INTRODUCTION

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Multiple downstream effectors of insulin action are shared in common by many receptor tyrosine kinases. This necessitates the existence of mechanisms for incorporating specificity at each step in the insulin signal transduction pathway, starting at the receptor and receptor substrate levels (16). Integration of signals generated by the well-known cross talk of the insulin receptor to different types of non-insulin receptor tyrosine kinases (e.g., insulin-like growth factor 1 receptor [IGF-1R]) or of the latter (e.g., platelet-derived growth factor receptor [PDGF-R]) to the insulin receptor substrate (IRS) proteins may contribute to the specificity of insulin action. Upon tyrosine phosphorylation, IRS proteins provide a common interface for the activated receptor and various downstream (Src homology 2 domain [SH2] containing) signaling proteins, including phosphatidylinositol-3'-kinase (PI 3K), p55^PIK, Grb-2, SHP2, Nck, and Crk (67, 71, 72).

Specificity of insulin action may also be determined by the external environment of the cells mediated through signal cross talk from integrins. Integrins, transmembrane proteins expressed in most tissues, including insulin-sensitive adipose and muscle cells, bind to particular extracellular matrix proteins. The key biological functions of integrins, including cell migration and adhesion, are mediated in part by focal adhesion kinase, pp125^FAK (2, 8). There is evidence that signaling pathways initiated by integrins synergize functionally with those triggered by growth factors (32, 55). Recent data imply that insulin potently augments ₅₁-integrin-mediated cell adhesion of insulin receptor-expressing CHO cells, while signaling via this integrin in turn enhances insulin receptor kinase activity and tyrosine phosphorylation and formation of complexes containing IRS-1 and PI 3K (15). The latter findings were extended to isolated rat adipocytes for artificial clustering of ₅₁-integrin (14). Thus, the insulin receptor may act synergistically with integrins to enhance cell adhesion, and, vice versa, the extracellular matrix surrounding the cell may influence signaling specificity by the insulin receptor.

A signaling pathway which also might sense information from the cellular environment or extracellular proteins and cross talk to various signal transduction cascades, such as insulin signaling, but is less well understood than the integrin system, emerges from glycosylphosphatidylinositol-anchored plasma membrane proteins (GPI proteins). The protein moiety of GPI proteins is attached to the extracellular face of the plasma membrane via a covalently attached glycolipid of the glycosylphosphatidylinositol (GPI) type that is embedded in the outer leaflet of the phospholipid bilayer (42). Two modes of initiation of signaling events through GPI proteins have been described so far. (i) Cross-linking of certain GPI proteins with antibodies in T cells and neutrophils elicits cell-specific responses via activation of non-receptor tyrosine kinases which are associated with the inner leaflet of the plasma membrane via their fatty acyl chains and form together with GPI proteins so-called glycolipid-enriched detergent-insoluble raft domains within the plasma membrane (5, 51, 56, 58, 59). (ii) Lipolytic cleavage of the GPI anchor of certain GPI proteins by a GPI-specific phospholipase C induces a range of insulin-mimetic metabolic effects in insulin-responsive cells (30, 35). The molecular mechanism(s) for signal transmission from GPI proteins via the plasma membrane to intracellular signaling cascades has not been elucidated for either mode; however, it has been linked to the generation of soluble phosphoinositolglycan (PIG) molecules in case of phospholipase C action (64).

