Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver ´ ´ ´ Bernard Desbuquois1,2, Veronique Bereziat3, Francois Authier4, Jean Girard1,2 and Anne-Francoise ¸ ¸ Burnol1,2 ´ Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), France Inserm, U567, Paris, France ´ ´ Centre de Recherche Saint-Antoine, UMR S893, Faculte de Medecine Pierre et Marie Curie, Paris, France ˆ ´ Inserm, U756, Faculte de Pharmacie Paris 11, Chatenay-Malabry, France Keywords endocytosis; insulin receptor; liver; molecular adaptor; tyrosine kinase activity Correspondence ´ B Desbuquois, Departement ´ d’Endocrinologie, Metabolisme, Cancer, Institut Cochin, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France Fax: +33 44 41 24 21 Tel: +33 53 73 27 08 E-mail: bernard.desbuquois@inserm.fr (Received 14 September 2007, revised 29 April 2008, accepted July 2008) doi:10.1111/j.1742-4658.2008.06583.x The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi ⁄ endosome fractions Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi ⁄ endosome fraction, which paralleled the increase in IR b-subunit tyrosine phosphorylation Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi ⁄ endosome fractions In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR b-subunit in these fractions Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14–IR complex in endosomes Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling Grb14 is a member of the Grb7 ⁄ Grb10 ⁄ Grb14 family of adaptor proteins, which lack intrinsic enzymatic activity and share a common multidomain structure These adaptors bind to several receptor tyrosine kinases and signaling proteins, and are involved in the regulation of various processes, including cell growth and Abbreviations BPS, between plekstrin homology and SH2; bpV(phen), bisperoxo(1,10-phenanthroline) oxovanadate; EGF, epidermal growth factor; ER, endoplasmic reticulum; GST, glutathione S-transferase; IR, insulin receptor; IRS-1, insulin receptor substrate-1; IRb, insulin receptor b-subunit; PDK-1, 3-phosphoinositide-dependent kinase-1; PI3-kinase, phosphoinositide-3-kinase; PIR, phosphorylated insulin receptorinteracting region; WGA, wheat germ agglutinin FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4363 Insulin-induced translocation of Grb14 in rat liver B Desbuquois et al metabolism, apoptosis, and cell migration [1–5] Grb14, which is selectively expressed in insulin target tissues, interacts with the phosphorylated insulin receptor (IR) through a region called BPS [between plekstrin homology (PH) and SH2] or phosphorylated insulin receptor-interacting region (PIR) [6] and inhibits the tyrosine kinase activity of the IR [7] On the basis of the crystal structure of the tyrosine kinase domain of the IR in complex with the PIR ⁄ BPS domain of Grb14, Depetris et al [8] have shown that Grb14 binds as a pseudosubstrate inhibitor to the peptide-binding groove of the kinase and thus acts as a selective inhibitor of insulin signaling Consistent with this observation, overexpression of Grb14 in CHO-IR cells impairs Akt and ERK insulin signaling pathways and inhibits distal effects of insulin on glycogen and DNA synthesis [6–9], and microinjection of Grb14 into Xenopus laevis oocytes inhibits insulin-induced oocyte maturation [10] Conversely, disruption of the Grb14 gene in mice ameliorates glucose tolerance in vivo and insulin signaling in both liver and skeletal muscle [11] However, although it improves the Akt insulin signaling pathway, depletion of Grb14 by RNA interference in mouse primary cultured hepatocytes inhibits the stimulatory effect of insulin on glycogen synthesis and on glycolytic and lipogenic gene expression, suggesting that Grb14 action on insulin signaling cannot be restricted to its inhibitory action on IR catalytic activity [12] In addition to the IR, three partners of Grb14 involved in insulin signaling have been identified: (a) protein kinase Cf interacting protein (ZIP), an adaptor protein that binds to the PIR domain of Grb14 and mediates the assembly of a protein kinase Cf–ZIP–Grb14 heterotrimer [10]; (b) insulin receptor substrate-1 (IRS-1), which binds through its phosphotyrosine-binding domain to an NPXY motif of Grb14 in a phosphorylation-independent manner [13]; and (c) 3-phosphoinositide-dependent kinase-1 (PDK1), which binds constitutively to a PDK-1 consensus binding motif of Grb14 [14] Although lacking a hydrophobic transmembrane domain, like many signaling proteins Grb14 is found in cells in both soluble and membrane-associated states In DU 145 human prostate cancer cells, endogenous Grb14 is predominantly associated with a lowdensity microsomal fraction, where it colocalizes with tankyrase [15] In indirect immunofluorescence studies, Grb14 is detected as a diffuse but also punctate cytoplasmic staining that is more concentrated around the nucleus, suggesting its association with the Golgi; a pool of Grb14 also localizes at the plasma membrane [15] In HEK 293 cells, epitope-tagged Grb14 is mainly expressed in the cytosol in the resting state, but 4364 redistributes in part to the membrane fraction upon insulin stimulation [14] In rat retina, endogenous Grb14 shows a perinuclear and nuclear localization, consistent with the identification of a functional nuclear localization signal in the Grb14 N-terminus [16] However, unlike with other insulin signaling proteins, Grb14 compartmentalization has not been characterized in physiological insulin target cells In the present study, subcellular fractionation and western blotting procedures have been used to assess the compartmentalization of Grb14 in rat liver, an organ that expresses both the IR and Grb14 at a high level, and where insulin-induced phosphorylation, activation and internalization of the IR have been previously documented [17–19] Our results show that, in the basal state, Grb14 is localized mainly in highdensity microsomal elements and cytosol Upon insulin stimulation, Grb14 is rapidly and dose-dependently translocated first to plasma membranes and then to endosomes, in which it associates with phosphorylated IR These results suggest that Grb14 is recruited by the activated IR at the plasma membrane and then undergoes internalization as a complex with the IR In addition, our results provide the first evidence for an in vivo inhibitory effect of endogenously recruited Grb14 on IR catalytic activity in both compartments Results Subcellular distribution of Grb14 and the IR in liver from control and insulin-injected rats The subcellular distribution of liver immunoreactive Grb14 was examined using preparative and analytical fractionation and compared to that of the IR Upon differential centrifugation (Fig 