Tài liệu Báo cáo khoa học: Insulin-dependent phosphorylation of DPP IV in liver Evidence for a role of compartmentalized c-Src ppt

Insulin-dependent phosphorylation of DPP IV in liver Evidence for a role of compartmentalized c-Src Nicolas Bilodeau1, Annie Fiset1, Guy G Poirier2, Suzanne Fortier1, Marie-Claude Gingras3, ´ Josee N Lavoie3 and Robert L Faure1 ´ Pediatric Research Unit, CRCHUL ⁄ CHUQ, Faculty of Medicine, Laval University, Quebec, Canada ´ Quebec Proteomic Center, CRCHUL ⁄ CHUQ, Faculty of Medicine, Laval University, Quebec, Canada ´ Cancer Research Center, CRHDQ ⁄ CHUQ, Faculty of Medicine, Laval University, Quebec, Canada Keywords c-Src; DPP IV; endosomes; tyrosine phosphorylation, subcellular fractionation Correspondence R.L Faure, Pediatric Research Unit (Cell Biology Laboratory), Room 9800, CHUL Medical Research Center, 2705 Laurier ´ Boulevard, Quebec, QC, G1V 4G2, Canada Fax: +1 418 654 2753 Tel: +1 418 656 4141, extn 48263 E-mail: robert.faure@crchul.ulaval.ca (Received 16 November 2005, revised 23 December 2005, accepted January 2006) doi:10.1111/j.1742-4658.2006.05125.x Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model aimed at elucidating protein sorting signals We identify here, by MS, several tyrosine-phosphorylated proteins in a rat liver Golgi ⁄ endosome (G ⁄ E) fraction including DPP IV We show that a pool of DPP IV is tyrosinephosphorylated Maximal phosphorylation was observed after following intravenous insulin injection DPP IV coimmunoprecipitated with the cellular tyrosine kinase Src (c-Src) with maximal association also observed after following insulin injection DPP IV was found phosphorylated after incubation of nonsolubilized G ⁄ E membranes with [c-32P]ATP The c-Src inhibitor PP2 inhibited DPP IV phosphorylation Oriented proteolysis experiments indicate that a large pool of c-Src is protected in G ⁄ E fractions Following injection of the protein-tyrosine phosphatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40% both in plasma membrane and G ⁄ E fractions In the fraction designated Lh, DPP IV levels decreased by 50% 15 following insulin injection Therefore, a pool of DPP IV is tyrosine-phosphorylated in an insulindependent manner The results suggest the presence of a yet to be characterized signalling mechanism whereby DPP IV has access to c-Srccontaining signalling platforms Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) is a type II membrane glycoprotein that is expressed in a variety of cell types [1] DPP IV belongs to a serine class of proteases exhibiting a restricted substrate specificity which favours release of Xaa–Pro or Xaa–Ala dipeptides from the N terminus of proteins [2,3] Within a cell, DPP IV is transported with high precision [4] and is synthesized with an uncleaved signal sequence that functions as a membrane-anchoring domain [5] It has been shown that cysteine residues and conformational changes are important components that facilitate sorting [6] Glycosylation is crucial [7,8] and recent data have highlighted the importance of both glycosylation and the lipid microenvironment [9] Among the proteins DPP IV may bind are: adenosine deaminase [10], the kidney Na+ ⁄ H+ exchanger [11], the protein-tyrosine phosphatase (PTP) CD45 [12] and the tyrosine kinase of the cellular Src (c-Src) family p56lck [13] In hepatocarcinoma cells, kinase activity was detected in DPP IV immunoprecipitates [14] In liver parenchyma, immunohistochemistry studies have shown that DPP IV is located mainly in the bile canalicular membrane [1] In the renal brush border, DPP IV is located in the microvilli and not in the Abbreviations bpV(phen), bisperoxovanadium 1,10-phenanthroline; c-Src, cellular tyrosine kinase Src; Cyt, cytosol; DPP IV, dipeptidyl peptidase IV; ER, endoplasmic reticulum; G ⁄ E, Golgi ⁄ endosome; Gi and Gh, Golgi intermediate and heavy endosomes; GLP-1, glucagon-like peptide-1; IR, insulin receptor; Li and Lh, light intermediate and heavy endosomes; PM, plasma membrane; PTP, protein-tyrosine phosphatase; PY, phosphotyrosine; WGL, wheat germ lectin 992 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS N Bilodeau et al Regulation of DPP IV trafficking coated pit microdomain [15] In hepatocytes, DPP IV is transported rapidly from the basolateral membrane to the apical membrane by endocytosis [16] In Madin–Darby canine kidney (MDCK) cells, a study of chimeric forms of DPP IV has shown that the luminal domain of DPP IV carries dominant apical sorting information while the short cytoplasmic tail and the transmembrane domain