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Analysis of insulin receptor substrate signaling dynamics on microstructured surfaces € glinger1, € ller1, Otmar Ho Peter Lanzerstorfer1,*, Yosuke Yoneyama2,*, Fumihiko Hakuno2, Ulrike Mu 2 1 Shin-Ichiro Takahashi and Julian Weghuber 1 School of Engineering and Environmental Sciences, University of Applied Sciences Upper Austria, Wels, Austria 2 Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Japan

Keywords fluorescence recovery after photobleaching; IGF-I receptor; insulin receptor; insulin receptor substrates; micropatterning Correspondence J. Weghuber, University of Applied Sciences Upper Austria, Stelzhamerstrasse 23, A-4600 Wels, Austria Fax: +43 (0)50804 44403 Tel: +43 (0)50804 44403 E-mail: [email protected] *These authors contributed equally to this work. (Received 14 August 2014, revised 20 January 2015, accepted 21 January 2015) doi:10.1111/febs.13213

Insulin receptor substrates (IRS) are phosphorylated by activated insulin/ insulin-like growth factor I receptor tyrosine kinases, with this comprising an initial key event for downstream signaling and bioactivities. Despite the structural similarities, increasing evidence shows that IRS family proteins have nonredundant functions. Although the specificity of insulin/insulin-like growth factor signaling and biological responses partly reflects which IRS proteins are dominantly phosphorylated by the receptors, the precise properties of the respective IRS interaction with the receptors remain elusive. In the present study, we utilized a technique that combines micropatterned surfaces and total internal reflection fluorescence microscopy for the quantitative analysis of the interaction between IRS proteins and insulin/insulin-like growth factor in living cells. Our experimental set-up enabled the measurement of equilibrium associations and interaction dynamics of these molecules with high specificity. We revealed that several domains of IRS including pleckstrin homology and phosphotyrosine binding domains critically determine the turnover rate of the receptors. Furthermore, we found significant differences among IRS proteins in the strength and kinetic stability of the interaction with the receptors, suggesting that these interaction properties could account for the diverse functions of IRS. In addition, our analyses using fluorescent recovery after photobleaching revealed that kinases such as c-Jun N-terminal kinase and IjB kinase b, which phosphorylate serine/threonine residues of IRS and contribute to insulin resistance, altered the interaction kinetics of IRS with insulin receptor. Collectively, our experimental setup is a valuable system for quantitifying the physiological interaction of IRS with the receptors in insulin/insulin-like growth factor signaling.

Introduction It is well established that insulin receptor substrates (IRS) are important mediators of insulin and insulinlike growth factor (IGF) signaling. Insulin and IGFs bind to their cognate receptors, insulin receptor (IR)

and IGF-I receptor (IGF-IR), leading to the activation of tyrosine kinases in the b subunits. Activated receptors then phosphorylate IRS proteins, which initiates the recruitment of Src homology 2 domain-containing

Abbreviations EGFR, epidermal growth factor receptor; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; IGF, insulin-like growth factor; IGF-IR, insulin-like growth factor-I receptor; IKK, IjB kinase; IR, insulin receptor; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; KRLB, kinase regulatory-loop binding; mRFP, monomeric red fluorescent protein; PH, pleckstrin homology; PTB, phosphotyrosine binding; TIRF, total internal reflection fluorescence; TNFa, tumor necrosis factor a.

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molecules such as the p85 phosphoinositide 3-kinase regulatory subunit and Grb2 to phosphorylated IRS and results in the activation of two main signaling pathways: the phosphoinositide 3-kinase/Akt pathway and the Ras-mitogen-activated protein kinase pathway [1,2]. Six members of IRS family proteins (IRS-1-6) have been identified to date. The IRS proteins share two highly homologous N-terminal regions: the pleckstrin homology (PH) domain and the phosphotyrosine binding (PTB) domain, respectively. The PTB domain binds to phosphorylated NPxY motif in the juxtamembrane region of IR/IGF-IR [3–8]. The PH domain is required for tyrosine phosphorylation of IRS [9,10], although whether this occurs via its phosphoinositide binding or protein–protein interaction has not been clearly defined [11–13]. Furthermore, in addition to the PTB domain, IRS-2 uniquely possesses a second IRassociated region termed the kinase regulatory-loop binding (KRLB) region [5,14–16]. The C-terminus of the IRS proteins contains multiple tyrosine-phosphorylation sites that bind to Src homology 2 domaincontaining proteins in an IR/IGF-IR-mediated phosphorylation-dependent manner. IRS is also regulated through phosphorylation of its serine/threonine residues by various kinases such as c-Jun N-terminal kinase (JNK) and IjB kinase b (IKKb), which are known to negatively affect IRS signaling, leading to the insulin resistance [17]. Despite the high structural homology, recent studies have indicated that IRS proteins show distinct roles in insulin/IGF signaling and bioactivities. Importantly, knockout mice studies confirmed that IRS-1 and IRS2 complementarily serve as the major substrates participating in insulin/IGF actions [18–20]. IRS-1 is shown to mainly regulate the phosphoinositide 3-kinase/Akt pathway and glucose uptake in skeletal muscle and adipose tissues, as well as growth, whereas IRS-2 is implicated in the regulation of the Ras-mitogen-activated protein kinase pathway and glucose metabolism in liver [21–24]. In addition, IRS-1 and IRS-2 have different contributions to hepatic gene expression related to glucose metabolism [25]. These two IRS proteins are also suggested to play distinct roles in cancer pathologies because IRS-2 specifically promotes aggressive tumor behavior, and both contribute to tumor initiation and primary tumor growth [26]. IRS3, for which expression is limited to brain and adipose tissue, is shown to function in insulin-dependent glucose uptake in adipocytes [27] and also to act negatively on IRS-1 and IRS-2 functions [28,29]. Molecular mechanisms that underlie these functional differences of IRS proteins have been proposed in several studies. For example, IRS-1 and IRS-2 have been shown to 988

