3 with the speckles.



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Fig. 4. GFP-IRS3 Colocalization with Components of the mRNA-Processing Machinery in Nuclear Speckles

L6 cells were transfected with GFP-IRS3 (A and D). After 24 h, cells were left untreated (A) or treated with 10 µg/ml of the transcriptional inhibitor {alpha}-amanitin for 4 h (D). After a brief preextraction with 0.2% Triton X-100, cells were fixed and incubated with an anti-Sm antibody, Y-12 antibody (B and E). Colocalization is represented by yellow in the overlays (C and F). Bar, 10 µm.

 
Requirement of PH Domain for Intracellular Localization of GFP-IRS3
It has been recently reported that the IRS3 PTB domain contains the targeting sequence for nuclear localization of the protein (10). However, as shown in Fig. 1Go and Fig. 2Go (panels B and D), we observed that the isolated PH domain fused to the GFP was also localized in the nucleus although it showed a different nuclear staining from the full-length protein. To clarify which domains are responsible for IRS3 targeting into the nucleus, L6 cells were transfected with cDNAs encoding either GFP-PH IRS3 or the N-terminal region, corresponding to both the PH and the PTB domains (GFP-PH+PTB IRS3, a.a. 20–310) fused to the GFP or the GFP fusion protein of full-length IRS3 lacking the entire PH domain (GFP-IRS3 {Delta}PH, a.a. 107–495) (a schematic diagram of the different constructs is shown in Fig. 5AGo). Confocal microscopy analysis confirmed the diffuse nuclear staining and the plasma membrane localization of GFP-PH IRS3 (Fig. 5BGo). GFP-PH+PTB IRS3 showed a strong plasma membrane localization and a diffuse nuclear staining (Fig. 5CGo). On the contrary, GFP-IRS3 {Delta}PH did not localize at the plasma membrane and was only weakly detected in the nucleus (Fig. 5DGo).



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Fig. 5. Intracellular Localization of GFP-IRS3 Deletion Mutants

L6 cells were transfected with cDNAs encoding the GFP-PH IRS3 (a.a. 20–107), or GFP-PH+PTB IRS3 (a.a. 20–310), or GFP-IRS3-{Delta}PH (a.a. 107–495). A, Schematic representation of the GFP fusion proteins compared with the full-length GFP-tagged protein. After 24 h, the intracellular localization of GFP-PH IRS3 (B), GFP-PH+PTB IRS3 (C), and GFP-IRS3 {Delta}PH (D) was assessed by confocal microscopy. Bar, 10 µm.

 
Taken together, these results suggest that the PH domain of IRS3 is necessary for IRS3 localization at the level of both nucleus and plasma membrane.

However, neither GFP-PH IRS3 nor GFP-PH+PTB IRS3 showed the nuclear punctuate pattern observed with GFP-IRS3, thus indicating that these fusion proteins do not localize at the level of the speckles. This observation indicates that PH domain is necessary for IRS3 targeting into the nucleus but not sufficient for its proper speckles localization.

Site-Directed Mutagenesis of PH Domain
To identify residues that are critical for IRS3 localization, we decided to perform site-directed mutagenesis analysis. Since IRS3 is the only member of the IRS family showing a constitutive nuclear and plasma membrane localization (10), sequences responsible for this localization are likely to reside in regions of IRS3 with no homology with IRS1 or IRS2. In addition, our data suggest that residues responsible for intracellular localization are within the PH domain. We then aligned sequences of the PH domains from IRS1, IRS2, and IRS3 and identified the region encompassing amino acids 47 and 103 in PH IRS3 as showing the lower homology (Fig. 6AGo).



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Fig. 6. Identification of Residues Critical for Phosphoinositide Binding within the PH Domain

A, Sequence alignment of PH domains from IRS1, IRS2, and IRS3. Identical amino acids are shown in bold; arrows mark the mutated residues. B, The following amount of the indicated phosphoinositides lipid was spotted in each row of nitrocellulose filters: 0.2, 0.4, 0.6, 0.8, 1.0 µg. 32P-labeled wt and mutated PH domains from IRS3, fused to the GST, were then used to probe the filters as described in Materials and Methods. The same amount of labeled proteins (0.5 µg/ml) was used for each phosphoinositide-containing nitrocellulose, and the specific activities of proteins were identical.