1A), Grb14 was detectable as a major protein of 60 kDa in both particulate and soluble fractions A minor component of slightly reduced electrophoretic mobility, which may represent a phosphorylated form of Grb14 [10], was also observed Under basal conditions, Grb14 content was three-fold to four-fold higher in the light mitochondrial–microsomal and cytosolic fractions than in the nuclear and mitochondrial fractions (P < 0.001) Analysis of separate light mitochondrial and microsomal fractions showed that Grb14 content was about 10-fold higher in the microsomal fraction than in the light mitochondrial fraction (results not shown) In recovery studies, the microsomal and cytosolic fractions accounted respectively for about 40% and 49% of total Grb14 contained in the whole homogenate Grb14 was present at relatively low levels in the plasma membrane and Golgi ⁄ endosome fractions, with FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS B Desbuquois et al Insulin-induced translocation of Grb14 in rat liver A H N B M LP S PM GE –ins Grb14 – ins Grb14 + ins –ins + ins + ins – ins EEA1 + ins Na+K+ATPase Calnexin + ins number: 10 11 12 13 14 Grb14 *** * 120 100 250 *** 200 * *** 150 * 80 60 100 Grb14 control i i ins ins 40 50 20 0 – ins + ins 800 *** 10 11 12 13 14 Fraction number 120 100 600 80 400 60 40 200 20 * * *** *** *** * H N M LP S PM GE 10 11 12 13 14 Fig Comparative expression of Grb14 and IR proteins in liver subcellular fractions of control and insulin-injected rats (A) Liver homogenates (H) prepared from five control ()ins) and five insulin-injected (+ins, postinjection) rats were fractionated into nuclear (N), mitochondrial (M), light mitochondrial–microsomal (LP), cytosolic (S), plasma membrane (PM) and Golgi ⁄ endosome (GE) fractions as described in Experimental procedures Aliquots (10 lg of protein) were analyzed by western blotting using antibodies against Grb14, IRb and phosphotyrosine to detect phosphorylated IRb (p-IRb) as indicated Upper panel: representative immunoblots No signal was detected on an anti-phosphotyrosine immunoblot performed in the absence of insulin Lower panel: quantitation of Grb14 and IRb signals by scanning densitometry, with results expressed as percentage of signal intensity in the homogenate (mean ± SEM of five determinations on fractions originating from separate livers) (B) Liver microsomal fractions prepared from three control and six insulin-treated rats (2 and postinjection, three rats per time point) were subjected to centrifugation through a continuous sucrose density gradient Fourteen subfractions with densities increasing linearly from 1.065 (fraction 1) to 1.195 gỈmL)1 (fraction 14) were collected, and aliquots (10 lg of protein) were analyzed by western blotting using antibodies directed against Grb14, IRb, EEA1 (endosomal marker), Na+ ⁄ K+-ATPase (plasma membrane marker) and calnexin (ER marker) as indicated Top: representative immunoblots in control ()ins) and insulin-injected (+ins, postinjection) rats Bottom: quantitation of Grb14 and IRb signals, with results expressed as percentage of maximum (mean ± SEM of three determinations on microsomal fractions originating from separate livers) Significant differences between control and insulin-treated liver fractions [2 in (A) and in (B)] using the LSD test are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001 recoveries of about 1% and 0.2%, respectively The subcellular distribution of Grb14 clearly differed from that of the IR, which was detected only in particulate fractions with a marked enrichment in the plasma membrane and Golgi ⁄ endosome fractions, and, to a lesser extent, in the light mitochondrial–microsomal fraction, in agreement with a previous report [17] Two minutes after insulin injection, consistent with the insulin-induced internalization and phosphorylation of the IR [17–19], IR content decreased in the plasma membrane fraction and reciprocally increased in the light mitochondrial–microsomal and Golgi ⁄ endosome fractions, and IR b-subunit (IRb) phosphorylation was detected in each of these fractions Concomitantly, a significant increase in Grb14 content was observed in the plasma membrane and Golgi ⁄ endosome fractions (twofold and 10-fold increase respectively, P < 0.001) and to a lesser extent in the light mitochondrial–microsomal fraction (30% increase, P < 0.05) As the Grb14 content in crude FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4365 Insulin-induced translocation of Grb14 in rat liver B Desbuquois et al IRb towards lower densities, which was more pronounced at than at The shift in Grb14 distribution resulted from an increased Grb14 content at low and intermediate densities (subfractions 3–8; density range, 1.08–1.14 gỈmL)1), whereas the shift in IRb distribution involved both a decreased content at intermediate and high densities (subfractions 8–12; density range, 1.13–1.18 gỈmL)1) and an increased content at low densities (subfractions 2–5; density range, 1.07–1.11 gỈmL)1) Insulin treatment also resulted in an increased content of phosphorylated IRb, the distribution of which was superimposable on that of IRb (data not shown) Taken together, these results extend those obtained with preparative procedures and confirm that, upon insulin-induced IR internalization and activation, Grb14 associates in part with plasma membranes and endosomes homogenates was unchanged, these results suggest a translocation of cytosolic Grb14 to IR-enriched compartments However, the increased recovery of Grb14 in IR-enriched fractions did not exceed 5% of the total Grb14 liver pool, explaining why no decrease in cytosolic Grb14 could be detected To further characterize the distribution of membraneassociated Grb14 and IR under basal and insulinstimulated conditions, the microsomal fraction, which contained the majority of both proteins, was subjected to analytical density gradient centrifugation The distribution of Grb14 and IRb was analyzed and compared to that of three organelle markers: EEA1 (endosomes); Na+ ⁄ K+-ATPase (plasma membrane); and calnexin [endoplasmic reticulum (ER)] (Fig 1B) In control rats, Grb14 was expressed predominantly in the high-density region of the gradient (subfractions 8–14; density range, 1.13–1.20 gỈmL)1), as was calnexin In contrast, IRb was expressed mainly at intermediate densities (subfractions 6–10; density range, 1.11–1.16 gỈmL)1), similarly to Na+ ⁄ K+-ATPase, and to a lesser extent at low densities (subfractions 2–5; density range, 1.07– 1.11 gỈmL)1), coinciding with EEA1 Insulin treatment caused a shift in the distribution of both Grb14 and 15 30 60 0.5 2 B 15 30 60 *** *** *** *** In time-course studies (Fig 2A), Grb14 content