PIG molecules represent the polar core glycan head groups of free GPI lipids or GPI protein membrane anchors. They consist of a cyclic phosphoinositol moiety coupled to nonacetylated glucosamine and an additional glycan structure, which in case of GPI protein membrane anchors, is built from three mannose residues in typical glycosidic linkages followed by a phosphodiester bridge to the terminal ethanolamine residue (20, 34, 36). During the past few years, we have demonstrated that chemically synthesized complete PIG molecules (Fig. 1) mimic a number of metabolic insulin effects (e.g., stimulation of glucose transport and nonoxidative glucose metabolism) in normal and insulin-resistant isolated fat and muscle cells at the micromolar range to up to the maximal insulin response (11). The complete glycan core structure (three mannose residues plus glucosamine) of typical GPI protein membrane anchors including a mannose side chain and the inositol(cyclic)phosphate moiety (Fig. 1) is required for maximal insulin-mimetic activity of PIG compounds, with some variations possible with regard to the type of residues coupled to the terminal mannose or inositol as well as the type of linkages involved (12). This potent insulin-mimetic metabolic activity of PIG compounds is not accompanied by stimulation of the insulin receptor tyrosine kinase; however, it correlates with dramatic tyrosine phosphorylation of IRS-1 as well as activation of PI 3K and its downstream-located cascade (12, 22, 39, 40). Thus, PIG compounds seem to mimic metabolic insulin action by insulin receptor-independent activation of the IRS-PI 3K pathway and thus circumvent defects at the level of the insulin receptor in muscle and adipose tissue (Fig. 1), which may represent (in part) the molecular basis for peripheral insulin resistance, the hallmark of non-insulin-dependent diabetes mellitus (28).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Structure of the PIG compounds 1, 7, 37, and 41 (with the activity-determining phosphate moiety marked by an asterisk) and their mode of action in fat and muscle cells according to references 12 and 22 (also see the introduction). PIG compounds induce tyrosine phosphorylation of IRS proteins and their association with PI 3K, which is thereby activated, as does insulin by stimulating the insulin receptor tyrosine kinase. Operation of the IRS-1-PI 3K pathway (and possibly membrane association of the regulatory p85 subunit of PI 3K) is a prerequisite for fusion of GLUT4-containing vesicles with the plasma membrane, the so-called GLUT4 translocation, but may not be sufficient (also see Discussion). In addition, PIG compounds modulate the phosphorylation state of a number of phosphoproteins, which are not affected by insulin (22), but may be required for PIG-dependent GLUT4 translocation.

The present study was performed to identify the tyrosine kinase(s) responsible for PIG-dependent IRS phosphorylation and to characterize the upstream-located signaling cascade. We found that insulin-mimetic signaling by PIG compounds depends on activation and direct interaction of the pp59^Lyn and pp125^FAK non-receptor tyrosine kinases, which is antagonized by integrin engagement. This raises the possibility for cross talk of a GPI protein-mediated signal transduction cascade to metabolic insulin signaling via components of the cell adhesion pathway.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. [-³²P]ATP (6,000 Ci/mmol) and 2-deoxy-D-[2,6-³H]glucose (60 Ci/mmol) were bought from NEN/DuPont (Bad Homburg, Germany). PIG 41, 37, 45, 7, and 1 and recombinant human insulin were made available by the Departments of Medicinal Chemistry and Pharma Synthesis of Aventis Pharma (Frankfurt am Main, Germany). Recombinant human IRS-1 was delivered by Upstate Biotechnology (Lake Placid, N.Y.). pp125^FAK (amino acids [aa] 385 to 405) Src docking site wild-type (Y397) peptide and mutant (F397) control peptide and pp125^FAK (aa 368 to 388) control peptide, pp125^FAK (aa 568 to 582) regulatory loop wild-type (Y576, Y577) peptide and mutant (F576, F577) control peptide, GRGDSP peptide, and GRADSP peptide were synthesized by Biotrend (Cologne, Germany). Human plasma fibronectin, rat plasma vitronectin, and poly-L-lysine were purchased from Sigma (Deisenhofen, Germany). Neutralizing anti-pp59^Lyn and anti-pp125^FAK antibodies were prepared in our laboratory by immunizing rabbits with the recombinant (insect cells) catalytic SH1 of human pp59^Lyn comprising aa 217 to 512 and catalytic domain of chicken pp125^FAK comprising aa 354 to 625, respectively, fused to amino-terminal glutathione S-transferase tags. Monoclonal antibodies against pp59^Lyn (clone 42), paxillin (clone 165), and pp125^FAK (clone 77) for immunoprecipitation were obtained from Transduction Laboratories (Lexington, Ky.). Monoclonal antiphosphotyrosine antibody (clone 4G10) for immunoblotting was purchased from Upstate Biotechnology. Anti-IRS-1 antibody for immunoprecipitation raised in rabbits against total human recombinant (insect cells) IRS-1 (purified by gel filtration) was provided by Biotrend. Anti-IRS-1 antibody for immunoblotting raised in rabbits against a synthetic peptide corresponding to the carboxy-terminal sequence comprising aa 1223 to 1235 of rat IRS-1 was a kind gift of Suzanne Dalle (Humboldt University, Berlin, Germany). Monoclonal anti-₁-integrin antibody (clone K20) for clustering was bought from Immunotech (San Francisco, Calif.). Monoclonal anti-₃-integrin antibody for clustering (clone F11) was delivered by PharMingen (Heidelberg, Germany). Polyclonal antirat immunoglobulin G (IgG) (whole molecule) from rabbit (used as nonimmune IgG) was bought from Sigma (Deisenhofen, Germany). Materials for cell culture (including sera) were obtained from Gibco/BRL (Eggenstein/Leopoldshafen, Germany). Antibiotics and proteinase inhibitors were from Roche Molecular Biochemicals (Mannheim, Germany). Detergents were bought from Calbiochem (Bad Soden, Germany). All other chemicals were provided by Merck (Darmstadt, Germany) unless indicated otherwise.