contain competing basolateral sorting information [17] From one cell type to another, DPP IV is sorted by different mechanisms Hence in hepatocytes, DPP IV reaches the apical membrane by transcytosis; while in MDCK cells, apical and basolateral proteins are segregated from each other in the trans-Golgi network [18] DPP IV exopeptidase activity is involved in a variety of regulatory processes including chemokine regulation [19] and maintenance of physiological glucose homeostasis [20] Knockout mice lacking the gene for DPP IV show enhanced insulin secretion and accelerated clearance of blood glucose coincident with increased endogenous levels of both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide [21] Pharmacological inhibition of DPP IV activity increases insulin production and improves glucose control in diabetic animals [20,22–24] as well as in humans [25] Apart from its proteolytic activity, DPP IV is also engaged in multiple functions depending on its ability to bind to extracellular matrix [26] Hence, DPP IV may be involved in normal tissue architecture and growth patterns [27] DPP IV binding to type collagen and fibronectin has been demonstrated [28,29] and DPP IV can be considered as a cell surface adhesion receptor for fibronectin [30] with possible implications in cell migration and metastasis [27,30,31] Also, DPP IV functions in triggering the immune response [19,32] Previously, we reported the presence of a series of tyrosine-phosphorylated proteins in a wheat germ lectin (WGL) subfraction prepared from a hepatic endosomal fraction [33] Using MS, we identified the most abundant tyrosine-phosphorylated proteins first We show here that one of these proteins, DPP IV, is tyrosine-phosphorylated in a ligand-dependent manner Results MS analysis of major proteins purified by antiphosphotyrosine (PY) affinity column chromatography We have reported previously the presence of several tyrosine-phosphorylated proteins in WGL affinity column chromatography eluates prepared from a combined fraction of endosomes and Golgi elements (G ⁄ E) isolated from liver parenchyma [33] Identification, by finger printing MS, of the nine major proteins purified by anti-PY affinity column chromatography, stained with Coomassie blue, indicates that of these proteins, four [insulin receptor (IR), LAR, ER-60, SAPAP-3] are related to signalling events (Table 1) Identification of the IR was expected, as it is readily concentrated by the WGL affinity column chromatography step [34] The PTP LAR is less well characterized However previously, it was reported as being a regulator of the IR [35–37] The cysteine protease ER-60 was purified first from the endoplasmic reticulum (ER) of rat liver [38] ER-60 was found to be regulated by insulin and PTP-1B [39] SAPAP-3 is a signalling protein found associated with tyrosine phosphorylation events in membrane subdomains [40–43] The other major proteins identified were: the chaperone BIP, the Table MS analysis of major endosomal proteins purified by anti-PY affinity column chromatography Endosomal glycoproteins were eluted from anti-PY affinity column Major proteins of: 220, 180, 117, 110, 106, 79, 61, 60, 38 kDa stained with Coomassie blue were excised from gels after SDS ⁄ PAGE and subjected to proteolysis and MALDI-TOF analysis Data were analysed using MASCOT; accession numbers for each scored protein in the NCBI nonredundant databank are listed; Sequence coverage indicates the percentage of the identified protein covered by the sequences of identified peptides; m indicates the molecular mass of each protein predicted from the sequence (pred.) or experimentally observed in the gel (expt.) Protein Accession no Name Sequence coverage (%) mpred ⁄ expt (Da) NP_062122 NP_058767 NP_019369 P97838 A39914 P06761 NP_059015 NP_445770 AAF31764 Protein-tyrosine phosphatase, receptor-type, F: LAR Insulin receptor (precursor) Inter-alpha-inhibitor H4 heavy-chain SAPAP-3 DPP IV, membrane-bound form precursor BIP ER-60 protease Hemopexin Beta-1 adducin 19 21 14 29 12 24 198 640 ⁄ 180 000 159 420 ⁄ 220 700 103 930 ⁄ 117 400 106 970 ⁄ 110 000 91 650 ⁄ 106 400 72 500 ⁄ 79 000 57 030 ⁄ 61 100 52 010 ⁄ 60 000 20 990 ⁄ 38 000 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 993 Regulation of DPP IV trafficking N Bilodeau et al protease inhibitor inter-alpha-inhibitor H4, the transporter hemopexin and beta-1 adducin—a component of the cytoskeleton DPP IV phosphorylation in the G/E fraction Assessment of DPP IV distribution in our hepatic fractions, using anti-DPP IV (26C), showed