 
Many PH domains interact directly with cell membranes by binding to phosphoinositides with a broad range of specificity and affinity (16). We have already demonstrated that an arginine in the ß2-strand is critical for lipid binding and plasma membrane localization, at least in PH domains known to bind PI 3-K products (12). We then identified arginine at position 48 (R48) in IRS3 PH domain as potentially involved in binding to phosphoinositides and in localization of the protein. In addition, since we have reported that IRS3 PH domain binds to PtdIns-3-P (12), we aligned the IRS3 PH domain with different FYVE domains (another structural module known to bind to PtdIns-3-P) and identified residues RH99–100 as potentially involved in binding to PtdIns-3-P.

The isolated PH domains carrying the mutations R48C and RH99–100CL were then expressed as glutathione-S-transferase (GST)-fusion proteins, andtheir lipid binding properties were analyzed by a lipid dot-blot assay (Fig. 6BGo). We observed that GST-IRS3 PH wild type (wt) bound PtdIns-3-P with high affinity, as previously described (12), whereas the mutant GST-IRS3-PH R48C bound PtdIns-3-P with much less affinity. On the contrary, mutation of residues RH99–100 did not affect the binding to PtdIns-3-P.

These results indicate that residue R48 is necessary for binding of PH domain to phosphoinositides while residues RH99–100 are not involved in phosphoinositides binding.

Intracellular Localization of IRS3 Mutants
To assess whether mutations within the PH domain result in a mistargeting of the IRS3, full-length proteins carrying either the mutations R48C or RH99–100CL were then expressed as myc-tagged fusion proteins in L6 cells. As shown in Fig. 7Go, myc-IRS3 showed both the typical punctuate nuclear staining and plasma membrane localization (Fig. 7AGo). On the contrary, the mutant myc-IRS3 R48C was not present on the plasma membrane but still localized in the nucleus (Fig. 7BGo). More interestingly, this mutant is dispersed in the nucleus, and it did not show punctuate nuclear staining. Finally, myc-IRS3 RH99–100CL showed a thin plasma membrane localization and was only partially localized in the nucleus (Fig. 7CGo). Subcellular fractionations of L6-transfected cells confirmed that myc-IRS3 and myc-IRS3 R48C were enriched in the nuclear fractions whereas myc-IRS3 RH99–100CL was barely detected in this fraction. Only myc-IRS3 was abundant in the membrane fraction (Fig. 7DGo) whereas myc-IRS3 RH99–100CL was weakly detectable in this fraction.



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Fig. 7. Intracellular Localization of IRS3 Mutants

A–C, L6 cells were transfected with cDNAs encoding either myc-IRS3 (A) or myc-IRS3 R48C (B) or myc-IRS3 RH99–100CL (C). After 24 h, cells were fixed, permeabilized, and incubated with an anti-myc antibody before confocal microscopy analysis. Bar, 10 µm. D, Cytosolic (C), nuclear (N), and membrane (M) fractions were prepared from L6 cells expressing myc-IRS3 or myc-IRS3 R48C or myc-IRS3 RH99–100CL. After SDS-PAGE, proteins were transferred into polyvinylidine difluoride and the fusion proteins were revealed by using an anti-myc antibody. Equal amounts of the different fractions were loaded on a separate gel and monitored by using anti-c-Jun, antiflotillin, and anti-GAPDH antibodies (lanes 1: fractions from myc-IRS3-expressing cells, lanes 2: fractions from myc-IRS3 R48C-expressing cells, lanes 3: fractions from myc-IRS3 RH99–100CL-expressing cells).

 
Similar results were obtained by using the corresponding GFP fusion proteins both in confocal microscopy and subcellular fractionation analyses (data not shown).

Taken together, these results demonstrate that, despite their different lipid binding properties, all three residues are necessary for IRS3 plasma membrane localization.

In addition, these results indicate that residues RH99–100 are important for IRS3 nuclear targeting whereas residue R48 is necessary for nuclear localization at the level of the speckles. Nevertheless, the observation that isolated wt PH domain is diffused in the nucleus and does not show punctuate staining suggests that R48 is necessary, but not sufficient, for localization in the speckles.

Effect of Overexpression of IRS3 wt and Mutants on Glucose Uptake
To check the hypothesis that the PH domain-mediated localization of IRS3 is crucial for its cellular functions, we tested the effect of PH domain mutations on IRS3-mediated glucose uptake. L6 cells were transfected with either GFP-IRS3 or GFP-IRS3 R48C or GFP-IRS3 RH99–100CL or GFP alone, as a control, and glucose uptake was assessed in serum-deprived cells. As shown in Fig. 8Go, overexpression of IRS3 wt induced a 2- to 2.5-fold increase in glucose uptake compared with GFP-transfected control cells. By contrast, overexpression of the mutant IRS3 R48C had no effect on basal uptake, whereas overexpression of IRS3 RH99–100CL partially increased glucose uptake (161 ± 17% control).