in the plasma membrane fraction reached a maximum (three GE PM A ins (min) 0.5 Grb14 Time course and dose-dependence of the insulin effect on Grb14 content in plasma membrane and endosomal liver fractions *** * ins (µg): Grb14 ** 0.3 PM 30 * *** *** 30 20 10 *** ** ** ** *** p- *** 20 *** 15 * 10 *** *** *** 0.5 *** *** 20 * * *** * ** *** 30 *** ** 10 0 * 10 p- 30 * 15 0 GE 0.3 *** 0 0.5 15 30 60 0.5 15 30 60 Fig Time course and dose-dependence of in vivo insulin effects on Grb14, phosphorylated IRb (p-IRb) and IRb content in liver plasma membrane (PM) and Golgi ⁄ endosome (GE) fractions PM and GE fractions were prepared from livers of rats studied at the indicated time points after injection of 30 lg of insulin (four to six rats per time point) (A), or studied after injection of the indicated dose of insulin (two rats per dose) (B) Aliquots (10 lg of protein) were analyzed by western blotting using antibodies against Grb14, phosphotyrosine and IRb, as indicated The figure shows representative immunoblots and quantitation of protein signals by scanning densitometry, with results expressed as fold change relative to basal value [mean ± SEM of four to six determinations for (A) or two determinations for (B), on fractions originating from separate livers] Significant changes relative to control (no insulin) using the LSD test are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001 4366 FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS B Desbuquois et al times basal level) as early as 30 s after insulin injection, and subsequently declined to almost basal values within 30 min, whereas Grb14 content in the Golgi ⁄ endosome fraction was maximal at (about six times basal level) and declined more slowly, remaining elevated at 60 The insulin-induced increase in Grb14 content in the plasma membrane and Golgi ⁄ endosome fractions was significantly correlated with the increase in phosphorylated IRb content in the same fractions (plasma membrane, r = 0.72, P < 0.001; Golgi ⁄ endosome, r = 0.60, P < 0.01) When normalized to IRb content, insulin-induced changes in Grb14 content were similar in the two fractions (twofold to threefold maximal increase), as were the changes in phosphorylated IRb content (10–20-fold maximal increase) These findings suggest that the ability of phosphorylated IRb to recruit Grb14 is the same in the plasma membrane and Golgi ⁄ endosome fractions In dose–response studies (Fig 2B), the insulininduced increase in Grb14 content was detectable for lg of insulin in the plasma membrane fraction and 0.3 lg in the Golgi ⁄ endosome fraction, and was maximal for 30 lg in both fractions, again in good agreement with the increase in phosphorylated IRb content Functional relationships between membrane association of Grb14 and IR tyrosine phosphorylation The parallel increase in Grb14 content and phosphorylated IRb content in the plasma membrane and Golgi ⁄ endosome fractions of insulin-treated rats suggested that IRb phosphorylation state was involved in the association of Grb14 with these compartments To gain further insight into the functional relationship between these two events, we used three complementary approaches First, we assessed the response of endosomal Grb14 to [GluA13,GluB10]insulin and [HisA8,HisB4,GluB10,HisB27]insulin, two high-affinity insulin analogs that were previously reported to induce prolonged tyrosine phosphorylation of the endosomal IR [20], administered in vivo As shown in Fig 3, these analogs also induced a more sustained association of Grb14 with the Golgi ⁄ endosome fraction, which paralleled their effects on phosphorylated IRb content Second, we examined the ability of Grb14 associated with the Golgi ⁄ endosome fraction to dissociate upon incubation at 37 °C, conditions under which the phosphorylated IR has been shown to be rapidly dephosphorylated by endogenous phosphatases [21] As shown in Fig 4, incubation of Golgi ⁄ endosome fractions from insulin-treated rats indeed resulted in a progressive, time-dependent dephosphorylation of Insulin-induced translocation of Grb14 in rat liver Grb14 A3 Grb14 H2 Grb14 WT : 15 30 60 Fig Time course of in vivo effects of human insulin analogs on Grb14 and phosphorylated IRb (p-IRb) content in liver Golgi ⁄ endosome fractions Golgi ⁄ endosome fractions were isolated from livers of rats studied at the indicated time points (one rat per time point) after injection of 30 lg of [GluA13,GluB10]insulin (A3), [HisA8,HisB4,GluB10,HisB27]insulin (H2) or wild-type human insulin (WT) Aliquots (30 lg protein) were analyzed by western blotting using antibodies against Grb14 and phosphotyrosine phosphorylated IRb Concurrently, Grb14 content decreased in sedimented membranes, while remaining unchanged in total incubation mixtures (data not shown), indicating a dissociation of membrane-bound Grb14 Membrane-bound Grb14 and phosphorylated IRb were significantly correlated (r = 0.56; P < 0.05) Similar results were obtained using the plasma membrane fraction (data not shown) Importantly, both processes were almost fully inhibited by bisperoxo(1,10-phenanthroline) oxovanadate [bpV(phen)], a potent inhibitor of endosomal phosphotyrosine phosphatases Finally, we examined the response of endosomal Grb14 to bpV(phen) administered in vivo; this was previously shown to increase IRb phosphorylation [22] and to prevent the dephosphorylation of activated IRb that occurs shortly after insulin injection [23] Our results confirm these observations, and further show that the changes in phosphorylated IRb content induced by bpV(phen) in the Golgi ⁄ endosome fraction are accompanied by somewhat parallel changes in Grb14 content (Fig 5) When injected alone, bpV(phen) led, at 15 and 45 postinjection, to a 12–16-fold increase in phosphorylated IRb content and to a nearly two-fold increase in Grb14 content in the Golgi ⁄ endosome fraction When injected 15 prior to an inframaximal dose of insulin, bpV(phen) FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4367 Insulin-induced translocation of Grb14 in rat liver B Desbuquois et al bpV A bpV (min) – 15 * 45 * 15 45 Grb14 p- + 6.2 12.5 16.5 B – + Grb14 + 10 20 30 bpV – Grb14 bpV (min) *** *** 10 40 *** – ** 100 + + ins (min) – 50 ins (min) Fig In vitro dissociation of membrane-bound Grb14 upon dephosphorylation of in vivo activated IRs in liver Golgi ⁄ endosome fractions Golgi ⁄ endosome fractions from five insulin-injected rats (2 postinjection) were incubated at 37 °C for the indicated times in the absence or presence of bpV(phen) (0.2 mM), and then subjected to high-speed centrifugation as described in Experimental procedures Total incubation mixtures and resuspended pellets were analyzed by western