Preparation and stimulation of isolated rat adipocytes with insulin or PIG. Adipocytes from epididymal fat pads of male Wistar rats prepared as described previously (39) were suspended in buffer S [Dulbecco's minimal essential medium (DMEM) containing 5 mM glucose, 0.5 mM sodium pyruvate, 4 mM L-glutamine, 200 nM 1-methyl-2-(phenylethyl)adenosine (PIA), 100 µg of gentamicin per ml, 1% bovine serum albumin (BSA), and 25 mM HEPES-KOH (pH 7.4)] at 5% cytocrit (corresponding to about 7 × 10⁵ cells/ml). For determination of the packed cell volume, small aliquots of the cell suspension were aspirated into capillary hematocrit tubes and centrifuged for 90 s in a microhematocrit centrifuge in order to measure the fractional occupation of the suspension by the adipocytes (cytocrit). A 20-ml portion of the (electroporated) adipocyte suspension (5% cytocrit) was added to 20 ml of buffer S containing PIG or human insulin as indicated. Incubations (20 min, 37°C) were performed under 5% CO₂ in 200-ml polyethylene vials with shaking at 110 cycles/min and with a stroke length of 3.5 cm.

Preparation and stimulation of adherent 3T3-L1 adipocytes with insulin or PIG. 3T3-L1 fibroblasts were seeded in 12-well (60,000 cells/well) plates and maintained in DMEM (high glucose) plus 10% fetal bovine serum (FBS), 5 mM L-glutamine, and 2% BSA. Following 3 days at 100% confluence, differentiation was initiated by the addition of DMEM containing 10% FBS, 400 nM human insulin, 1 µM dexamethasone, and 1 mM isobutylmethylxanthine (Sigma, Deisenhofen, Germany). Three days later, the medium was replaced with DMEM plus 10% FBS and 100 nM insulin. After an additional 2 days, the medium was changed to DMEM (low glucose) plus 10% FBS. Adipocytes were used 5 to 12 days after completion of the differentiation protocol, when more than 85% of the cells expressed the adipocyte phenotype. Prior to experiments, the cells were rinsed two times with low serum medium (DMEM containing 5 mM glucose, 0.5% BSA, 0.1% FBS, 25 mM HEPES [pH 7.4] 10 mM glutamine, 100 U of streptomycin or penicillin per ml) and then incubated (12 to 14 h) in this medium and finally washed twice with phosphate-buffered saline (PBS) containing 2 mM sodium pyruvate prior to incubation (30 min, 37°C) with PIG or insulin as indicated in 4 ml of buffer S.

Preparation and stimulation of nonadherent 3T3-L1 adipocytes with insulin or PIG. Adherent 3T3-L1 adipocytes were serum starved for 12 h in a mixture of serum-free DMEM, 10 mM glutamine, 0.5% BSA, and 50 U of streptomycin or penicillin per ml; thereafter washed twice with PBS containing 1 mM EDTA; and then removed gently from the dishes by using a rubber policeman. After being washed once in PBS, the cells were maintained in suspension (30 min, 37°C) in 4 ml of PBS containing 5 mM glucose, 1% BSA, and 200 nM PIA before the addition of PIG or insulin as indicated and further incubation (30 min, 37°C).