differ in their abilities to interact with signaling proteins and other proteins that can modulate IRS functions [1,30]. In addition, IRS proteins were reported to differ in their subcellular localization that allows compartment-specific functions [31–33]. These distinct roles of IRS proteins indicate that the specificity of insulin/IGF signaling and biological responses in part reflects the availability of IRS proteins to IR/IGF-IR. Many of the interactions between IR/IGF-IR and IRS have been inferred from the studies using yeast two-hybrid assay [5,6,34]. However, this technique is not well suited for investigating the protein–protein interaction quantitatively because detection is established in the yeast nucleus and not in the mammalian cell environment and has a high frequency of false-positives. On the other hand, their interaction has been analyzed by in vitro pull-down or co-immunoprecipitation assays [14,35]. However, these data also do not reflect interaction in the live cell context. In the present study, we studied the interaction properties of IRS proteins with IR/IGF-IR by the use of l-patterned surfaces in combination with total internal reflection fluorescence (TIRF) microscopy. Live cell l-patterning has been shown to be a suitable tool for detection and quantitation of protein-protein events in cell membranes [36–41]. It is a system used to quantify interactions between a fluorophore-labeled protein (‘prey’) and a membrane protein (‘bait’) in living cells. Cells are plated on l-patterned surfaces functionalized with antibodies to the bait exoplasmic domain. Bait– prey interactions are assayed via the redistribution of the fluorescent prey in cell membranes using TIRF microscopy. Using the l-patterning assay, we enriched endogenous bait IR/IGF-IR in HeLa cells into microdomains and monitored the co-recruitment of fluorescent prey IRS proteins. We were able to confirm and quantitate the recruitment of different IRS proteins to the IR/IGF-IR and could unravel the role of conserved IRS protein domains in receptor binding. Furthermore, using photobleaching experiments, significant differences in the kinetic recruitment of IRS proteins and altered kinetic behavior by JNK and IKKb serine/threonine kinases were discovered.

Results Set-up for analysis of interaction properties between IR/IGF-IR and IRS on l-patterned surfaces To investigate the interaction properties of IR/IGF-IR with intracellular IRS proteins in a living cell context, we first attached capture antibodies targeting extracellular a FEBS Journal 282 (2015) 987–1005 ª 2015 FEBS

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subunits of the receptors (bait proteins) in characteristic l-patterns on the surface of functionalized glass coverslips (Fig. 1A,B). This should lead to the redistribution of IR/IGF-IR and the co-recruitment of IR/ IGF-IR interacting proteins such as IRS (prey proteins) within the same l-patterned regions on the plasma membrane. A similar experimental set-up has been used to analyze the interaction of the epidermal growth factor receptor (EGFR) with Grb2 [42]. In the present study, we used the HeLa cell line, which expresses sufficient levels of endogenous IR and IGFIR. In addition, live cell l-patterning experiments require a flat interface of the cell membrane with the l-biochip to exclude false-positive signals in TIRF illumination. HeLa cells were shown to fulfill those requirements as confirmed by staining with the lipophilic tracer DiD (Fig. 1C). IGF-I-induced tyrosine phosphorylation of green fluorescent protein (GFP)-fused IRS proteins Next, we constructed the plasmids expressing IRS-1, IRS-2, or IRS-3 tagged with enhanced GFP at the Nor C-terminus to examine whether the fluorescently

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labeled IRS proteins could interact with IR/IGF-IR. To this end, HeLa cells were transiently transfected with the plasmids expressing IRS-1 fused with N-terminal GFP (GFP-IRS-1) or the one fused with C-terminal GFP (IRS-1-GFP) and IGF-I-dependent tyrosine phosphorylation of these proteins was examined by immunoprecipitation and immunoblotting. As shown in Fig. 2C, tyrosine phosphorylation of GFPIRS-1, but not IRS-1-GFP, was significantly increased in response to IGF-I addition. These results indicated that C-terminal GFP fusion of IRS proteins interfered the interaction with IR/IGF-IR and the tyrosine phosphorylation of IRS. We therefore used the constructs expressing N-terminal GFP fused IRS-1/-2/-3 in the present study (Fig. 2A). In HeLa cells, endogenous expression of IRS-1 and IRS-2 was detected by immunoblotting, especially a remarkably high expression of IRS-2 (Fig. 2B). By contrast, endogenous IRS-3 was not detected, in agreement with the functional homologue of IRS-3 not having been found in the human genome [43]. Ectopic expression of GFP-IRS-1/-2/-3 did not affect the expression levels of endogenous IR and IGF-IR (Fig. 2B). Similar to GFP-IRS-1, tyrosine phosphorylation of GFP-IRS-2 and GFP-IRS-3 was

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Fig. 1. Principle of the applied l-patterning assay. (A) Schematic illustration of IR/IGF-IR-IRS interaction in living cells grown on a functionalized l-biochip. Cells attach within 3–4 h after seeding on the l-structured glass surface and endogenous IR/IGR-IR is captured by anti-IR/IGF-IR antibodies. Specific interaction of fluorescently labeled IRS proteins with the IR/IGF-IR is characterized by enhanced co-patterning in anti-IR/IGF-IR antibody-enriched regions. Antibody-free regions were blocked with a BSA-Cy5 grid. (B) Hela cells transiently expressing GFP-IRS-3 were grown on an anti-IR antibody-coated l-biochip. Co-localization of the fluorescently labeled IRS-3 in anti-IR antibody positive regions indicates specific protein–protein interactions. (C) HeLa cells transiently expressing GFP-IRS-3 were grown on an anti-IR antibody-coated l-biochip for 4 h. Sufficient cell attachment to the micropatterned glass surface was confirmed by cell membrane staining using the lipophilic tracer DiD. Scale bars = 10 lm.

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significantly increased in response to IGF-I (Fig. 2D, E,F), suggesting that these IRS constructs are available for analysis of the interaction with IR/IGF-IR. Specificity of the interaction between IR/IGF-IR and IRS on l-patterned surfaces Using the experimental set-up described above, we analyzed the redistribution of IRS-1 on anti-IR/IGFIR antibody-coated l-biochips. HeLa cells were transiently transfected with IRS-1 fused with GFP, and the fluorescent distribution was observed by TIRF microscopy. As shown in Fig. 3A, GFP-IRS-1, but not IRS-1-GFP, which was not phosphorylated, accumulated in the anti-IR/IGF-IR antibody-positive regions. We next examined whether the distribution of GFP-IRS-1 on the l-patterned surface is dependent on 990