blotting using antibodies against phosphotyrosine and Grb14 as indicated Incubation did not affect the intensity of Grb14 signals in total incubation mixtures (data not shown) Top: blots representative of five experiments carried out on Golgi ⁄ endosome fractions originating from separate livers Bottom: quantitation of Grb14 (gray bars) and phosphorylated IRb (p-IRb) signals (white bars) in the absence of bpV(phen), with results expressed as percentage of the time value (mean ± SEM of five determinations) Both membrane-bound Grb14 and p-IR show a significant, time-dependent decrease according to ANOVA (P < 0.0001) prevented the decrease in phosphorylated IRb and Grb14 content that occurred between and 15 after insulin injection A significant correlation was observed between phosphorylated IRb and Grb14 (r = 0.81, P < 0.01) Comparable effects of bpV(phen) treatment on IRb phosphorylation and Grb14 content, albeit less marked, were also observed in the plasma membrane fraction of untreated and insulin-treated rats (data not shown) Taken together, these data strongly suggest that the phosphorylation status of IRb is implicated in the in vivo association of Grb14 with IR-containing subcellular compartments 4368 15 15 Fig In vivo effects of bpV(phen), alone or in association with insulin, on Grb14, phosphorylated IRb (p-IRb) and IRb content in liver Golgi ⁄ endosome fractions Golgi ⁄ endosome fractions were prepared from livers of rats studied at the indicated time points after injection of 1.2 lmol of bpV(phen) (A) (three to four rats per time point) or lg of insulin (B) In (B), rats were pretreated or not with 1.2 lmol of bpV(phen) 15 prior to insulin injection (two to three rats per time point and per condition) Fractions (10 lg of protein) were analyzed by western blotting using antibodies against Grb14, phosphotyrosine and IRb The blots (left part) are representative of experiments carried out on Golgi ⁄ endosome fractions originating from separate livers, and densitometric measurements of Grb14, p-IRb and IRb signals are shown on the right In (A), results are expressed as fold increase in Grb14 (white bars) and p-IRb (numbers under the blot) above the bpV(phen) control (mean ± SEM of the three or four determinations) In (B), results are expressed as fold increase above the insulin control, with (gray bars) or without (white bars) bpV(phen) pretreatment (mean ± SEM of the two or three determinations) A significant effect of bpV(phen) treatment using the LSD test is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001 Nature of the association of Grb14 with liver membrane fractions from control and insulin-treated rats As expected for a peripheral protein lacking a transmembrane domain, Grb14 associated with the microsomal, plasma membrane and Golgi ⁄ endosome fractions was partially extracted by treatment with KCl at concentrations above m On the basis of the comparative quantitation of sedimentable Grb14 in untreated and KCl-treated fractions, the proportion of Grb14 removed by m KCl was about 60–75% in FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS B Desbuquois et al Insulin-induced translocation of Grb14 in rat liver fractions of control rats and 40–60% in fractions isolated shortly after (30 s to min) insulin injection (data not shown) Under these conditions, at least 90% of the IR remained membrane-associated as assessed by both western blotting and insulin binding NaCl, at m, was less effective than KCl at the same concentration, and so was urea at m On the other hand, treatment with 0.1 m Na2CO3 (pH 11.5) removed at least 60–70% of Grb14, again leaving the IR membrane-associated (data not shown) These findings indicate that Grb14 recruited under insulin stimulation is tightly associated with membranes To determine whether Grb14 translocated to the plasma membrane and Golgi ⁄ endosome fractions in insulin-treated rats interacts with the phosphorylated IR, fractions were solubilized with Triton X-100 and soluble extracts were subjected to immunoprecipitation using antibodies against IRb and Grb14, followed by western blotting using antibodies against Grb14 and phosphotyrosine, respectively As shown in Fig 6, insulin treatment increased Grb14 content in IR immunoprecipitates, and phosphorylated IRb content in Grb14 immunoprecipitates, in a time-dependent manner As in crude lysates, these changes were maximal at 30 s in the plasma membrane fraction and at in the Golgi ⁄ endosome fraction, and subsequently declined These findings strongly favor a direct interaction between Grb14 and the phosphorylated IRb induced by in vivo insulin stimulation Furthermore, they suggest that Grb14 recruited by the phosphorylated IR at the plasma membrane may undergo cointernalization along with the IR Specificity of insulin-induced changes in Grb14 content in liver subcellular fractions The IR also binds to Grb7 and Grb10, two adaptor proteins that are structurally related to Grb14, and besides the IR, the epidermal growth factor (EGF), fibroblast growth factor and platelet-derived growth factor receptors also can bind Grb14 [5] As liver expresses both Grb7 and the EGF receptor at high levels, we performed a comparative analysis, in time studies, of the in vivo effects of insulin and EGF on the contents of Grb7 and Grb14 in the plasma membrane and Golgi ⁄ endosome fractions As shown in PM ins (min) 0.5 15 GE 30 60 0.5 15 30 60 Grb14 IP 100 80 60 40 20 100 80 60 40 20 100 80 60 40 20 100 80 60 40 20 IP Grb14 Fig Time course of in vivo insulin-induced coimmunoprecipitation of Grb14 with phosphorylated IRb (p-IRb) Plasma membrane (PM) and Golgi ⁄ endosome (GE) fractions were prepared from livers of rats killed at the indicated time points after injection of 30 lg of insulin (two rats per time point) Following solubilization by Triton X-100, fractions were immunoprecipitated with monoclonal antibody against IRb or polyclonal antibodies against Grb14 as indicated Immunoprecipitates (IP) were analyzed by western blotting using antibodies against Grb14 and phosphotyrosine, and polyclonal antibodies against IRb Immunoblots are representative of two experiments, carried out on fractions originating from separate livers Densitometric measurements of Grb14 (white bars) and p-IRb (gray bars) signals are expressed as percentage of maximum (mean ± SEM of these duplicate determinations) FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4369 Insulin-induced translocation of Grb14 in rat liver PM A ins (min): 0.5 B Desbuquois et al GE 0.5 15 30 60 15 30 60 Grb14 Grb7 2 0 PM GE 0.5 15 30 0.5 15 30 B EGF (min) : Grb14 Effect of insulin-induced association of Grb14 on IR tyrosine kinase activity Grb7 4 