that  80% of the amount of DPP IV detected was located in the G ⁄ E fraction No DPP IV signal was observed in the cytosol (Cyt) The remaining portion ( 20%) was present in the plasma membrane (PM) fraction (Fig 1) We then verified DPP IV phosphorylation in the G ⁄ E fraction by use of an anti-PY IgG (4G10) Following insulin injection (1.5 lgỈ100 g)1 body weight), the IR was readily internalized as originally described [44] (Fig 2A, upper panel) The IR tyrosine phosphorylation and autophosphorylation activity were both maximal before 15 postinsulin injection Under these circumstances, analysis of DPP IV immuno-complexes revealed a signal detected by the anti-PY IgG This signal increased after postinjection (Fig 2A, lower panel) Sequence analysis (LC-MS ⁄ MS) of the excised immunoprecipitated 110-kDa band (SYPRO Ruby staining) confirmed unambiguously that this protein was DPP IV (Fig 2B) Previously, DPP IV was thought to be associated with c-Src-like kinases in lymphocytes [45] In addition, another study has shown that c-Src is present in endosomes of fibroblasts [46] We detected c-Src in DPP IV immunocomplexes, with an increased signal observed 15 following insulin injection (Fig 2A, lower panel) We then verified DPP IV phosphorylation in vitro Following incubation of the nonsolubilized G ⁄ E fraction in the presence of [c-32P]ATP, and immunoprecipitation with anti-DPP IV IgG, a phosphorylated protein of appropriate apparent molecular mass (110 kDa) was observed (Fig 3A) DPP IV 32P phosphorylation was alkali resistant Insulin injection (1.5 lgỈ100 g)1 body weight) also increased DPP IV phosphorylation by twofold above basal levels by 15 When the PTP inhibitor bisperoxovanadium 1,10-phenanthroline [bpV(phen)] was added to the incubation medium, DPP IV phosphorylation was enhanced at (control) and min, but not at 15 postinsulin injection (Fig 3A) In order to link this phosphorylation event with c-Src catalytic activity, samples were incubated either in the presence or absence of the c-Src inhibitor PP2 [47] prior to DPP IV immunoprecipitation The results show that DPP IV phosphorylation was readily abolished when PP2 was added to the incubation medium Also, we note the presence of an associated band around 56 kDa, presumably c-Src itself or a putative substrate (Fig 3B) Localization of c-Src in G/E fractions Fig Distribution of DPP IV in hepatic subcellular fractions The Cyt, PM and G ⁄ E fractions were submitted directly to immunoblot analysis (80 lg protein; 7.5% resolving gel) using the anti-DPP IV (26C) IgG Results are also presented as a percentage of the total amount of DPP IV detected, calculated from the yields measured for each fraction (see Experimental procedures) This experiment was repeated three times with similar results; mean ± SD are shown 994 We assessed further c-Src localization in our fractions Permeabilization of the G ⁄ E membranes with Triton X-100 resulted in loss of several proteins, most notably albumin (66 kDa), indicating that during permeabilization, soluble luminal proteins were washed out (Fig 4B) A number of proteins ‘disappeared’ following treatment with proteinase K alone while the 66-kDa albumin was protected The 66-kDa albumin band almost completely disappeared when permeabilized membranes were treated with proteinase K along with other bands including the 110-kDa band (presumably DPP IV) (Fig 4B) The quality of the permeabilization step was also assessed by electron microscopy The G ⁄ E fraction mainly contains typical lipoproteinfilled tubulovesicular elements as well as 70–400-nm diameter vesicles [48] (Fig 4C) The permeabilization step resulted in empty vesicular elements (Fig 4D) Therefore, while partial solubilization of membrane FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS N Bilodeau et al Fig Insulin-dependent tyrosine phosphorylation of DPP IV and its association with c-Src in the G ⁄ E fraction (A) Rats were injected with insulin [1.5 lgỈ100 g)1 body weight (bw)] The G ⁄ E fraction was isolated at the indicated times postinjection (Upper panels) Proteins were separated by SDS ⁄ PAGE (80 lg, 7.5% resolving gel); the IR was detected by using either the anti-IR b-subunit IgG or the anti-PY IgG (4G10) Autophosphorylation of the IR (95 kDa 32 P panel) was achieved by incubating aliquots (30 lg protein) with [c-32P]ATP Following centrifugation, the pellet was solubilized and proteins immunoprecipitated using the anti-IR b-subunit IgG Immunoprecipitates were separated by SDS ⁄ PAGE and gels were subjected to alkali treatment and autoradiography (Lower panels) DPP IV immunoprecipitation: Aliquots of G ⁄ E fraction (200 lg protein) were immunoprecipitated using an anti-DPP IV IgG (MA-2607) Immunoprecipitated proteins were separated by SDS ⁄ PAGE (10% resolving gel) Membranes were incubated with anti-DPP IV (26C), anti-PY (4G10) or anti-c-Src IgG (pieces of the same membrane) The signals were submitted to densitometric analysis, and the results were expressed as a percentage of the maximum signal Each value represents the mean ± SD of three independent experiments (B) Amino acid sequence of rat DPP IV (NCBI accession number NP_36921, Swiss-Prot P14740) The immunoprecipitated 110-kDa band, stained with SYPRO Ruby (left panel), was excised and subjected to proteolysis Rat DPP IV peptide sequences that were identified by LC-MS ⁄ MS are boxed Hashed boxes indicate that a common sequence is present in two different peptides Results are representative of three independent experiments elements by 0.1% Triton X-100 is possible, we were still in a position to address the question of the orientation of c-Src We used the endosomal fraction G ⁄ E as well as the Golgi intermediate and heavy endosomes (Gi ⁄ Gh) and the light intermediate and heavy endosomes (Li ⁄ Lh) fractions [49] Li is a homogeneous fraction containing late endosomes and a negligible amount of the marker enzymes sialyl transferase and galactosyl transferase The Gi fraction representing early endosomes is contaminated ( 50%) by Golgi elements The other fractions (Lh, Gh) contain less characterized endosomes and are rich (80%) in Golgi elements As above, the fractions were subjected to proteinase K digestion after a membrane permeabilization step (Triton X-100 0.1%) In the G ⁄ E, Lh and Gh fractions, c-Src was detected easily in all conditions (control, Triton 0.1%, proteinase K) except for the permeabilized membranes subjected to proteolysis (Triton 0.1% + proteinase K) (Fig 4A) In contrast, c-Src was not protected from proteinase K degradation in the PM fraction for both conditions (nonpermeabilized or permeabilized) (Fig 4A) Using the same assay, we also determined that DPP IV signal disappeared only when permeabilized membranes (G ⁄ E) were submitted to proteolysis (Fig 4A) Therefore, the results indicate that in endosomal fractions, c-Src is largely protected from exogenously added proteinase K Regulation of DPP IV trafficking A B DPP IV levels following stimulation with insulin and bpV(phen) In order to examine changes in DPP IV levels following insulin stimulation, rats were injected with the PTP inhibitor bpV(phen) 16 h and 30 prior to insulin injection and isolation of the G ⁄ E and PM fractions No effect on DPP IV level was detected following insulin injection However, DPP IV levels, as detected by immunoblotting (26C), decreased by  40% when bpV(phen) was injected (Figs 5A and C) Such a FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 995 Regulation of DPP IV trafficking N Bilodeau et al A Fig In vitro phosphorylation of DPP IV (A) Rats were injected with insulin (1.5 lgỈ100 g)1 body weight) The G ⁄ E fraction was isolated at the indicated times postinjection and aliquots (100 lg protein) were incubated with [c-32P]ATP in the presence or absence of 100 lM bpV(phen) They were solubilized and proteins immunoprecipitated using the anti-DPP IV IgG (MA-2607) The immunoprecipitates were separated by SDS ⁄ PAGE (10% resolving gel) and gels were subjected to autoradiography before and after alkali treatment (B) The G ⁄ E fraction was isolated 15 following insulin injection and was subjected to phosphorylation, in the presence or absence of the c-Src inhibitor PP2 (10 lM), and then immunoprecipitated as above Results are representative of three independent experiments B decrease was also observed for the PM fraction (Figs 5B and C) In order to assess whether this effect on DPP IV level was coincident with a phosphorylation event, we used an antibody which reconizes residues phosphorylated by Src kinases (aPY-42 antibody) The results revealed the coincident hyperphosphorylation of a 100-kDa band This is consistent with the view that a c-Src-dependent phosphorylation event had occurred (Fig 5A) Further fractionation was then used to refine our assessment of DPP IV and c-Src distribution Following insulin injection, IR accumulation and tyrosine phosphorylation was observed for all examined fractions, most evidently for the Lh, Gi and Gh fractions (Fig 6A) The results show that both c-Src and DPP IV are also located mainly in the Lh and Gh fractions No changes in DPP