Fig. 2. IGF-I-induced tyrosine phosphorylation of GFP-fused IRS proteins. (A) Diagrams of the GFP-fused IRS-1/-2/-3 constructs used. Two white boxes located at the N-terminus represent the PH and PTB domains. (B) The lysates of HeLa cells expressing GFP-IRS-1/-2/-3 or GFP only were analyzed by immunoblotting with the indicated antibodies. (C–E) HeLa cells expressing GFP-IRS-1 or IRS-1-GFP (C), GFP-IRS-2 (D), or GFP-IRS-3 (E) were treated with or without IGF-I (100 ng
mL 1) for 5 min. Cell lysates were subjected to immunoprecipitation with anti-IRS-1/-2/-3 antibodies followed by immunoblotting with the indicated antibodies. (F) Protein amounts of phosphorylated IRS proteins were quantified, and phospho-Tyr IRS/total IRS was calculated from at least three independent experiments. Data are the mean  SE. *P < 0.05.

the activation of IR/IGF-IR. Unfortunately, our experimental set-up does not allow for serum depletion because this leads to weak cell attachment [42]. Instead, we performed several control experiments. We constructed IRS-1 mutants lacking PH or PTB domains (DPH or DPTB), both of which are required for the tyrosine phosphorylation by activated IR/IGFIR (Fig. 3B). Biochemical analyses showed that the deletion of PH or PTB domains diminished IGF-Idependent tyrosine phosphorylation of IRS-1 (Fig. 3C). Consistent with this, on l-patterned surfaces coated with anti-IR or anti-IGF-IR antibody, both GFP-IRS-1 DPH and DPTB mutants showed reduced patterning with low contrast (Fig. 3D). For quantitation of the co-enrichment, we used an algorithm that allows for automatic analysis of l-patterns by calculating the fluorescence contrast , as described previously [44]. FEBS Journal 282 (2015) 987–1005 ª 2015 FEBS

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Fig. 3. l-patterning of different IRS-1 mutants. (A) HeLa cells were transiently transfected with GFP-IRS-1 or IRS-1-GFP and grown on antiIR antibody-coated l-biochips. Representative images on a l-biochip are shown. (B) Diagrams of the GFP-IRS-1 mutant constructs used. Three tyrosine residues (Tyr 608, 628, and 658) critical for IRS-1 binding to AP-1 complex are also depicted in full-length IRS-1 structure. (C) HeLa cells expressing GFP-IRS-1 mutants were treated with or without IGF-I (100 ng
mL 1) for 5 min. Cell lysates were subjected to immunoprecipitation with anti-IRS-1 antibody followed by immunoblotting with the indicated antibodies. (D) Representative images of the cells expressing indicated GFP-IRS-1 mutants in a l-biochip are shown. (E) Contrast quantification of fluorescent images. Error bars are based on the SE of 40 analyzed cells from three individual experiments for each IRS-1 mutant. *P < 0.05. (F) Representative images of HeLa cells expressing GFP-IRS-1 grown on biotinylated-IGF-I and insulin-coated l-biochips. Scale bars = 10 lm.

Analysis of more than 200 cells resulted in a mean contrast of 0.32  0.02 (IR) and 0.28  0.04 (IGF-IR) for full-length GFP-IRS-1 (Fig. 3E). The calculated of ~ 0.3 for GFP-IRS-1 is a reasonable and prominent value compared to other interaction pairs analyzed by the l-patterning technique [41,42,45]. By contrast, DPH and DPTB mutants resulted in significant lower contrast values: 0.13  0.01 (IR) and 0.12  0.01 (IGF-IR), and 0.13  0.01 (IR) and 0.13  0.01 (IGF-IR), respectively (Fig. 3E). In addition, when biotinylated IGF-I and insulin were bound on the l-patterned surface instead of an anti-IR/IGF-IR antibody, a similar FEBS Journal 282 (2015) 987–1005 ª 2015 FEBS

co-recruitment of GFP-IRS-1 was observed (Fig. 3F). These data suggest that the anti-IR/IGF-IR antibody used in the present study induced activation of the receptor on the l-patterned surface, leading to the co-recruitment of GFP-IRS-1. Next, we investigated whether co-recruitment of IRS-1 observed on the l-patterned surface reflected its subcellular localization. We examined the distribution of IRS-1 mutant lacking the region interacting with the AP-1 clathrin adaptor complex, which is required for the IRS-1 targeting to intracellular signaling membrane compartments [33]. IRS-1 DAPBR (AP-1 binding region) lacks the region containing critical tyrosine

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residues (Tyr608, 628 and 658) for AP-1 binding. Similar to the DPH and DPTB mutants, IGF-I-dependent tyrosine phosphorylation was significantly decreased in this mutant (Fig. 3C). In addition, the co-recruitment of GFP-IRS-1 DAPBR on the l-patterned surface was found to be significantly weaker, with a contrast of 0.17  0.01 (IR) and 0.17  0.02 (IGF-IR) (Fig. 3D,E), again supporting the practicability of this l-patterned technique to evaluate the interaction between IR/IGF-IR and IRS in living cells. Different interaction properties of IRS proteins with IR/IGF-IR on l-patterned surfaces We then analyzed the redistribution of IRS-1, -2 and -3 on anti-IR or anti-IGF-IR antibody-coated l-biochips, respectively. We could detect remarkable differences in the co-recruitment of these IRS proteins. As shown in Fig. 4A,B, GFP-IRS-3 showed significantly higher contrast of the co-recruitment compared to GFP-IRS-1 and IRS-2. Quantitative analysis resulted in a mean contrast of 0.32  0.02 (IR) and 0.28  0.04 (IGF-IR) for IRS-1, 0.33  0.02 (IR) and 0.25  0.03 (IGF-IR) for IRS-2 and 0.59  0.06 (IR) and 0.53  0.05 (IGF-IR) for IRS-3 (Fig. 4B). Interestingly, the differences in the interaction of the respective IRS proteins with both receptors were only minimal, suggesting similar binding characteristics of IRS proteins to IR and IGF-IR. Role of IRS-2 KRLB region in its interaction property Among IRS proteins, IRS-2 is shown to possess a second (in addition to the PTB domain) IR-interacting region called KRLB region. To investigate the role of this region in IRS-2 interaction properties, we constructed an IRS-2 mutant lacking the region corresponding to amino acid residues 546–753, which