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Fig. 8. Effect of Overexpression of wt and Mutated IRS3 on Glucose Uptake

L6 cells were transfected with cDNAs encoding the GFP alone, GFP-IRS3, GFP-IRS3 R48C, or GFP-IRS3 RH99–100CL, respectively. 2-Deoxy-(3H)-D-glucose uptake was determined as described in Materials and Methods. Data were obtained from three independent experiments performed in duplicate. In the same conditions insulin induced a 1.5- to 2-fold increase in glucose uptake. *, P < 0.01.

 
These results indicate that PH domain-mediated localization of IRS3 is crucial for cellular functions of the protein.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Among the different members of IRS family, IRS3 is quite peculiar with respect to its tissue and cell distribution (5, 6, 17). Expression of IRS3 is developmentally regulated such that it is high in the first part of embryonic life (when IRS1 mRNA is barely detected) and is markedly reduced during the remainder of embryonic as well as adult life (6). Although detectable levels of IRS3 mRNA have been observed in adipose tissue, liver, heart, kidney, and lung, the only tissue in which the expression of IRS3 protein has been definitively demonstrated is white adipose tissue (6). Furthermore, the precise role of IRS3 in insulin signaling has not yet been precisely assessed whereas several lines of evidence have recently suggested novel intracellular functions for this isoform (10, 11).

We have previously reported that the PH domains from IRS1 and IRS2 are localized exclusively in the cytoplasm and translocate to the plasma membrane specifically upon insulin stimulation in a PI 3-K-dependent manner. In contrast, IRS3 PH domain is constitutively associated to the plasma membrane (12), thus suggesting a different mechanism of intracellular localization and, furthermore, different roles for this IRS isoform. In this study, we analyze the intracellular localization of the full-length IRS3 and observe that this protein is localized both at the plasma membrane and in the nucleus. This is in agreement with the recent observation that GFP-IRS3 localizes both at the plasma membrane and in the nucleus of transfected-COS7 cells (10). In addition, we demonstrate that the nuclear localization of the protein is restricted to specific nuclear sites known as nuclear speckles. By using different mutants of IRS3 protein, we show that the PH domain is necessary for both plasma membrane and nuclear localization, i.e. the isolated domain itself is able to localize to these regions whereas the full length protein lacking the entire PH domain is not localized at the plasma membrane and only weakly detected in the nucleus. It has been well established that the PH domain may target the host protein to membranes but, to our knowledge, this is the first report of a PH domain localizing in the nucleus and being responsible for a nuclear targeting. Interestingly, the nuclear localization of an isolated PH domain is quite different from that of full-length protein, being more dispersed in the nucleus and lacking the speckles localization. This observation suggests that the PH domain is necessary for IRS3 to enter the nucleus but not sufficient to localize the protein at the level of the speckles and that probably the C-terminal region of the protein is also required for the speckles localization. It has been proposed that speckles function as temporary storage sites or recycling centers for multiple factors required for mRNA biogenesis (13). Our data might then be in agreement with the observation that the IRS3 C-terminal region possesses transcriptional activity (10).

By site-directed mutagenesis we have identified residues within the PH domain that are critical for localization.

The full-length IRS3 carrying the mutation RH99–100CL within the PH domain is only weakly detected both at the plasma membrane and in the nucleus, suggesting that these residues are involved in plasma membrane localization and are responsible for IRS3 targeting into the nucleus. Mutation of these residues does not alter the lipid binding properties of IRS3 PH domain, indicating that the mistargeting of IRS3 RH99–100CL is not due to an impaired phosphoinositides binding, Taken together, these observations suggest that residues RH99–100 are involved in IRS3 localization by interacting with different, yet unidentified, factors.

Full-length IRS3 carrying a mutation in residue R48 is not localized at the plasma membrane, thus indicating that not only residues RH99–100, but also residue R48, are required for plasma membrane localization of the protein.

In addition, IRS3 R48C is localized in the nucleus, thus confirming the crucial role of residue RH99–100 for IRS3 to enter the nucleus. However, although present in the nucleus, IRS3 R48C does not show the peculiar punctuate staining indicating that residue R48 is necessary for nuclear localization at the level of the speckles. It is noteworthy that this residue is critical for PH domain binding to PtdIns-3-P. Because it is well established that both plasma membrane and speckles are cellular regions enriched in phosphoinositides (14, 18), this observation suggests that correct binding to phosphoinositides is necessary for R48-mediated IRS3 proper localization. However, we have also observed that isolated wt PH domain, although present in nucleus and containing the critical residue R48, does not show the speckles localization. These observations indicate that R48-mediated binding of PH domain to phosphoinositides is necessary, but not sufficient, for proper localization at the level of the speckles.