2 0 C EGF ( (min) : ) PM 0.5 15 GE 0.5 15 p-EGFR EGFR Fig Comparative in vivo effects of insulin and EGF on Grb7 and Grb14 content in liver subcellular fractions Plasma membrane (PM) and Golgi ⁄ endosome (GE) fractions were isolated from livers of rats studied at the indicated time points after injection of 30 lg of insulin (A) (four rats per time point) or 100 lg of EGF (B, C) (three rats per time point) Aliquots (10 lg of protein) were analyzed by western blotting using antibodies against Grb14 and Grb7 as indicated (A, B) Following EGF treatment, aliquots were also immunoblotted with antibodies against phosphotyrosine and EGF receptor (EGFR) (C) The blots are representative of experiments carried out on fractions originating from separate livers Densitometric measurements of Grb14 (white bars) and Grb7 (gray bars) signals are expressed as fold increase above control (mean ± SEM of four determinations for insulin and three determinations for EGF) See text for statistical analysis of insulin and EGF effects on Grb7 and Grb14 content in the PM and GE fractions p-EGFR, phosphorylated EGFR Fig 7A, insulin induced an increase in Grb7 content that paralleled the increase in Grb14 content, with a maximal effect at 30 s in the plasma membrane fraction and at in the Golgi ⁄ endosome fraction The response of Grb7 to insulin was similar to that of Grb14 in the plasma membrane fraction but somewhat greater in the Golgi ⁄ endosome fraction EGF did not significantly affect Grb14 content in the plasma mem4370 brane and Golgi ⁄ endosome fractions, except for for a three-fold increase at 15 (P < 0.001) in the Golgi ⁄ endosome fraction It did, however, significantly increase Grb7 content in these two fractions, with a maximal effect at 0.5 in the plasma membrane fraction (3.5-fold increase, P < 0.001) and at 5– 15 in the Golgi ⁄ endosome fraction (sixfold increase, P < 0.001) (Fig 7B) These changes were temporally correlated with the increase in phosphorylated EGF receptor content in both the plasma membrane and Golgi ⁄ endosome fractions and in EGF receptor content in the Golgi ⁄ endosome fraction (Fig 7C) We have previously shown that glutathione S-transferase (GST)–Grb14 inhibits the in vitro tyrosine kinase activity of the recombinant IR as measured using poly(Glu,Tyr) as substrate [7] Consistent with this observation, GST–Grb14 also inhibited the ability of the endogenous liver IR partially purified from a microsomal fraction to phosphorylate RCAM-lysozyme, a high-affinity substrate of the receptor (Fig 8) The inhibitory effect of GST–Grb14 on insulin-stimulated RCAM-lysozyme phosphorylation by the IR was dose-dependent, being detectable at 0.15 lgỈmL)1 (1.5 nm) and almost complete at lgỈmL)1 (50 nm) Inhibition by exogenous Grb14 of the IR activated in vitro suggested that removal of endogenous Grb14 from the IR activated in vivo would elicit an opposite effect To address this question, we first assessed the ability of insulin administered in vivo to increase the level of RCAM-lysozyme in cell fractions, and then assayed the fractions of insulin-injected rats for Grb14, IR and phosphorylated IR content and RCAM-lysozyme phosphorylation following treatment or no treatment with m KCl As shown in Fig 9A, intact Golgi ⁄ endosome fractions isolated after insulin injection displayed a four-fold increase (4.03 ± 0.32, mean ± SEM, n = 4) in the extent of RCAM-lysozyme phosphorylation relative to control fractions KCl treatment of the Golgi ⁄ endosome fractions from insulin-injected rats affected neither the content nor the tyrosine phosphorylation state of the IR, but led to a 60% decrease in Grb14 content (Fig 9B) Removal of Grb14 from these fractions led to a nearly two-fold concomitant increase in the extent of RCAM-lysozyme phosphorylation (Fig 9C) KCl treatment of the plasma membrane and microsomal fractions isolated and after insulin injection induced a similar increase in the extent of RCAM-lysozyme phosphoryla- FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS B Desbuquois et al Insulin-induced translocation of Grb14 in rat liver A p-RCAM-L – ins p-RCAM-L + ins Time (min): GST-Grb14: 10 20 40 10 20 – 40 + B p-RCAM-L 0.15 0.5 1.5 µg GST-Grb14 15 15 GST 100 80 60 40 20 Fig In vitro effects of GST–Grb14 on the tyrosine kinase activity of liver IRs IRs were partially purified from a liver microsomal fraction by adsorption on WGA–Sepharose, and examined for their ability to phosphorylate RCAM-lysozyme as described in Experimental procedures (A) Time course of RCAM-lysozyme phosphorylation in the presence (+ins) or absence ()ins) of 0.5 lM insulin, and in the presence (+) or absence ()) of lgỈmL)1 GST–Grb14 (B) Dosedependent effect of GST–Grb14 on insulin-stimulated RCAM-lysozyme phosphorylation (20 incubation of the receptor with RCAM-lysozyme) The blots in (A) and (B) are representative of two experiments on separate microsomal fractions, and the densitometric measurements in (B) are expressed as percentage of the value in the absence of GST–Grb14 (mean ± SEM of two determinations) p-RCAM-L, phosphorylated RCAM-lysozyme tion by subsequently prepared wheat germ agglutinin (WGA)-purified insulin receptors (data not shown) Altogether, these results suggest that Grb14 endogenously recruited by the phosphorylated IR after in vivo insulin stimulation exerts an inhibitory action on IR catalytic activity Discussion Insulin signaling proteins in adipocytes [24–27] and liver [28,29] have been shown to be compartmentalized and to undergo activation and ⁄ or redistribution to specific subcellular compartments in response to insulin In liver, plasma membranes and endosomes are major sites to which IRS-1, phosphoinositide-3-kinase (PI3-kinase) and Akt1 redistribute upon in vivo insulin stimulation, and where IRS-1 and PI3-kinase interact with phosphorylated IRs [28,29] Our results extend these observations to the molecular adaptor Grb14, thus reinforcing its role as an insulin signaling protein Specifically, we show that following insulin treatment, endogenous Grb14 undergoes a time-dependent and reversible translocation to plasma membranes and endosomes, in which it is recruited by phosphorylated IRb Furthermore, we present evidence that Grb14 exerts a physiological negative feedback control on IR catalytic activity in these compartments Under basal conditions, liver Grb14 was recovered mainly in the cytosolic and microsomal fractions, and about 80% of microsomal Grb14 was recovered in high-density subfractions, as was calnexin, a marker of the ER Although final evidence that a pool of Grb14 is localized in the ER awaits morphological confirmation, this localization is not unprecedented Many peripheral membrane