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contains the KRLB region (amino acid residues 591– 741 in human IRS-2) (Fig. 5A). Full-length and DKRLB mutant of GFP-IRS-2 were transiently transfected into HeLa cells, and these cells were stimulated by insulin or IGF-I, followed by immunoprecipitation with anti-IRS-2 antibody. As shown in Fig. 5B,C, in the cells expressing full-length GFP-IRS-2, insulin/ IGF-I-induced tyrosine phosphorylation of both the endogenous (lower band) and exogenous (GFP-tagged) IRS-2 (upper band) was observed, whereas, in the cells expressing GFP-IRS-2 DKRLB, only endogenous IRS2 was detected, suggesting that deletion of KRLB in IRS-2 resulted in the decrease in ligand-dependent tyrosine phosphorylation. Furthermore, GFP-IRS-2 DKRLB also showed significantly lower contrast of the co-recruitment in the IR/IGF-IR captured l-patterned surface with a contrast of 0.18  0.02 (IR) and 0.18  0.01 (IGF-IR) (Fig. 5D,E). These results indicated that KRLB functions in the interaction of IRS-2 with IR/IGF-IR in living cells. Diverse interaction kinetics of IRS proteins As shown in previous studies, the l-patterning assay is a superior tool for studying interaction kinetics in a live cell context [37,42]. Using the fluorescence recovery after photobleaching (FRAP) technique, we bleached single patterns on the l-biochip and determined the recovery of the IRS fluorescence (Fig. 6A). Figure 6B shows the respective fluorescence recovery curves for the indicated IRS protein determined using cells grown on anti-IR- or IGF-IR-antibody-coated lbiochips. Calculated kinetic FRAP parameters are shown in Table 1. Because the expression rate is known to influence the recovery rates of bleached molecules, we quantitated the expression levels of the analyzed IRS proteins. As shown in Fig. 6C, the fluorescence intensities of IRS-1, -2 and -3 were found to be in an identical range. Similar to the fluorescence

B Fig. 4. l-patterning of different full-length IRS proteins. (A) HeLa cells were transfected with the indicated GFP-fused IRS proteins and grown on anti-IR or IGFIR antibody-coated l-biochips. Representative images are shown. Scale bar = 10 lm. (B) Contrast quantification of fluorescent images. Error bars are based on the SE of 40 analyzed cells from three individual experiments for each IRS protein. *P < 0.05.

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B Fig. 5. Role of KRLB region on IRS-2 interaction behavior. (A) Diagrams of GFPIRS-2 full-length and DKRLB constructs are shown. KRLB region corresponding to amino acids 591–741 (human IRS-2) are highlighted in red on the full-length structure of IRS-2. (B,C) HeLa cells expressing GFP-IRS-2 full-length or DKRLB were treated with or without insulin (100 nM) (B) or IGF-I (100 ng
mL 1) (C) for 5 min. Cell lysates were subjected to immunoprecipitation with anti-IRS-2 antibody followed by immunoblotting with the indicated antibodies. (D) HeLa cells were transfected with the indicated GFPfused IRS-2 mutants and grown on anti-IR or IGF-IR antibody-coated l-biochips. Representative images are shown. Scale bar = 10 lm. (E) Contrast quantification of fluorescent images. Error bars are based on the SE of 40 analyzed cells from three individual experiments for each IRS-2 protein. *P < 0.05.

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contrast analysis, we found significant differences in the recovery rates of the three IRS proteins (Fig. 6D). In general, the recovery process proved to be very fast, with IRS-1 and -2 showing significantly higher exchange rates than IRS-3. In addition, minor differences of the IRS exchange rates with IR and IGF-1R were also found. The mobile fraction was also remarkably higher for IRS-1 and -2 (~ 70% within 10 s) compared to IRS-3 (~ 40% within 10 s). The described recovery behavior was similar on IR and IGF-IR lbiochips. These results suggest an increased retention time of IRS-3 bound to IR and IGF-IR compared to IRS-1 and -2. Furthermore, we quantified the recovery process in the cells expressing GFP-IRS-1 or -2 grown on the anti-IR antibody-coated biochips in the presence or absence of insulin (Fig. 6E). Calculated interaction values from the respective fluorescence recovery curves showed that the treatment of insulin significantly decreased the mobile fraction of IRS-1 and -2 with minor effects on the exchange rates (Fig. 6F and Table 3). These data strongly support that these FRAP analyses are suitable for detecting the physiologically relevant interaction between IRS and the receptors. FEBS Journal 282 (2015) 987–1005 ª 2015 FEBS

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Roles of IRS functional domains in the interaction dynamics Next, FRAP experiments were performed to study the exchange rates of the generated IRS mutants (Fig. 7A, B). Calculated kinetic FRAP parameters are shown in Table 2. Again, we quantitated the expression levels of the analyzed IRS proteins, which were found to be in a similar range (Fig. 7D). Our experiments indicated that the mobile fractions of all four analyzed IRS mutants were significantly higher than the mobile fractions of wild-type IRS-1 and -2 (between ~ 80% and 90% compared to ~ 70%, respectively) (Tables 1 and 2). This finding is consistent with a reduced binding of the mutant IRS proteins to the receptor. When comparing the recovery rates, we could not detect any significant differences among the IRS mutants and receptors, except for IRS-1 DAPBR (Fig. 7C). Taking these data together, we conclude that deletion of the PH, PTB and APBR domains of IRS-1 and KRLB domain of IRS-2 results in the reduced interaction of these proteins with IR and IGF-IR, which subsequently leads to reduced phosphorylation of these signaling adaptor proteins.

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Effects of serine/threonine kinases on IRS interaction behavior with IR Serine/threonine phosphorylation of IRS observed in insulin resistance is considered to reduce the insulindependent tyrosine phosphorylation of IRS. Among various kinases, JNK and IKKb, both of which are activated by the inflammatory cytokine tumor necrosis 994

Fig. 6. Photobleaching experiments of fulllength IRS proteins. (A) HeLa cells were transfected with the indicated IRS protein and grown on anti-IR and anti-IGF-IR antibody-coated l-biochips, respectively. Individual patterns were selected for the FRAP experiment. Images show a representative cell on anti-IGF-IR lbiochips with a single bleached spot before and at the indicated time points after photobleaching for IRS-1 and IRS-2 (high exchange kinetic) and IRS-3 (low exchange kinetic), respectively. Scale bar = 10 lm. (B) Normalized mean fluorescence recovery curves of analyzed IRS proteins. (C) Mean fluorescence intensity of cells used for photobleaching experiments. (D) Calculation of Kslow. (E) Normalized mean fluorescence recovery curves of untreated or insulin-stimulated (100 nM, 5 min) cells expressing GFP-IRS-1 or GFP-IRS-2, respectively. (F) Calculation of the mobile fraction. Error bars are based on the SE of 15 analyzed cells from three individual experiments. *P < 0.05.