IV levels are observed following insulin injection, except at 15 postinjection where the signal declines significantly by more than 50% in the Lh fraction (n ¼ 4; P < 0.001) (Fig 6B) No significant changes in c-Src levels were observed Discussion Previously, we have reported the presence of a series of tyrosine-phosphorylated proteins partially purified from hepatic endosomes [33] Following anti-PY affinity column chromatography, a systematic identification performed first on the more abundant protein species reveals here that one of these is DPP IV (Table 1) 996 DPP IV is well represented in the G ⁄ E fraction where it is found even more abundantly than in the PM fraction (Fig 1) At the cell surface, DPP IV is located mainly in the bile canalicular domain This relative abundance in the G ⁄ E fraction may be explained by the diverse representation of the three major domains (sinusoidal, lateral, bile canalicular) of the hepatocytes present in the PM fraction [50] To the best of our knowledge, tyrosine phosphorylation of DPP IV has not been reported before In addition, the results show that DPP IV phosphorylation is regulated, thus defining a new insulin-dependent effect The observation that maximal DPP IV phosphorylation (after postinsulin injection) does not correspond with maximal IR tyrosine phosphorylation is consistent with the fact that DPP IV is not phosphorylated by the IR Previous studies performed with immune cells have shown that DPP IV is associated with the c-Src related tyrosine kinase p56lck [13], despite a short (6 residues) cytoplasmic tail Investigation here for a role of c-Src in DPP IV phosphorylation indeed reveals that not only is c-Src associated with DPP IV, but its association is dependent of insulin with maximal association coincident with maximal tyrosine phosphorylation of DPP IV in vivo (Fig 2) Insulin-dependent DPP IV phosphorylation is also readily detected in vitro and is furthermore inhibited by the c-Src inhibitor PP2 This confirms that DPP IV is tyrosine-phosphorylated and further supports the idea that c-Src is involved in DPP IV phosphorylation FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS N Bilodeau et al A C Regulation of DPP IV trafficking B D Fig Oriented proteolysis: The tyrosine kinase c-Src is protected from exogenously added protease in endosomal fractions (A, upper panels) The G ⁄ E, Lh, Gh and PM fractions (100 lg protein) were incubated in the presence or absence of Triton X-100 and proteinase K before immunoblotting with the anti-c-Src IgG The same experiment (lower panel) was performed using the G ⁄ E fraction and the anti-DPP IV (26C) IgG Results shown are typical of three independent experiments (B) The G ⁄ E fraction (100 lg protein) was incubated in the presence or absence of Triton X-100 (0.1%) and proteinase K before staining with SYPRO Ruby The 66-kDa (albumin) and 110-kDa bands are shown with arrows (C) Electron microscopy of the G ⁄ E fraction that was purified and processed as described in Experimental procedures Note the presence of typical tubulovesicular structures as well as lipoprotein-filled vesicles (70–400 nm) (Scale bar ¼ 200 nm) (D) Electron microscopy of the G ⁄ E fraction treated with 0.1% Triton X-100 as described in Experimental procedures Note the absence of typical lipoproteinfilled (dark) vesicles (Scale bar ¼ 200 nm) FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 997 Regulation of DPP IV trafficking N Bilodeau et al A A B B C Fig Effect of the PTP inhibitor bpV(phen) on DPP IV levels in G ⁄ E and PM fractions (A) bpV(phen) was injected (0.3 mgỈ100 g)1 body weight) 16 h and 30 before the injection of insulin (1.5 lgỈ100 g)1 body weight) Endosomes (G ⁄ E) were isolated at the noted times and were submitted directly to immunoblot analysis (100 lg protein; 7.5% resolving gel) using the anti-DPP IV (26C) IgG or the aPY-42 antibody (B) bpV(phen) was injected into rats 16 h and 30 before liver excision The PM fraction was prepared as described and immunoblotted as in (A) (C) DPP IV signals obtained in (A) and (B) were submitted to densitometric analysis, and the results were expressed as a percentage of the maximum signal, respectively Means ± SD are shown (n ¼ 11 in G ⁄ E fraction, n ¼ in PM fraction) Fig DPP IV level is decreased by insulin in the Lh subfraction (A) Following insulin injection (1.5 lgỈ100 g)1 body weight), fractions (Li, Lh, Gi and Gh) were isolated at the indicated times Aliquots were immunoblotted (40 lg protein; 7.5% resolving gel) using an anti-IR b-subunit IgG (95 kDa, b-subunit