proteins, including signaling proteins such as Shc [30], mammalian target of rapamycin [31] and tyrosine phosphatase PTP-1B [32], have been shown to be localized on the cytosolic surface of the ER membrane [33] Localization of Grb14 to the ER may involve the interaction of its PH domain with membrane phosphoinositides, possibly phosphatidylinositol 4,5-bisphosphate, which was ultrastructurally identified in intracellular membranes [34], and shown to bind to Grb14 in an insulin-independent manner [16] The changes in the subcellular distribution of Grb14 induced by insulin are consistent with a model whereby cytosolic Grb14 is recruited to the phosphorylated IR at the plasma membrane and is then translocated to endosomes as a protein complex with the receptor First, as expected from insulin-induced internalization of the IR, insulin led to an increase in Grb14 level earlier in plasma membrane fractions than in endosomal fractions Second, in kinetics and dose– response studies with insulin and superactive insulin analogs, a striking correlation between the IR phosphorylation state and the content of Grb14 in the plasma membrane and ⁄ or endosomal fractions was observed Third, a similar correlation was found upon in vitro dephosphorylation of the activated IR by endogenous phosphatases and, reciprocally, in vivo phosphorylation of the IR by bpV(phen), an inhibitor of tyrosine phosphatases Finally, coimmunoprecipitation experiments showed that, following insulin treatment, Grb14 was associated with the phosphorylated IR in both the plasma membrane and endosomal fractions On the other hand, although at least part of the Grb14–IR complex present in endosomes may derive from internalization of a complex formed at the plasma membrane, direct recruitment of cytosolic Grb14 to activated IR internalized in the endosomes FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4371 Insulin-induced translocation of Grb14 in rat liver B Desbuquois et al AU 150 A – ins ** * + ins p-RCAM-L – ins p-RCAM-L + ins ** * 100 50 min: 16 24 32 ** * ** * min: 16 24 32 B 100 50 – KCl ** * + KCl p- Grb14 C AU p-RCAM-L –KCl p-RCAM-L + KCl – KCl ** + KCl * min: 10 20 30 min: 10 20 cannot be excluded It is noteworthy that the recovery of phosphorylated IRb was about two to four times lower in Grb14 immunoprecipitates than in IR immunoprecipitates (data not shown) As Grb14 is less effectively immunoprecipitated by antibodies against Grb14 than is the IR by antibodies to IR, these data would be compatible with a high proportion of phosphorylated receptors having Grb14 bound to them Structural studies of the kinase domain of the IR in complex with the PIR–BPS of Grb14 and of the Grb14 SH2 domain have allowed us to propose a model for the Grb14–IR interaction The PIR–BPS binds as a pseudosubstrate inhibitor in the substratebinding groove of the kinase, whereas the SH2 domain interacts with phosphorylated tyrosine residues of the IR kinase loop, which help position the PIR–BPS and increase binding affinity [8] Although the interaction of Grb14 with the IR is probably the major determinant of the insulin-induced membrane translocation of Grb14, several lines of evidence suggest that the association of Grb14 via its PH domain with locally produced phosphatidylinositol 3,4,5-trisphosphate may also contribute to this process First, insulin stimulates the association of the regulatory p85 subunit of PI3kinase with both plasma membranes and endosomes 4372 30 Fig Effect of Grb14 recruited in vivo on IR kinase activity in the Golgi ⁄ endosome fraction (A) Golgi ⁄ endosome fractions isolated from three control rats and four rats studied after insulin injection were assayed for RCAM-lysozyme (RCAM-L) phosphorylation over an 8–32 incubation (B, C) Golgi ⁄ endosome fractions from four insulin-injected rats (2 postinjection) were divided into two identical aliquots, one of which was treated with M KCl as described in Experimental procedures Untreated and KCl-treated fractions were assayed for Grb14, IRb and phosphorylated IRb (p-IRb) content (B) and for ability to phosphorylate RCAM-L (C) The figure shows representative immunoblots and results of densitometric measurements expressed as arbitrary units (AU) for phosphorylated RCAM-L (p-RCAM-L) and percentage of control (no KCl treatment) for Grb14, IRb and p-IRb [mean ± SEM of three or four determinations in (A), four to six determinations in (B), four determinations in (C)] Significant effects of insulin in (A) and KCl in (B) and (C) are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001 [28] Second, full-length Grb14, as well as its PH domain, bind D3 phosphoinositides in vitro in a protein–lipid overlay assay, and in retina lysates Grb14 is coimmunoprecipitated by antibodies to phosphatidylinositol 3,4,5-trisphosphate in an insulin-dependent manner [16] Finally, insulin-induced membrane translocation of epitope-tagged Grb14 in HEK 293 cells is inhibited by wortmanin, a PI3-kinase inhibitor [14] Previous studies have shown that the adaptor protein Grb7 interacts with the IR in two-hybrid, GST pull-down and coimmunoprecipitation assays [35] On the other hand, although the EGF receptor binds both Grb14 and Grb7 in cloner of receptor targets (CORT) and GST pull-down assays, neither endogenous Grb14 in DU145 cells nor epitope-tagged Grb14 in transfected HEK 293 cells is recruited by the EGF receptor [36] Consistent with the association of Grb7 with the activated IR in crude liver lysates [35], insulin treatment led to an increase in Grb7 content in liver plasma membrane and endosomal fractions, the kinetics and extent of which were comparable to those observed with Grb14 These findings suggest that the relative affinities of Grb7 and Grb14 for the IR are similarly increased by insulin, and that, like Grb14, Grb7 is recruited by the activated IR at the plasma membrane FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS B Desbuquois et al and undergoes internalization as a complex with the receptor Like insulin, EGF induced an increased content of Grb7 in liver membrane fractions that was temporally correlated with the increased tyrosine phosphorylation of the EGF receptor However, such treatment affected Grb14 only marginally, suggesting a preferential association of the EGF receptor with Grb7, as previously documented [36] Whether the membrane translocation of Grb14 induced by EGF involves the direct interaction of this protein with the activated EGF receptor or the association of the Grb14 PH domain with membrane phosphoinositides remains to be established Inhibition by Grb14 of IR catalytic activity was previously found using GST–Grb14 and IR purified from CHO-IR cells, and