factor a (TNFa), are the best characterized kinases that phosphorylate serine/threonine residues of IRS and inhibit IRS signaling [46–48]. We thus examined the effects of these kinases on the interaction between IRS and IR using the l-patterning technique. To investigate the effect of JNK on the tyrosine phosphorylation of IRS by IR, HeLa cells expressing

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1.36  0.22 1.36  0.28 10.74  5.25 2.94  0.59 2.98  0.64 6.08  0.88 0.09  0.03 0.07  0.04 0.44  0.37 0.18  0.02 0.17  0.04 0.21  0.03 0.50  0.09 0.50  0.11 0.06  0.04 0.23  0.04 0.23  0.05 0.11  0.01 8.07  4.71 9.78  6.45 1.55  1.30 0.72  0.01 0.70  0.02 0.60  0.13 IRS-1 IRS-2 IRS-3

0.71  0.01 0.74  0.01 0.63  0.02

3.84  0.57 3.94  0.99 3.18  0.55

IGF-IR IGF-IR IR IGF-IR

IR

IGF-IR

IR

IGF-IR

IR

Slow half-life [s] Fast half-life [s] Kslow [s 1] Kfast [s 1] Plateau

Table 1. Kinetic FRAP parameters for full-length IRS proteins obtained from two-phase exponential fit of fluorescence recovery. Results are shown as the mean  SE.

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GFP-IRS-1 or -2 were pretreated with anisomycin, a potent activator of JNK, followed by insulin, and the tyrosine phosphorylation of these proteins was assessed. As expected, treatment of anisomycin partly reduced the insulin-dependent tyrosine phosphorylation of GFP-IRS-1 and -2, with delayed migration of IRS during SDS/PAGE indicating anisomycin stimulates the serine/threonine phosphorylation of IRS (Fig. 8A,B, lane 4). We also examined the effect of TNFa and found that TNFa reduced the tyrosine phosphorylation of GFP-IRS-2 but had minor effects on that of GFP-IRS-1 (Fig. 8A,B, lane 6). We then studied the IRS interaction with the receptor under anisomycin or TNFa treated conditions in comparison with untreated cells using the l-patterning system (Fig. 8C). For this purpose, GFP-IRS-1 or GFP-IRS-2 transfected cells were pretreated with anisomycin or TNFa, respectively, and grown on anti-IR antibody-coated l-biochips. Although there were no differences in the contrast between untreated and treated cells, photobleaching experiments unraveled great differences in the calculated interaction values (Fig. 8D,E and Table 3): We found a significant decrease in the exchange rates in anisomycin or TNFa treated cells, respectively (exchange rate [s 1] GFPIRS-1: 0.098  0.011 and 0.012  0.050; GFP-IRS-2: 0.112  0.052 and 0.061  0.032). Furthermore, TNFa resulted in a significantly reduced mobile IRS fraction (Fig. 8E). These results were unexpected because a reduction of exchange rates and the mobile fraction of IRS indicates an enhanced interaction and a prolonged retention time of the IRS at the receptor sites. To further clarify whether other serine/threonine kinases of IRS might alter the similar interaction behavior of IRS, we examined the effects of IKKb, another kinase that phosphorylates serine/threonine residues of IRS [47,49]. To this end, we constructed two IKKb mutants fused with monomeric red fluorescent protein (mRFP) for two-color FRAP experiments: a constitutively active IKKb-Ser177Glu/Ser181Glu (EE) and catalytically inactive IKKb-Lys44Met (KM) [50]. Analysis using a luciferase reporter of NF-jB, a downstream transcription factor of IKKb, confirmed that IKKb-EE-mRFP served in constitutive active manner irrespective of the presence of TNFa, whereas IKKb-KM-mRFP had only minor effects on the reporter activity (Fig. 9A). We found that the coexpression of IKKb-EE-mRFP, but not IKKb-KMmRFP, significantly reduced the insulin-dependent tyrosine phosphorylation of GFP-IRS-1 and -2, indicating the activation of IKKb inhibits the tyrosine phosphorylation of IRS by IR (Fig. 9B,C). For FRAP

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experiments, the fluorescence of IRS and IKKb was bleached and the fluorescence recovery was monitored in both color channels (Fig. 10A,B). Co-expression of IKKb-EE-mRFP resulted in a significantly decreased for both GFP-IRS-1 exchange rate [s 1] 996

Fig. 7. Photobleaching experiments of various IRS mutants. (A) HeLa cells were transfected with the indicated IRS mutants and grown on anti-IR and antiIGF-IR antibody-coated l-biochips, respectively. Individual patterns were selected for the FRAP experiment. Images show a representative cell on anti-IGF-IR l-biochips with a single bleached spot before and at the indicated time points after photobleaching. Scale bar = 10 lm. (B) Normalized mean fluorescence recovery curves of analyzed IRS proteins. (C) Calculation of Kslow. (D) Mean fluorescence intensity of cells used for photobleaching experiments. Error bars are based on the SE of 15 analyzed cells from three individual experiments.

(0.097  0.016) and GFP-IRS-2 (0.181  0.027) compared to the co-expression of IKKb-KM-mRFP (GFP-IRS-1: 0.229  0.051; GFP-IRS-2: 0.442  0.106) (Fig. 10C). Furthermore, we found a reduced mobile fraction for GFP-IRS-1 (0.66  0.02) and FEBS Journal 282 (2015) 987–1005 ª 2015 FEBS

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2.24 4.18 1.41 2.19 0.58 0.38 0.22 0.49     2.92 2.30 0.86 2.30 0.19 0.24 0.10 0.07     0.12 0.04 0.08 0.08 0.31 0.17 0.49 0.31     0.23 0.30 0.81 0.30 3.65 2.88 6.62 9.88 5.80 18.85 8.32 8.32 0.92 0.92 0.88 0.89 IRS-1 IRS-1 IRS-1 IRS-2

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0.84 0.79 0.81 0.87

   

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IR

   

0.05 0.06 0.03 0.02

IGF-IR

Slow half-life [s] Fast half-life [s] Kslow [s 1] Kfast [s 1] Plateau

Table 2. Kinetic FRAP parameters for IRS-mutant proteins obtained from two-phase exponential fit of fluorescence recovery. Results are shown as the mean  SE.

IR

   

0.57 2.82 0.23 0.78

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GFP-IRS-2 (0.77  0.01) only upon co-expression of IKKb-EE-mRFP (Fig. 10D). Detailed kinetic parameters are shown in Table 3.