Taken together, these results suggest the existence of two different cooperative mechanisms, both involving the PH domain and responsible for proper IRS3 intracellular localization. Plasma membrane localization of the protein is likely to be the result of a cooperative mechanism of PH domain binding to phosphoinositides via residue R48 and to other still unidentified factors via residues RH99–100CL.

On the contrary, a cooperative mechanism involving binding to phosphoinositides of residue R48 in the PH domain and interaction of the C-terminal region with yet unknown factors is likely to be required for proper nuclear localization at the level of the speckles. Residues RH99–100CL are required for targeting into the nucleus.

The majority of PH domains bind phosphoinositides with a low specificity and/or a low affinity, thus raising the question of how specificity in membrane targeting can be achieved. We have recently proposed that a weak PH domain-phosphoinositide interaction might be stabilized either by a cooperative binding with a protein within the same PH domain or by a simultaneous action of PH domain with other domains of the protein to achieve a stable recruitment of the host protein to the plasma membrane (16). Both mechanisms have been proposed alternatively for different PH domains. This study suggests that both cooperative mechanisms might be responsible for IRS3 localization. Furthermore, they suggest that the same PH domain may be able to mediate two specific intracellular localizations through two different mechanisms. Future investigation will focus on identifying the molecules involved in cooperation with PH domain for IRS3 proper localization.

The observation that overexpression of mutated versions of the protein, improperly localized, inhibited the IRS3-mediated glucose uptake further underlines the crucial role of the PH domain in localization of IRS3 that, in turn, leads to correct intracellular functions of the protein.

Taken together, these data indicate that IRS3 has a very specific PH domain-mediated intracellular localization and regulation when compared with other insulin receptor substrates that probably may account for the different intracellular roles of this isoform.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
DMEM, fetal bovine serum, and calf serum were obtained from Invitrogen, Life Technologies, Inc. (San Diego, CA). Fluorescent dye-conjugated secondary antibodies, Rhodamine Reda-X1,2-dihexadecanoyl-sns-glycero-3-phosphoethanolamine, and Vybrant DiI were from Molecular Probes (Eugene, OR). Antibodies were from CLONTECH (anti-GFP), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (anti-c-Jun and anti-GAPDH), and Transduction Laboratories, Inc. (Lexington, KY) (antiflotillin-1). All other biochemicals were of the highest possible purities and were obtained from Sigma (St. Louis, MO). Y-12 antibody was a kind gift of Dr. G. W. Zieve (SUNY Stony Brook, New York, NY).

Plasmid Construction
For the construction of the GFP-IRS3 fusion plasmid, the entire coding sequence of the murine IRS3 gene was amplified by PCR with specific primers. The BamHI/EcoRI DNA fragment was subcloned into the BglII/EcoRI sites of the pEGFP-C1 expression vector (CLONTECH). This construct was then used to generate the following deletion mutants: 1)GFP-PH IRS3, containing only the PH domain (a.a. 20–107); 2) GFP-PH+PTB IRS3, containing both the PH and the PTB domains (a.a. 20–310); 3) GFP-IRS3 {Delta}PH, lacking the PH domain (a.a. 107–495). All these PCR products were cloned (in frame with GFP) into the BglII/EcoRI restriction site of the pEGFP-C1. The QuikChange Mutagenesis System (Stratagene, La Jolla, CA) was used to generate the myc-and GFP-tagged mutant forms of IRS3 using the mIRS3 gene (9), as a template. DNA sequence of the constructs was confirmed by nucleotide sequencing.

To express the GST fusion proteins of wt and mutated IRS3 PH domain, the PCR products corresponding to the PH domains of IRS3 wt, IRS3 R48C, and IRS3 RH99–100CL were digested with BamHI plus EcoRI and were ligated into the appropriately digested pGEX-2TK (Amersham, Arlington Heights, IL). The DNA sequence of each PH domain insert was verified by dideoxynucleotide sequencing. The GST fusion proteins were then expressed and purified as previously described (19).