Attachment of 3T3-L1 adipocytes to fibronectin-coated dishes. Cell culture dishes (12-well plates) were coated (4°C, overnight) with fibronectin (10 µg/ml) or poly-L-lysine (10 µg/ml) together with vitronectin (2 µg/ml) in PBS containing 2.7 mM KCl, 6.5 mM Na₂HPO₄, and 1.5 mM KH₂PO₄ (pH 7.4), blocked (1 h) with 0.1% BSA in PBS, and then dried (1 h, 37°C) prior to plating of the cells. Confluent 3T3-L1 adipocytes were serum starved for 12 h in serum-free DMEM, 10 mM glutamine, 0.5% BSA, and 50 U of streptomycin-penicillin per ml and then detached by adding EDTA-trypsin (0.05 mM, 0.025%). The detached adipocytes were washed three times with PBS containing 1% BSA and then held in suspension (30 min, 37°C) in 4 ml of buffer S prior to addition of PIG or insulin as indicated. After incubation (10 min, 37°C), the cells were replated on dishes coated with fibronectin or poly-L-lysine in the absence or presence of 25 µg of peptide per ml as indicated and further incubated (20 min, 37°C) under 5% CO₂.

Electroporation of isolated rat adipocytes. A 0.4-ml portion of buffer E (4.74 mM NaCl, 118 mM KCl, 0.38 mM CaCl₂, 1 mM EGTA, 1.19 mM MgSO₄, 1.19 mM KH₂PO₄, 25 mg of BSA per ml, 3 mM sodium pyruvate, 25 mM HEPES-KOH, pH 7.4) was placed in a 0.4-cm gap-width electroporation cuvette (Bio-Rad, Munich, Germany) together with the antibodies or peptides. A 0.4-ml portion of the adipocyte suspension (50% cytocrit in buffer E) was added to each cuvette and gently mixed. Electroporation was performed with a Gene Pulser Transfection Apparatus (Bio-Rad), which was set at a capacitance of 25 µF and voltage of 800 V (2 kV/cm), at 25°C for six shocks (46, 57). After the third treatment, the adipocyte suspension was gently stirred with a plastic stick, and the electric polarity was reversed. The time constant of electroporation was typically 0.6 ms during the final shock. Routinely, 4 ml of adipocyte suspension (25% cytocrit) was electroporated in five cuvettes. The time required for treatment of the five cuvettes was about 3 min. After electroporation, the cells from five electroporations were pooled and transferred to 50-ml polystyrene tubes (Falcon). After incubation (30 min, 37°C) in 5% CO₂-95% O₂, the cells were centrifuged (200 × g, 1 min, swing-out rotor) and the infranatant was aspirated. Thereafter the cells were washed once with 40 ml of buffer S containing 4% BSA, suspended in 20 ml of buffer S, and then incubated (1 h, 37°C) under 5% CO₂ prior to stimulation with PIG or insulin (see above).

Clustering of adipocytes. Isolated rat adipocytes or nonadherent 3T3-L1 adipocytes were washed with buffer S containing 2% BSA and 2 mM pyruvate and then suspended in the same medium at 10% cytocrit. After 30 min of rest, the cells were incubated (30 min, 37°C) in the absence or presence of anti-₁-integrin or anti-₃-integrin antibody (4 µg/ml) together with plasma human fibronectin or poly-L-lysine (40 µg/ml) with or without further additions and then stimulated (20 min, 37°C) with PIG as indicated under 5% CO₂.