Discussion Ligand-dependent interaction of IR/IGF-IR with IRS proteins is a critical cue triggering the activation of downstream signaling, thereby expressing various insulin/IGF bioactivities. Evidence demonstrating the interactions of IR/IGF-IR with respective IRS proteins was obtained using in vitro phosphorylation and yeast two-hybrid assays [5,6,34,35]. There is, however, little published information directly comparing the kinetics of IRS protein recruitment to IR/IGF-IR. Recently, the interaction between IR and its binding partners including IRS was examined in the live cell context by a bioluminescence resonance energy transfer technique, which is based on an energy transfer between different luminescent dipoles tagging the target proteins [51]. These methods revealed the functional domains and the dependence on IR/IGF-IR tyrosine kinase activities for IRS interactions with the receptors. Compared to the previous assays, our l-patterning method provides several advantages. First, the interaction of transmembrane receptors with binding partners is monitored in living mammalian cells, rather than in an artificial environment. Second, the differences in the interaction property can be evaluated quantitatively among IRS family proteins and mutants when the expression levels are comparable. Third, the application of FRAP technique enables us to measure precise interaction kinetics of IRS proteins with IR/IGF-IR that were not evaluated by any previous assays. In the present study, we utilized capture antibodies against extracellular domains of IR or IGF-IR, and detected a significant redistribution of IRS proteins to the antibody-coated areas. Co-recruitment of IRS proteins to the IR/IGF-IR was already detected without any additional agonist addition, suggesting that the capture antibodies have an agonistic effect on the receptors. It is well known that some antibodies against membrane receptors function as agonists [52]. This conclusion is also confirmed by the finding that biotinylated IGF-I and insulin coating recruited IRS proteins to receptor-enriched patterns similar to the anti-IR/IGF-IR coating (Fig. 3F). Previous studies demonstrated that IRS is recruited to the juxtamembrane region containing phosphorylated tyrosine in the receptors in a ligand-dependent manner [6,8]. To probe the dependence of the interaction on IR/IGF-IR activation in our assay, we performed several control experiments. Deletion of PTB domain, which mediates

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E

the interaction with the juxtamembrane-positioned phosphotyrosine of the receptor, greatly inhibited the redistribution of IRS-1 to the antibody-coated regions. In addition, we demonstrated the requirement of the PH domain, which is also critical for ligand-dependent IRS phosphorylation [9,10] and for IRS-1 recruitment to the receptor. We further investigated the impact of intracellular IRS localization on the interaction with the receptor in our assay. It has previously been reported that IRS-1 is mainly localized to intracellular membrane compartments and that the clathrin adaptor AP-1 complex is a crucial regulator of IRS-1 localization [31,33]. In addition, IRS-1 targeting to intracellular membrane compartments is required for liganddependent tyrosine phosphorylation of IRS-1. We thus utilized an IRS-1 mutant that does not bind to the AP-1 complex and found a significant decrease in the redistribution to the antibody-coated regions, again supporting the specificity of our method. Taking these data together, we concluded that, in our system, we have analyzed the interaction of activated IR/IGF-IR. Although differences in response to insulin and IGFs have been reported in various cell types, it is difficult to identify the specific contribution of IR and IGF-I because of the existence of heterodimeric IR/ IGF-IR hybrid receptors [53]. HeLa cells used in the present study express both IR and IGF-IR (Fig. 2B), 998

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Fig. 8. Effects of anisomycin and TNFa on IRS interaction behavior. (A, B) HeLa cells expressing GFP-IRS-1 (A) or GFP-IRS-2 (B) were treated with anisomycin (5 lg
mL 1) or TNFa (25 ng
mL 1) for 2 h followed by insulin (100 nM) for 5 min. Cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. (C) Normalized mean fluorescence recovery curves of indicated proteins on anti-IR antibody-coated lbiochips. Cells were pretreated with anisomycin (5 lg
mL 1) and TNFa (25 ng
mL 1) for 4 h or left untreated. (D) Calculation of Kslow. (E) Calculation of the mobile fraction. Error bars are based on the SE of 15 analyzed cells from two individual experiments. *P < 0.05.

indicating that a possible contribution of the hybrid receptors cannot be excluded in our experimental system. However, on comparing the redistribution and interaction kinetics of IRS proteins, we found no significant differences when anti-IR or IGF-IR antibodies were used to capture the bait receptors. We conclude that IR and IGF-IR have similar binding properties for IRS proteins. Thus, the different response to insulin and IGFs does not rely on the selectivity of the substrate proteins but rather on varying interaction kinetics and downstream signaling events. However, we could not completely rule out the possibility that our results were affected by IR/IGF-IR hybrid receptor to some extent. Utilizing the advantages of our method, we investigated the role of conserved IRS domains that have been reported to be required for the interaction with the receptors and tyrosine phosphorylation reactions. We showed that deletion of PH and PTB domains significantly decreased the redistribution of IRS-1 to antiIR/IGF-IR antibody-coated regions. Although there is no biochemical evidence for the interaction of the PH domain with IR/IGF-IR, PH and PTB domains show high structural similarities [54]. Our results indicate that the PH domain of IRS proteins has an important role for receptor binding in living cells. Furthermore, our method confirmed the significance of the KRLB FEBS Journal 282 (2015) 987–1005 ª 2015 FEBS

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C

Fig. 9. Effect of IKKb on IRS phosphorylation. (A) HeLa cells were transfected with the NF-jB-luciferase reporter construct together with IKKb-mRFP EE or KM mutant. The cells were treated with TNFa (25 ng
mL 1) for 6 h and the lysates were subjected to the luciferase assay. Data are the mean  SE. (B, C) HeLa cells expressing GFP-IRS-1 (B) or GFP-IRS-2 (C) together with IKKbmRFP EE or KM mutant were treated with or without insulin (10 nM) for 5 min. Cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies.

region in IRS-2, which interacts with the kinase regulatory loop of IR, for proper binding to the receptor [14,15]. In agreement with a previous study [5], the IRS-2 DKRLB mutant displayed significantly lower redistribution to antibody-coated areas. We also found that this mutant failed to redistribute to anti-IGF-IRcoated regions. In both cases, IRS-2 DKRLB showed faster exchange rates in FRAP experiments. These data suggest a critical role of KRLB in the interaction with IR and IGF-IR in living cells. FEBS Journal 282 (2015) 987–1005 ª 2015 FEBS