poly(Glu,Tyr) as a substrate [7] The inhibitory effect of the GST–Grb14 fusion protein was confirmed in the present study using IR prepared from rat liver and RCAM-lysozyme as a substrate Importantly, partial removal of endogenous Grb14 by KCl treatment from the plasma membrane and endosomal fractions of insulin-treated rats led to a substantial increase in IR tyrosine kinase activity This observation argues for a role of endogenous Grb14, physiologically recruited to the IR after in vivo insulin stimulation Although KCl treatment removed Grb7 from cell fractions to the same extent as Grb14 (data not shown), this is unlikely to contribute significantly to the increased kinase activity of the IR, as Grb7 is both less potent and less efficient than Grb14 in its ability to inhibit in vitro activation of IR tyrosine kinase [7] At the present time, we cannot assess whether the kinase activity of the IRs before KCl treatment represents residual activity of receptors with Grb14 bound to them, the activity of a fraction of receptors lacking bound Grb14, or a combination of both The finding that, following insulin treatment, the majority of liver Grb14 remains associated with the microsomal and cytosolic fractions suggests that, besides its ability to interact with the activated IR and to inhibit IR catalytic activity, Grb14 may play additional roles in the liver As a molecular adaptor, Grb14 interacts constitutively in cells with several partners, including ZIP [10], PDK-1 [14], IRS-1 [13], and tankyrase [15] The binding of ZIP to Grb14, by recruiting protein kinase Cf, enhances the serine phosphorylation of Grb14 and its inhibitory effect on IR kinase and insulin signaling [10] The interaction of Grb14 with PDK-1, an upstream kinase that activates Akt in response to insulin, appears to be required for insulin-induced membrane translocation of PDK-1 and Akt activation in transfected HEK cells [14] On the other hand, the functional significance of the inter- Insulin-induced translocation of Grb14 in rat liver action of Grb14 with tankyrase 2, a poly(ADPribose)polymerase closely related to tankyrase, is still unclear [15] Conceivably, it could explain the colocalization of these two proteins in a low-density microsomal fraction in DU 145 prostate cancer cells and in transfected HEK 293 cells Future identification of other Grb14 partners should lead to interesting new information on the role of this adaptor protein The endosomal colocalization and ⁄ or cointernalization of the activated IR and Grb14 raises the question as to whether Grb14 could, directly or via its partners, regulate IR traffic and ⁄ or degradation of the IR However, despite a recent report showing the involvement of Grb10 in the ubiquitination and proteasomal degradation of the IR in transfected cells [37], there is so far no evidence to support a physiological role of Grb10 and Grb14 in the regulation of IR degradation [5] Additional studies are thus needed to determine whether Grb14 is involved in insulin-induced endocytosis and ⁄ or degradation of the IR In summary, our results show that, whereas Grb14 is located in the basal state mainly in high-density microsomal elements and the cytosol, upon insulin stimulation endogenous Grb14 is in part translocated to plasma membranes and endosomes, where it is associated with phosphorylated IRb Our data also suggest that Grb14 recruited by the IR at the plasma membrane undergoes cointernalization with the receptor Finally, evidence is presented that Grb14 exerts negative feedback control on IR catalytic activity in these compartments, indicating that it is a physiological regulator of liver insulin signaling, and that it may represent an interesting molecule with which to regulate liver insulin action Experimental procedures Materials Porcine insulin, recombinant human insulin, [GluA13, GluB10]insulin and [HisA8,HisB4,GluB10,HisB27]insulin were gifts from Novo Nordisk A ⁄ S (Bagsvaerd, Denmark) Mouse EGF (receptor grade) was from Collaborative Biomedical (Bedford, MA, USA) Monoclonal and polyclonal antibodies against IRb, polyclonal antibodies against EGF and polyclonal antibodies against Grb7 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Monoclonal antibody against phosphotyrosine (clone 4G10) was from Upstate Biotechnology Incorporated (Lake Placid, NY, USA) Monoclonal antibody against EEA1 (clone 14) was from BD Transduction Laboratories (San Jose, CA, USA) Monoclonal antibody against Na+ ⁄ K+-ATPase a-subunit (clone M7-PB-E9) and polyclonal antibodies against calnex- FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4373 Insulin-induced translocation of Grb14 in rat liver B Desbuquois et al in were from Sigma-Aldrich (St Louis, MO, USA) GST– Grb14 and rabbit polyclonal antibodies against Grb14 have been described previously [6] Horseradish peroxidase-conjugated goat anti-(rabbit IgG) and goat anti-(mouse IgG) were from Biorad (Hercules, CA, USA) bpV(phen) was a gift from B Posner (McGill University, Montreal, Quebec, Canada) Reduced and carboxyamidomethylated lysozyme was a gift from R Kohanski (Mount Sinai School of Medicine, New York, NY, USA) [125I]TyrA14 insulin, receptor grade, was from Perkin Elmer Life Sciences (Waltham, MA, USA) Nitrocellulose membranes were from BioRad and Schleicher and Schull (Duren, Germany) The enhanced ă ă chemiluminescence detection kit was from Amersham-Pharmacia (Little Chalfont, UK) and Pierce Biotechnology Inc (Rockford, IL, USA) WGA–Sepharose 6MB (WGA– Sepharose), protein G–Sepharose, protein A–Sepharose and other chemicals were purchased from Sigma-Aldrich Animals and injections Animal studies were performed according to the French Guidelines for Use and Care of Experimental Animals Male Sprague–Dawley rats (body weight, 190–210 g) were obtained from Elevage Janvier (Le Genest Saint Isle, France) Animals were housed in an animal facility with 12 h light cycles, fed ad libitum, and fasted overnight (16–18 h) before study After pentobarbital anesthesia, rats received an intravenous injection (penis vein) of insulin or insulin analogs (30 lg unless otherwise stated), EGF (100 lg) or bpV(phen) (1.2 lmol), diluted in 0.5 mL of NaCl ⁄ Pi containing 0.1% BSA At the indicated times, the liver was rapidly excised through a median incision, and minced in ice-cold 0.25 m sucrose Subcellular fractionation Livers were homogenized in 0.25 m sucrose containing 2.5 mm NaF, mm Na3VO4, mm benzamidine and 0.5 mm phenylmethanesulfonyl fluoride (about mL per gram of tissue) using a Dounce homogenizer (12 up-anddown strokes of the loose-fitting pestle) Following dilution to about 70 mL and removal of fibrous and undisrupted tissue by centrifugation at 250 gav