Preparation of cell lysates. Stimulated adherent or nonadherent 3T3-L1 cells were placed on ice and then washed twice with a mixture of 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM EDTA, 10 mM glycerol-3-phosphate, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄, 100 mM NaF (in the case of nonadherent cells by flotation [200 × g, 2 min] and aspiration of the infranatant). Cells were solubilized in the above buffer containing 1% (vol/vol) Triton X-100, 0.1% sodium deoxycholate, 10% glycerol and protease inhibitors (20-µg/ml leupeptin, 10-µg/ml pepstatin A, 50-µg/ml aprotinin, 10 µM E-64, 0.5 mM phenylmethylsulfonylfluoride [PMSF] [lysis buffer]) for 30 min on ice (nonadherent cells) or by scraping with a Teflon policeman (adherent cells). Total lysates were centrifuged (25,000 × g, 20 min, 18°C). The infranatant was aspirated, taking care to avoid contamination by the upper fat layer, and recentrifuged to obtain the defatted cell lysate. Stimulated normal or electroporated rat adipocytes were washed once with PBS containing 2 mM sodium pyruvate by flotation (200 × g, 2 min) and aspiration of the infranatant and immediately homogenized in lysis buffer containing 4 mM benzamidine by 10 strokes/200 rpm in a medium-fitting Teflon-in-glass homogenizator (2-ml vessel volume) at 18°C. Defatted cell lysate was prepared as described above.

Immunoprecipitation. Defatted cell lysates (standardized for 5 to 10 µg of protein) were precleared (1 h, 4°C) with protein G or A-Sepharose (Pharmacia, Freiburg, Germany) and then supplemented with the appropriate antibodies (pp125^FAK, 2 µg/sample; IRS-1, 1:50 serum dilution; pp59^Lyn, 5 µg/sample; paxillin, 0.7 µg/sample) preadsorbed on protein G-Sepharose (monoclonal antibodies) or protein A-Sepharose (rabbit antibodies) in a total volume of 100 µl of lysis buffer. After incubation (4 h, 4°C, end-over-end rotation) and centrifugation (13,000 × g, 2 min, 4°C), the collected immune complexes were washed twice with 1 ml each of immunoprecipitation buffer (50 mM HEPES-KOH [pH 7.4], 500 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM Na₄P₂O₇, 1 mM Na₃VO₄) containing 1% NP-40 (omitted for sequential immunoprecipitation), and then twice with 1 ml each of immunoprecipitation buffer containing 150 mM NaCl and 0.1% NP-40 and once with 1 ml of immunoprecipitation buffer lacking salt and detergent and finally suspended in 50 µl of Laemmli buffer (2% sodium dodecyl sulfate [SDS], 5% 2-mercaptoethanol), heated (95°C, 2 min), and centrifuged. The supernatant samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE; 4 to 12% Bis-Tris precast gel [pH 6.4], morpholinoethanesulfonic acid [MES]-SDS running buffer; Novex, San Diego, Calif.) under reducing conditions. For sequential immunoprecipitation, the supernatant samples (50 µl) were supplemented with 1 ml of immunoprecipitation buffer containing 1% NP-40 and 5 µl of anti-IRS-1 antiserum. After incubation (12 h, 4°C), 50 µl of protein A-Sepharose (100 mg/ml in immunoprecipitation buffer) was added, and the incubation continued (4 h, end-over-end rotation). The immune complexes were collected, washed, and processed for SDS-PAGE as described above.

Immunoblotting. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Eschborn, Germany). The blocked membrane was incubated (2 h, 25°C) with antibodies against pp125^FAK (1:200), IRS-1 (1:500), or phosphotyrosine (2 µg/ml), washed (four times with Tris-buffered saline [TBS] containing 1% [vol/vol] NP-40 and 0.5% Tween 20, twice with TBS containing 0.5% Tween 20, and twice with TBS) and then incubated (1 h, 25°C) with the appropriate horseradish peroxidase-coupled detection (antimouse or antirabbit) antibodies (1:15,000 dilution; enhanced luminescence; Pierce, Rockford, Ill.) in TBS containing 5% (wt/vol) BSA, washed (five times with TBS containing 1% NP-40 and 0.5% Tween 20, three times with TBS containing 0.05% Tween 20), and finally processed with chemiluminescent reagents (Renaissance Chemiluminescence Detection System; NEN/DuPont, Bad Homburg, Germany) and subjected to phosphorimaging.