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Interestingly, contrast analysis and FRAP experiments indicated that IRS-3 has the strongest interaction with IR and IGF-IR. Several studies have revealed that overexpression of IRS-3 impaired IRS1/-2-mediated signaling [28,29]. A possible explanation for this finding might be that the low exchange rates of IRS-3 inhibit the recruitment of other substrates to the receptor. Furthermore, it has been reported that the PH domain of IRS-3 is permanently localized to the plasma membrane, which may also contribute to a prolonged retention time at the receptor [13]. As already noted, we did not detect a significant difference in the binding properties of IRS-1 and IRS-2 (Fig. 4 and Table 1). Exogenous IRS-1 and IRS-2 fusion proteins were expressed at comparable rates (Fig. 6D). However, we cannot rule out the possibility that endogenous IRS-1 and IRS-2 influence the determined interaction kinetics with IR/IGF-IR. Future studies should aim to clarify whether the differences in the interaction properties between IR and IGF-IR with IRS-1 and IRS-2 influence the downstream signaling events. We have recently analyzed the interaction kinetics of Grb2 with the EGFR, another important receptor tyrosine kinase, on l-patterned surfaces [42]. Compared to this previous study, the interaction of IRS-1 and IRS-2 with IR/IGF-IR appears to be highly transient with fast exchange rates. Those of IRS-3 were found to be in a similar range as the one of Grb2 and the EGFR. Compared to IRS-1 [Kslow = 0.23  0.04 (IGF-IR), 0.5  0.09 (IR)] and IRS-2 [Kslow = 0.23  0.05 (IGF-IR), 0.5  0.11 (IR)], Grb2 shows a three- to six-fold lower recovery rate (Kslow = 0.084  0.01). Because Grb2 has also been found to interact with the IR via IRS-1 [55], further studies that analyze the interaction properties of Grb2 and IR/IGF-IR in comparison to those of Grb2 and EGFR will be of interest. Phosphorylation of multiple serine/threonine residues of IRS is considered to contribute to the desensitization of insulin signaling through a number of different mechanisms [17]. Using a yeast three-hybrid system consisting of IR, IRS-1 and JNK, it was proposed that the phosphorylation of a serine residue adjacent to the PTB domain disrupts the IR-IRS-1 interaction [46]. However, the generalized inhibitory mechanisms of the serine/threonine phosphorylation governing IRS signaling remain largely unknown. In the present study, we combined the l-patterning assay with FRAP analysis and revealed that activation of JNK (via anisomycin or TNFa treatment) and IKKb, both of which have been reported to phosphorylate serine/threonine residues of IRS, leads to a strong

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delay in IRS recovery and a reduced mobile fraction of IRS (Figs 8 and 10). These results were unexpected because the reduction in insulin-induced tyrosine phosphorylation of IRS was assumed to result in the faster exchange caused by decreased receptor binding. However, biochemical analyses have shown that anisomycin inhibits the insulin-induced translocation of IRS between cell membranes and cytosol, suggesting that 1000

Fig. 10. Role of IKKb co-expression in IRS kinetic. (A) HeLa cells were co-transfected with GFP-IRS-1 or GFP-IRS-2 and IKKbmRFP EE or KM, respectively, and grown on anti-IR antibody-coated l-biochips. Individual patterns were selected for the FRAP experiment. Images show a representative cell with a single bleached spot before and at the indicated time points after photobleaching. Scale bar = 10 lm. (B) Normalized mean fluorescence recovery curves of indicated proteins. (C) Calculation of Kslow. (D) Calculation of the mobile fraction. Error bars are based on the SE of 15 analyzed cells from two individual experiments. *P < 0.05.

the serine/threonine phosphorylation status of IRS also regulates the shuttle of IRS proteins between the cytosol and the plasma membrane [56]. Thus, serine/ threonine based inhibition of IRS translocation also leads to a significantly reduced pool of cytosolic IRS molecules that can return to the plasma-membrane after deactivation during the recycling process. This effect may well explain the observed reduced exchange FEBS Journal 282 (2015) 987–1005 ª 2015 FEBS

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Table 3. Kinetic FRAP parameters for IRS-1 and IRS-2 obtained from two-phase exponential fit of fluorescence recovery under indicated conditions. Results are shown as the mean  SE. Kfast [s 1]

Kslow [s 1]

Fast half-life [s]

Slow half-life [s]

 0.01  0.02

7.25  3.53 6.04  4.08

0.38  0.05 0.44  0.11

0.09  0.05 0.11  0.08

1.81  0.22 1.57  0.37

 0.01  0.01

5.73  2.57 4.11  0.96

0.33  0.03 0.39  0.03

0.12  0.05 0.17  0.04

2.10  0.22 1.77  0.17

 0.02  0.04

1.48  0.24 1.15  0.60

0.09  0.01 0.12  0.05

0.47  0.07 0.60  0.31

7.06  0.78 5.68  2.41

 0.06  0.08

0.43  0.16 0.78  0.72

0.01  0.05 0.06  0.03

1.59  0.60 0.89  0.82

57.42  15.78 11.44  6.06

 0.01  0.01

1.89  0.34 3.71  0.71

0.09  0.01 0.21  0.02

0.37  0.07 0.19  0.04

7.08  1.11 3.25  0.35

 0.01  0.01

2.82  0.46 6.01  1.81

0.23  0.05 0.44  0.11

0.24  0.04 0.11  0.03

3.01  0.67 1.56  0.37

Plateau Basal IRS-1 0.76 IRS-2 0.78 Insulin 100 nM IRS-1 0.69 IRS-2 0.69 Anisomycin 5 lg
mL 1 IRS-1 0.80 IRS-2 0.76 TNFa 25 ng
mL 1 IRS-1 0.61 IRS-2 0.56 Ikkb-EE coexpression IRS-1 0.66 IRS-2 0.77 Ikkb-KM coexpression IRS-1 0.81 IRS-2 0.86

properties as estimated by photobleaching experiments described in the present study. Our results emphasize the complexity with respect to serine/threonine kinase regulation on IRS signaling, which demands for further studies beyond the scope of the present study. Our approach using l-patterned surfaces revealed a precise signature of IRS interaction properties with IR and IGF-IR in a live cell context. From a pathological point of view, an impaired interaction between IR and IRS is considered to be a common feature in insulin resistance and diabetes. We anticipate the utility of our assay for monitoring the cellular response to insulin and IGFs and for identifying the compounds that modulate the interaction of IRS with the receptors.