Cell Culture and Transfection
L6 myoblasts and NIH-IR fibroblasts were maintained in DMEM supplemented with 10% fetal calf serum and 10% calf serum, respectively. For transfection, cells were seeded onto 24-mm circular glass coverslips in wells of a six-well plate and transfected with 1 µg of GFP fusion protein. LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) was used according to the manufacturer’s suggestions. For inhibition of transcription, L6 cells were treated with 10 µg/ml {alpha}-amanitin for 4 h.

Immunofluorescence and Microscopy
For immunofluorescence studies, cells were rinsed in PBS 24 h after transfection and then fixed for 15 min in PBS containing 4% paraformaldehyde at room temperature. Where indicated, cells were preextracted with 0.2% Triton X-100 in PBS for 3 min on ice. For incubation with antibodies, cells were permeabilized with a BSA solution in PBS (50 mM NH4Cl, 0.5% BSA, 0.05% saponin) for 30 min at room temperature and then incubated with primary antibodies in 0.5% BSA solution for 1 h at room temperature (5 µg/ml Y-12 antibody; anti-myc, 1:400). After washing five times with PBS, coverslips were incubated with a secondary antimouse IgG antibody (1:1000 dilution) conjugated with rhodamine red or Alexa 488 (Molecular Probes, Eugene, OR). Microscopy was performed using a Zeiss laser confocal microscope system (LSM 510) connected to an Axiovert 100 M (Zeiss) and a Zeiss x63 objective (Carl Zeiss, Inc., Thornwood, NY).

Subcellular Fractionations and Western Blot Analysis
Subcellular fractionations were performed as described (10). Fifteen micrograms of each fraction were separated on sodium dodecyl sulfate/polyacrylamide gel and then transferred to a polyvinylidine difluoride membrane. Filters were blocked by incubation with 5% low-fat dry milk in PBST (PBS containing 1% Tween 20) for 1 h and then incubated with the primary antibody in PBST overnight at 4 C or for 2 h at room temperature. Filters were extensively washed in PBST and then incubated with horseradish peroxidase-conjugated secondary antirabbit or antimouse antibody (Amersham), and proteins were visualized by enhanced chemiluminescence (Amersham).

Dot-Blot Assay
Phosphoinositides (Matreya, Inc., Pleasant Gap, PA; and Echelon, Salt Lake City, UT) in 1:1 chloroform-methanol were spotted onto nitrocellulose filters. 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 GST-PH domain fusion proteins were labeled with 32P as previously described (19) and then added to TBS/1% BSA to a final concentration of 0.5 µg/ml. 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.

Glucose Uptake
L6-transfected cells were serum deprived overnight, in serum-free media, and then washed twice with Krebs-Henseleit bicarbonate buffer [116 mM NaCl, 4.6 mM KCl, 1.2 mM KH2PO4, 25.3 mM NaHCO3, 2.5 mM CaCl2, 1.16 mM MgSO4, (pH 7.2) containing 0.1% BSA] and incubated with 10 µM 2-deoxy-D-glucose mixed with 2-deoxy-(3H)-D-glucose (Perkin-Elmer Corp., Norwalk, CT; 0.25 µCi per well), for 10 min. Uptake was terminated by washing three times with ice-cold PBS, and cells were solubilized with 1 M NaOH for 20 min. Radioactivity was then quantitated by liquid scintillation counting. Nonspecific uptake was determined in the presence of 10 µM cytochalasin B and was subtracted by total uptake. As a control, nontransfected cells were left untreated or stimulated with 300 nM insulin for 20 min before incubation with 2-deoxy-D-glucose.


    ACKNOWLEDGMENTS
 
We thank Dr. G. A. Rodrigues for advice and helpful suggestions, and Dr. G. W. Zieve for the gift of the Y-12 antibody.


    FOOTNOTES
 
This work was supported by Diabetes UK Grant BDA:RD02/0002388. M.F. is supported by an endowment from the Dr. Mortimer and Mrs. Theresa Sackler Trust.

T.M. and G.R. contributed equally to this work.

Present address for G.R.: Department of Biomedical Sciences, Università degli Studi di Modena e Reggio Emilia, Via Campi 287, 41100 Modena, Italy.

Abbreviations: a.a., Amino acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione-S-transferase; IRS, insulin receptor substrate; PBST, PBS containing 1% Tween 20; PH domain, pleckstrin homology domain; PI 3-K, phosphatidylinositol 3-kinase; PTB domain, phosphotyrosine-binding domain; PtdIns-3-P, phosphatidylinositol 3-phosphate; TBS, Tris-buffered saline; wt, wild-type.

Received for publication August 28, 2001. Accepted for publication April 22, 2003.


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