Immune complex kinase assays. pp59^Lyn or pp125^FAK immune complexes were washed in kinase buffer (50 mM HEPES-KOH [pH 7.4], 100 mM NaCl, 5 mM MnCl₂, 1 mM MgCl₂, 0.5 mM dithiothreitol [DTT], 1 mM Na₃VO₄), and then suspended in 50 µl of kinase buffer. The kinase reactions were started by addition of ATP (unlabeled or ³²P-labeled; final concentrations: pp59^Lyn, 40 µM; 0.2 mCi/ml; pp125^FAK, 100 µM, 0.5 mCi/ml) and incubated (pp59^Lyn, 15 min; pp125^FAK, 3 min [22°C]) in the absence (autophosphorylation) or presence of recombinant human IRS-1 (0.3 µg) or heat-denatured (10 min, boiling waterbath) rabbit muscle enolase (1 µg; Sigma, Deisenhofen, Germany). Autophosphorylation reactions were terminated by addition of 50 µl of ice-cold stop buffer (50 mM HEPES-KOH [pH 7.4], 150 mM NaCl, 100 mM ATP, 0.05% Triton X-100) and washing of the beads twice with 1 ml of stop buffer prior to addition of 20 µl of Laemmli buffer and boiling (95°C, 5 min). Substrate phosphorylation reactions were terminated by addition of 10 µl of fourfold-concentrated Laemmli buffer and boiling. The phosphoproteins were separated by SDS-PAGE (10% Bis-Tris resolving gel, MES-SDS running buffer) and analyzed by phosphorimaging directly (use of [³²P]ATP) or after immunoblotting with antiphosphotyrosine antibody (use of unlabeled ATP). Under these conditions, the kinase reactions were linear with time for the assay period.

Glucose transport. Glucose transport was assayed after two washing cycles of the cells with glucose-free and serum-free DMEM by using 2-deoxy-D-[2,6-³H]glucose (50 µM, 0.33 µCi/ml; 20 min at 37°C) in the absence or presence of 20 µM cytochalasin B for isolated rat adipocytes and nonadherent 3T3-L1 adipocytes by using the oil-centrifugation method (39) and for adherent 3T3-L1 adipocytes by washing the cells twice with ice-cold PBS containing 5 mM D-glucose and subsequent addition of 0.2% SDS-0.1 N NaOH prior to liquid scintillation counting (61).

Miscellaneous. Protein concentration was determined by the bicinchoninic acid protein assay protocol from Pierce (Rockford, Ill.) with crystalline BSA as a standard. Phosphorimaging was performed with a Storm 860 PhosphorImager (Molecular Dynamics) and quantitatively evaluated with ImageQuant software (Molecular Dynamics). Differences in recovery in the amounts of immunoprecipitated protein during a specific experiment were corrected in each case (data on fold or percent stimulation) for the amount of protein actually applied to the gel by homologous immunoblotting. All of the results reported herein were confirmed by running independent experiments with different batches of adipocytes several times (as indicated in the figure legends), each with two to five parallel independent immunoprecipitation, kinase assay, and immunoblotting analyses.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Involvement of pp59^Lyn in insulin-mimetic signaling by PIG in adipocytes. Our previous studies unequivocally have demonstrated that insulin-mimetic signaling by PIG critically depends on insulin receptor-independent tyrosine phosphorylation of IRS-1, -2, and -3 (39, 40). Differential sensitivity of insulin and PIG stimulation of lipogenesis toward tyrosine kinase inhibitors (11) and the reported association of members of Src-class kinases with GPI proteins (see Introduction) led us to speculate on the involvement of non-receptor rather than receptor tyrosine kinases in PIG signaling (34). Furthermore, Lebrun and coworkers found that expression of a constitutive active pp60^Src in 293 EBNA cells results in strong IRS-1 tyrosine phosphorylation (27). In addition, during an effort to identify new IRS-1 binding proteins by screening a mouse embryo expression library with recombinant [³²P]IRS-1, a specific association between IRS-1 and pp60^Fyn via its SH2 domain and Tyr⁸⁹⁵ and Tyr¹¹⁷² of IRS-1 was detected (60). These results suggested a role for Src-class kinases during insulin signaling. pp59^Lyn kinase is the predominant Src-class kinase in isolated rat adipocytes (47) and was therefore selected as a candidate non-insulin receptor tyrosine kinase for PIG-dependent IRS phosphorylation.