panel) or an anti-PY IgG (95 kDa aPY panel) (B) Immunoblot analysis of c-Src and DPP IV (80 lg protein; 7.5% resolving gel) using anti-c-Src and anti-DPP IV (26C) IgG (pieces of the same membrane) DPP IV signals are expressed as a percentage of the maximal value; means ± SD are shown (DPP IV: *Lh: P < 0.01, vs 15 min, n ¼ 4; Student’s t-test) c-Src is known to be located in endosomes [46] and Golgi elements [51] where it is thought to regulate retrograde transport [52] In lipid raft compartments, c-Src plays a role in signalling events [53] It is also clear, either by immunofluorescence microscopy of endogenous or transfected c-Src (data not shown) or by immunoblotting of endosomal fractions (G ⁄ E, Lh, Gh and Gi, Li) (Figs 2, and 6), that c-Src is distributed in the vacuolar system Our results also provide the first evidence that c-Src has access to the lumen Indeed, oriented proteolysis indicates that a pool of c-Src is protected from exogenous proteolysis There are no known translocation motifs in c-Src, and the mechanism of c-Src translocation is yet to be characterized However, the dynamic role of the translocons [54,55] and membrane restructuring enzymes [56] in protein targeting is beginning to be perceived For instance, one striking example of c-Src location inside one organelle has been reported for the inner membrane of mitochondria of osteoclasts [57] Moreover, there are 50 tyrosine residues in the sequence of rat DPP IV, all of which are located in the lumen 998 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS N Bilodeau et al The results show also that DPP IV levels are affected following stimulation with a potent PTP inhibitor [bpV(phen)] The observation that this effect coincides with the hyperphosphorylation of a 110-kDa band revealed by the aPY-42 antibody, supports the idea that DPP IV tyrosine phosphorylation is related to DPP IV levels (Fig 5) Hence, in the presence of bpV(phen) a relatively small pool of DPP IV can be continuously diverted towards lysosomal compartments for degradation Alternatively, a soluble form of DPP IV can be released from the cell surface In this regard, an increased circulating concentration of the soluble form of DPP IV may result in an increased proteolysis of GLP-1 peptides and thus decreased insulin secretion [20] The insulin-dependent phosphorylation of DPP IV observed here may thus provide a regulatory loop among a target organ (liver) and the insulin secreting cells Deregulation of this DPP IV phosphorylation mechanism may have implications for the homeostasis of circulating GLP-1 levels and diabetes However, we did not detect significant changes in circulating DPP IV activity, using Gly-Pro-p-nitroanilide as a substrate in our acute conditions of stimulation (insulin dose: 15 lgỈ100 g)1, control: 0.122 ± 0.013 mL)1, n ẳ 25; bpV(phen): 0.124 0.0045 UặmL)1, n ẳ 27; U ¼ amount of enzyme which hydrolyses lmol substratmin)1) It remains possible that more chronic alteration of circulating insulin results in significant changes of circulating DPP IV Indeed, further fractionation demonstrated the presence of a significant decrease in DPP IV levels in the Lh fraction thus revealing that DPP IV is subject to ligand control in a precise microenvironment This is also consistent with the fact that the IR, DPP IV and c-Src are present mainly in the same fractions (Lh and Gh) This therefore points to the importance of subjecting these fractions to further purification and biochemical characterization in order to gain a more detailed understanding of this process In conclusion, the results presented here demonstrate that DPP IV is tyrosine-phosphorylated in an insulindependent manner in hepatic endosomal fractions The possible involvement of luminal c-Src in this process suggests the presence of a mechanism whereby DPP IV en route along with the endocytosed IR can reach compartments where c-Src is present Regulation of DPP IV trafficking Transduction Laboratories (rabbit polyclonal, 188430) The hybridoma (26C, clone 287) expressing the monoclonal antibody against DPP IV was kindly provided by M.G Farquhar (University of California, San Diego, CA) and was used for immunoblotting experiments The antiDPP IV used for immunoprecipitation experiments was from Endogen (Woburn, MA) A mixture of antibodies against c-Src : (N-16, sc-19 and SRC2, sc-18, Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) was used for immunoblots The monoclonal anti-PY IgG (4G10) used to detect IR b-subunit phosphorylation was purchased from Upstate (Lake