for min, the homogenate was subjected to differential centrifugation, generating nuclear (1500 gav for 10 min), mitochondrial (3200 gav for 10 min), light mitochondrial–microsomal (150 000 gav for 45 min) and supernatant fractions In some experiments, an additional centrifugation step was introduced after sedimentation of the mitochondrial fraction, generating separate light mitochondrial (25 000 gav for 10 min) and microsomal (150 000 gav for 45 min) fractions Flotation through discontinuous sucrose density gradients was performed to isolate plasma membranes from the nuclear fraction (1.42 ⁄ 0.25 m sucrose interface) and Golgi ⁄ endosomes from the light mitochondrial–microsomal fraction (1 ⁄ 0.25 m 4374 sucrose interface), essentially as described in [38] and [18], respectively Fractions were assayed for protein content according to the method of Lowry et al [39], using BSA as standard Measurement of organelle marker enzymes showed that enrichments of the plasma membrane fraction in 5¢-nucleotidase and alkaline phosphodiesterase (plasma membrane markers) and of the Golgi ⁄ endosome fraction in galactosyltransferase (Golgi marker), ATP-dependent acidification (endosomal marker) and in vivo internalized [125I]insulin were as described in previous reports [17,38,40,41] Fractions were analyzed for Grb14, IRb and phosphorylated IRb by western blotting In some experiments, the microsomal fraction was subfractionated by analytical centrifugation (85 000 gav for 15 h) through a linear sucrose density gradient (1.03–1.25 gỈmL)1) Subfractions were assayed for density by refractometry and for content of the following organelle markers: calnexin (ER); Na+ ⁄ K+-ATPase (plasma membrane); and EEA1 (endosomes) Extraction and immunoprecipitation of membrane-associated Grb14 and insulin receptor Subcellular fractions prepared as described above were resuspended in 25 mm Hepes buffer (pH 7.6) containing mm Na3VO4, 2.5 mm NaF, mm phenylmethanesulfonyl fluoride, mm benzamidine, lgỈmL)1 pepstatin A, lgỈmL)1 leupeptin, lgỈmL)1 aprotinin, and, when indicated, 1–2 m KCl, m NaCl, m urea or 0.1 m Na2CO3 (pH 11.5) After 60–75 at °C, suspensions were centrifuged at 100 000 g for 45 Pellets were resuspended into Laemmli sample buffer and analyzed for Grb14 and IRb content by western blotting For immunoprecipitation, cell fractions were resuspended in Hepes buffer containing 100 mm NaCl, phosphatase and protease inhibitors as indicated above, and 0.5–1% Triton X-100 After 20 at °C, suspensions were centrifuged at 150 000 g for 60 min, and supernatants were incubated for 16 h at °C with polyclonal antibodies against Grb14 or monoclonal antibodies against IRb Protein A–agarose or protein G–Sepharose, respectively, was then added, and after rotatory shaking for h, beads were sedimented and washed four times in 25 mm Hepes buffer, 100 mm NaCl, mm Na3VO4 and 0.1% Triton X-100 Immunoprecipitated Grb14 and IRb were analyzed by western blotting In vitro dephosphorylation of IR phosphorylated in vivo Plasma membrane and Golgi ⁄ endosome fractions of insulininjected rats (2 postinjection) prepared in the absence of phosphatase inhibitors were resuspended in 25 mm Hepes buffer (pH 7.6) containing 0.15 m NaCl, mm EDTA, mm dithiothreitol, 0.5 mm phenylmethanesulfonyl fluoride, FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS B Desbuquois et al 0.2 mgỈmL)1 BSA, 0.05 mgỈmL)1 bacitracin, lgỈmL)1 aprotinin, lgỈmL)1 leupeptin, and 2.5 lgỈmL)1 pepstatin A After incubation at 37 °C for 5–40 in the absence or presence of 0.2 mm bpV(phen), suspensions were supplemented with KCl (0.5 m final concentration) at °C and immediately centrifuged at 150 000 g for 40 The total incubation mixture and the pellet were analyzed for phosphorylated IRb and Grb14 content by western blotting IR purification and quantitation IR was partially purified from Triton X-100-soluble extracts of cell fractions by adsorption on WGA–Sepharose Extracts (0.5–1 mL, 2–5 mg of protein) were incubated on a rotatory shaker with WGA–Sepharose (1 : 10, v ⁄ v) for h at °C, and glycoproteins were eluted with 0.3 m N-acetylglucosamine in 25 mm Hepes buffer (pH 7.6), 0.1% Triton X-100, and 0.2 mm phenylmethanesulfonyl fluoride IR contents in crude extracts and WGA eluates were quantitated by measurements of specific [125I]insulin binding (0.02 nm) as previously described [17] Assay of IR tyrosine kinase activity This assay was based on the ability of IR to phosphorylate RCAM-lysozyme, a high-affinity substrate [42] For assay of in vitro insulin-stimulated kinase activity, WGA eluates from a light mitochondrial–microsomal fraction prepared from control rat liver (about fmol of insulin bound) were preincubated in the absence or presence of insulin (0.5 lm) for h at 23 °C A phosphorylation mix (50 mm ATP, mm CTP, mm MnCl2, mm MgCl2) was then added in the presence or absence of GST–Grb14 at the indicated concentration After 30 at 23 °C, RCAM-lysozyme (6 lm) was added and incubations were allowed to proceed further for 10–40 min, until the reaction was stopped by the addition of 4· Laemmli sample buffer For assay of in vivo insulin-stimulated kinase activity, intact Golgi ⁄ endosome fractions or WGA eluates of light mitochondrial– microsomal and plasma membrane fractions prepared from insulin-injected rat liver and containing equal amounts of IR (0.5–2 fmol of insulin bound) were incubated with the phosphorylation mix and RCAM-lysozyme added together at the same concentrations as above for 10–40 at 23 °C RCAM-lysozyme phosphorylation was monitored by western blotting using antibody against phosphotyrosine Western blotting Equal amounts of cell fraction proteins (10 lg unless otherwise indicated) were supplemented with Laemmli sample buffer, subjected to SDS ⁄ PAGE analysis (15% acrylamide for RCAM-lysozyme, 8–10% acrylamide for other proteins), and immunodetected with the indicated antibod- Insulin-induced translocation of Grb14 in rat liver ies Immunoreactive bands were revealed using an enhanced chemiluminescence detection detection kit The signals identified on the 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in transfected HEK cells [14] On the other hand, the functional significance of the inter- Insulin-induced translocation of Grb14 in rat liver action of Grb14. .. residues of the IR kinase loop, which help position the PIR–BPS and increase binding affinity [8] Although the interaction of Grb14 with the IR is probably the major determinant of the insulin-induced. .. assessed the ability of insulin administered in vivo to increase the level of RCAM-lysozyme in cell fractions, and then assayed the fractions of insulin-injected rats for Grb14, IR and phosphorylated