Materials and methods Reagents RPMI, fetal bovine serum and antibiotics were purchased from PAA Laboratories GmbH (Pasching, Austria). Dulbecco’s modified Eagle’s medium and Hank’s buffered salt solution were purchased from Nissui (Tokyo, Japan). Human recombinant IGF-I was a kind gift from Astellas Pharma, Inc. (Tokyo, Japan). Bovine insulin, anisomycin and TNFa were purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-GFP antibody (B-2), anti-IGF-IRb antibody (C-20) and anti-IRb antibody (C-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phosphotyrosine antibody (4G10) was purchased from

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Millipore (Billerica, MA, USA). Anti-a-tubulin antibody (DM1A) was purchased from Sigma-Aldrich. Anti-IKKb antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-IRS-1 and anti-IRS-2 antibodies were raised in rabbits as described [30]. Anti-IRS-2 antibody (H-205) was also obtained from Santa Cruz Biotechnology for immunoblotting. Anti-IRS-3 antibody was a generous gift from Tomoichiro Asano (Hiroshima University, Hiroshima, Japan). Biotinylated anti-IR and anti IGF-IR antibodies for l-patterning experiments were purchased from Antibodies Online (Herford, Germany). Biotinylated insulin and IGF-I were obtained from IBT (Binzwangen, Germany).

Plasmids Full-length and deletion mutant lacking the amino acid residues 443–663 of rat IRS-1, and full-length human IRS-2 and rat IRS-3 were cloned into pEGFP-C1, as described previously [32,33]. IRS-1 mutants lacking the amino acid residues 12–113 (corresponding to PH domain) and 155– 258 (corresponding to PTB domain) and IRS-2 mutant lacking the amino acid residues 546–753 were amplified by PCR and these fragments were cloned into pEGFP-C1. pCS2-mRFP4 and mouse IKKb cDNA were generous gifts from Masanori Taira and Kazuhiro Chida (The University of Tokyo, Tokyo, Japan), respectively. To generate the plasmids expressing IKKb mutants (S177E/S181E and K44M) fused with mRFP in their C-terminus, the fulllength of IKKb was cloned into pCS2-mRFP4, and the corresponding mutations were constructed by site-directed mutagenesis.

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Cell culture and transfection Human HeLa cells from the American Type Culture Collection were used. An electroporation unit (Nucleofector) and electroporation cuvettes were obtained from Lonza (Basel, Switzerland). HeLa cells were cultured in RPMI medium supplemented with 10% fetal bovine serum and penicillin/streptomycin and grown at 37 °C in a humidified incubator (≥ 95%) with 5% CO2. For transient transfection, cells were subcultured the day before. Cells were then transfected with 0.8 lg of DNA at 50–70% confluence using the Nucleofector device. Cells were directly plated into 60-mm culture dishes and grown for 24–48 h and were then used for l-patterning experiments. For biochemical experiments, HeLa cells were transfected with plasmids using poly(ethylenimine). Linear poly(ethylenimine) (MW 25 000; Polysciences, Inc., Warrington, PA, USA) was dissolved in water at the concentration of 1 mg
mL 1. Cells were subcultured in 60-mm dishes at 50–75% confluence. Next day, 4 lg of plasmid and 12 ll of poly(ethylenimine) solution were diluted into 400 lL of Opti-MEM (Life Technologies, Tokyo, Japan) and incubated for 15 min at room temperature. The mixture was added over the culture dish, and the cells were grown for 24 h and then used for immunoprecipitation and immunoblotting.

Immunoprecipitation and immunoblotting Cells were rinsed twice with Hank’s buffered salt solution and starved overnight in Dulbecco’s modified Eagle’s medium containing 0.1% BSA, followed by treatment with IGF-I or insulin for 5 min at the final concentration of 100 ng
mL 1 or 10–100 nM, respectively. After rinsing twice with PBS, cells were harvested in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 100 Kallikrein inhibitor units
mL 1 aprotinin, 20 lg
mL 1 phenylmethylsulfonyl fluoride, 10 lg
mL 1 leupeptin, 5 lg
mL 1 pepstatin, 500 lM Na3VO4, 10 mg
mL 1 p-nitrophenyl phosphate, pH 7.4). After brief sonication, the supernatant was obtained by the centrifugation at 15 000 g for 10 min at 4 °C. The resulting lysate was incubated with anti-IRS-1, IRS-2, or IRS-3 antibody overnight, followed by precipitation with Protein A sepharose (GE Healthcare, Japan, Tokyo) for 1 h at 4 °C. The beads were then washed three times with lysis buffer, and proteins were eluted with Laemmli’s sample buffer. Samples were analyzed by immunoblotting, as described previously [48].

Then, the reporter activities were measured by Dual-Luciferase Reporter Assay System (Promega, Tokyo, Japan) in accordance with the manufacturer’s instructions.

Live cell TIRF microscopy, l-contact printing and contrast analysis Live cell TIRF microscopy was carried out on a microscopy set-up as used in a previous study, with an additional 568 nm DPSS laser (Toptica Photonics, Munich, Germany) for selective fluorescence excitation of red fluorescent proteins (RFP) [42]. l-contact printing and contrast analysis were performed as described previously [42]. Furthermore, FRAP images were analyzed using the Spotty framework [57].

Statistical analysis Data are expressed as the mean  SE. Comparisons between two groups were performed using Student’s t-test. P < 0.05 was considered statistically significant.

Acknowledgements This work was funded by the program ‘Regionale € 2007–2013’ with the finanWettbewerbsf€ ahigkeit OO cial support of the European Fund for Regional Development, as well as the Federal State of Upper Austria, and the Austrian Research Promotion Agency (FFG; project number 842379). This work was also partially supported by a Grant-in-Aid for Scientific Research (S)#25221204, the Core-to-core program from JSPS and the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food industry (to S-IT). We thank Nevin Lambert (Georgia Regents University, USA) for critically reading the manuscript. We also acknowledge Dr Susan Hall (The University of North Carolina at Chapel Hill) for help with writing the manuscript.

Author contributions PL, YY, SIT, OH and JW conceived and designed the experiments. PL, YY, FH, UM and JW performed the experiments. PL, YY and FH analyzed the data. PL, YY, SIT and JW wrote the paper.

References Luciferase assay HeLa cells were co-transfected with pNF-jB-Luc and pRLSV40 (Stratagene, La Jolla, CA, USA) together with the plasmids expressing mRFP only or IKKb-mRFP mutants. At 24 h after transfection, cells were starved overnight followed by the stimulation with TNFa (25 ng
mL 1) for 6 h.

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