Placid, NY) The antiphospho-E4orf4 (Y42) 42-2 was produced by injecting rabbits with a chemically synthesized peptide comprising phosphorylated Tyr42 (HEGVY[PO3H2]IEPEARGRLC) coupled to mcKLH (Imject Mariculture Keyhole Limpet Hemocyanin, Pierce Biotechnology Inc., Rockford, IL), following recommendations of the manufacturer This phosphosite is the major residue phosphorylated by Src kinases on the adenoviral protein E4orf4 [58,59] The serum was absorbed on immobilized phosphorylated peptide using a SulfoLink Kit (BioLynx, Brockville, ON) and blocked against an excess of nonphosphorylated peptide during immune detection The specificity of the purified 42-2 antibody was tested by western blot analysis of E4orf4 immune complexes and total cell lysates from cells transfected with wild type Flag-E4orf4 as compared to mutant Flag-E4orf4 (Y42F) alone, or together with c-Src or v-Src to induce maximum tyrosine phosphorylation of Ad2 E4orf4 and of Src substrates as well The antibody reacted specifically with wild-type Ad2 E4orf4 but not with mutant E4orf4 (Y42F) and the signal was proportional to the level of tyrosine phosphorylation This antibody does not react with the tyrosine-phosphorylated IR but it does react against other PY proteins that are selectively modulated by E4orf4 and whose phosphorylation is modulated by Src (data not shown) E4orf4 is itself a Src substrate, which acts as a modifier of Src-dependent phosphorylation [59,60] For western blot studies we used the enhanced chemiluminescence kit Western Plus (Perkin Elmer Life Sciences Inc., Boston, MA) and Immobilon-P transfer membrane (Millipore, Bedford, MA) [c-32P]ATP (1000–3000 CiỈmmol)1) was from New England Nuclear Radiochemicals (Lachine, QC) The c-Src inhibitor PP2 was from EMD Biosciences (La Jolla, CA) Reagents for SDS ⁄ PAGE were obtained from Bio-Rad (Mississauga, ON) bpV(phen) was synthesized as described [61] All other chemicals were of analytical grade and were purchased from either Fisher (Sainte-Foy, QC) or Roche Laboratories (Laval, QC) Experimental procedures Subcellular fractionation Reagents and antibodies Porcine insulin was from Sigma (St Louis, MO) The antibody directed against the b-subunit of the IR was from BD Sprague–Dawley rats (female, 140–150 g body weight) were purchased from Charles River Ltd (St Constant, QC) Work was conducted with the approval of the Laval FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 999 Regulation of DPP IV trafficking N Bilodeau et al University Animal Care committee Rats were fasted overnight, anesthetized with Nembutal (6.7 mgỈ100 g)1 body weight) and injected with insulin via the left jugular vein (1.5 lgỈ100 g)1 body weight) When bpV(phen) was used in vivo, a peritoneal injection (0.3 mgỈ100 g)1 body weight) was made 16 h and 30 before insulin injection Livers were excised rapidly at the noted times postinjection and minced in ice-cold homogenization buffer (250 mm sucrose, 50 mm Hepes pH 7.4, 40 mm sodium fluoride, mm MgCl2, mm benzamidine, mm phenlymethylsulphonyl fluoride) The G ⁄ E fraction was prepared immediately as described previously [44] This fraction has been characterized by transmission electron microscopy, enzyme markers, silver staining and receptor-mediated endocytosis SYPRO Ruby (Eugene, OR) staining of SDS ⁄ PAGE 1-D gels is also shown here (Fig 4B) The endosomal fractions previously designated Li ⁄ Lh and Gi ⁄ Gh were prepared from the parent light mitochondrial (L) and microsomal (P) fractions, respectively, by a flotation method as originally described elsewhere [62] The yield of the G ⁄ E fraction was 0.38 ± 0.015 mg proteinỈg)1 liver weight (n ¼ 36) The yields from the other fractions were: Li, 0.18 ± 0.036 mg proteinỈg)1 liver weight; Lh, 0.09 ± 0.01 mg proteinỈg)1 liver weight; Gi, 0.015 ± 0.004 mg proteinỈg)1 liver weight; Gh, 0.034 ± 0.005 mg proteinặg)1 liver weight, n ẳ 36 The PM fraction was prepared according to the method of Hubbard with modifications [44] and used directly A yield of 1.18 ± 0.61 mg proteinặg)1 liver weight (n ẳ 22) was obtained The Cyt fraction was generated by centrifuging the homogenate at 100 000 g for h and the supernatant was collected Protein content of fractions was determined by a modification of the Bradford method using BSA as a standard Statistical analysis was performed with statview (Abacus Concepts Inc., Berkeley, CA) Electron microscopy The G ⁄ E fraction was immediately fixed with 2.5% glutaraldehyde, and 100 mm sodium cacodylate pH 7.4 Samples were rinsed and postfixed in 1% ferrocyanide osmium tetroxide, dehydrated in a graded series of ethanol and then processed for embedding in EPON Ultrathin sections of each block were cut and placed on copper grids, stained with uranyl acetate and lead citrate [48] Sections were examined with a Philips EM 301 electron microscope (Philips, Eindhoven, the Netherlands) in 40 mm para-nitrophenylphosphate (pNPP) for 120 at room temperature The columns were spun to yield the eluates and protein contents in the fractions were determined as above Eluates were then separated by SDS ⁄ PAGE and the major bands, stained with Coomassie blue, were excised and subjected to alkylation and digestion procedures using lysyl endopeptidase C [63] Digestion products were spotted on a stainless steel MALDI plate (Applied Biosystems, Foster City, CA) Analyses utilized an automated acquisition procedure on a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems) operated in a delayed extraction mode mascot (Matrix Science Inc., Boston, MA) [64] was used for searches in the nonredundant NCBI database For identification of the immunoprecipitated DPP IV, a 110-kDa band, stained with SYPRO Ruby, was excised from the gel and subjected to trypsin digestion [63] The resulting peptides were separated by a capillary HPLC reverse phase C18 column (Picofrit BioBasic, 10 cm length, 0.075 mm internal diameter New Objective, Woburn, MA) and analysed by tandem MS using a LC-MS ⁄ MS quadrupole ion trap mass spectrometer (Finnigan LCQ Deca XP, Thermo Electro Corporation, San Jose, CA) mascot was used for searches in the nonredundant NCBI database [64] Phosphorylation and immunoprecipitation assays IR autophosphorylation and KOH treatment (hydrolysis of phosphorylated serine and threonine residues) of gels were conducted as reported previously [65] with minor modifications Aliquots of intact endosomes (G ⁄ E fraction) were incubated at 37 °C for 15 in the kinase buffer (50 mm Hepes pH 7.4, mm benzamidine, 40 mm MgCl2, mm MnCl2, 0.05% Triton X-100), in the presence of [c-32P]ATP (25 lm, 3000 CiỈmmol)1) When indicated, an inhibitor [100 lm bpV(phen) or 10 lm PP2] was added Samples were then solubilized in 1% Triton X-100 for 60 and centrifuged at 250 000 g for 30 The resulting supernatants were immunoprecipitated for IR (1 lg affinity purified antibodml)1; 100 lg protein) or DPP IV (5 lg affinity purified antibodml)1; 500 lg protein) The resulting immuno-complexes were separated by SDS ⁄ PAGE (7.5% resolving gel) and gels were subjected to autoradiography Unlabelled DPP IV immuno-complexes were analysed directly by immunoblotting using anti-DPP IV (26C), antiPY or anti-c-Src IgG Oriented proteolysis MS Tyrosine-phosphorylated proteins were recovered from the WGL subfraction of the G ⁄ E fraction prepared as described previously [33] The WGL subfraction was applied to an anti-PY affinity column (PY-20-agarose) and phosphoproteins were eluted by re-suspension of each column 1000 Oriented proteolysis experiments were performed essentially as described [33] Endosomes (G ⁄ E, Lh and Gh fractions) or PM fraction were incubated in 50 mm Hepes pH 7.4, at °C for 30 in the presence or absence of 0.1% Triton X-100 and proteinase K (60 ngỈ100 lg)1 protein of fractions) The membranes were then diluted FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS N Bilodeau et al (1 : 10) in ice-cold buffer without Triton X-100 and proteinase K and centrifuged for 30 at 250 000 g (TL-100, Beckman Coulter, Fullerton, CA) The resulting pellet was analysed by transmission electron microscopy or re-suspended in Laemmli sample buffer and subjected to SDS ⁄ PAGE (10 lg protein) Gels were stained with SYPRO Ruby for examination or immunoblotted with anti-c-Src and anti-DPP IV (26C) IgG Regulation of DPP IV trafficking 10 Acknowledgements This work is supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (RF: OGPO157551), by the Canadian Diabetes Association (CDA) and a 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In Madin–Darby canine kidney (MDCK) cells, a study of chimeric forms of DPP IV has shown that the luminal domain of DPP IV carries dominant apical sorting information while the short cytoplasmic... immunoprecipitation with anti -DPP IV IgG, a phosphorylated protein of appropriate apparent molecular mass (110 kDa) was observed (Fig 3A) DPP IV 32P phosphorylation was alkali resistant Insulin injection (1.5... can be continuously diverted towards lysosomal compartments for degradation Alternatively, a soluble form of DPP IV can be released from the cell surface In this regard, an increased circulating