View larger version (62K): [in this window] [in a new window]   FIG. 2.   Effect of anti-pp59Lyn antibody on PIG-induced IRS-1 tyrosine phosphorylation in adipocytes. Isolated rat adipocytes were electroporated with nonimmune IgG or neutralizing anti-pp59Lyn antibody (1:25) and then incubated in the absence or presence of increasing concentrations of PIG 41 or human insulin. IRS-1 was immunoprecipitated (IP) with anti-IRS-1 antibody from defatted cell lysates and then immunoblotted (IB) with antiphosphotyrosine antibody. Phosphorimages of a typical experiment are shown (left section) repeated four times with similar results. Quantitative evaluation is given as fold stimulation (mean ± standard deviation [right section]). Basal tyrosine phosphorylation is set at 1 in each case.

View larger version (83K): [in this window] [in a new window]   FIG. 3.   Effect of PIG compounds on pp59Lyn activity. Isolated rat adipocytes were incubated in the absence or presence of increasing concentrations of PIG compounds or human insulin. pp59Lyn was immunoprecipitated (IP) with anti-pp59Lyn antibody from defatted cell lysates and then subjected to immune complex kinase assays for autophosphorylation by incubation with unlabeled ATP (A) or phosphorylation of recombinant human IRS-1 by incubation with either [32P]ATP or unlabeled ATP (B). Phosphorimages of a typical experiment are shown (left sections) repeated three times with similar results. Basal pp59Lyn autophosphorylation and IRS-1 tyrosine phosphorylation were rather low and comparable to those observed in the presence of up to 0.01 µM PIG 37, 7, and 1 (which are shown instead of the basal phosphorylations only). Quantitative evaluation is given as fold stimulation (mean ± standard deviation [right sections]). Basal phosphorylation is set at 1 in each case. Insulin increased autophosphorylation of pp59Lyn and tyrosine phosphorylation of IRS-1 (according to antiphosphotyrosine immunoblotting [IB]), respectively, 1.6- to 1.4-fold at 0.3 nM, 2.2- to 2.7-fold at 1 nM, and 3.1- to 2.9-fold at 3 nM versus the basal level.

Involvement of pp125^FAK in insulin-mimetic signaling and action by PIG in adipocytes. In many cell types, Src-class kinase family members are activated by the cytosolic focal adhesion kinase, pp125^FAK, which is involved in integrin signaling, cytoskeletal reorganization, and signal transduction by a number of growth factors (13, 48, 76). Recently it was found that in nonattached cells, insulin promotes pp125^FAK phosphorylation and activation and pp125^FAK is a direct substrate of the insulin receptor tyrosine kinase (3). Furthermore, the interaction of IRS-1 with pp125^FAK using a mammalian two-hybrid system or coimmunoprecipitation and extensive IRS-1 tyrosine phosphorylation upon expression of pp125^FAK in 293 EBNA cells were described (27).

View larger version (70K): [in this window] [in a new window]   FIG. 4.   Effect of PIG compounds on tyrosine phosphorylation of pp125FAK and paxillin and pp125FAK-IRS-1 association. Isolated rat adipocytes (A) or nonadherent and adherent 3T3-L1 adipocytes (B) were incubated in the absence or presence of increasing concentrations of PIG compounds. pp125FAK and paxillin were immunoprecipitated (IP) with corresponding antibodies from defatted cell lysates and immunoblotted (IB) with anti-IRS-1, anti-pp125FAK (pp125FAK only), or antiphosphotyrosine antibodies. Phosphorimages of typical experiments are shown (A) repeated two to four times with similar results. Quantitative evaluation is given as fold stimulation (mean ± standard deviation [B]). Basal tyrosine phosphorylation and association are set at 1 in each case.

View larger version (40K): [in this window] [in a new window]   FIG. 5.   Effect of cell attachment on PIG-induced tyrosine phosphorylation of pp125FAK, paxillin and IRS-1, and pp125FAK and IRS-1 association. Serum-starved adherent 3T3-L1 adipocytes were detached, held in suspension (30 min), and then stimulated (10 min) in the absence or presence of increasing concentrations of PIG 41, replated on fibronectin-coated culture dishes in the absence or presence of 0.5 mM GRGDSP or GRADSP peptide, and further incubated (20 min). pp125FAK, paxillin, and IRS-1 were immunoprecipitated (IP) with corresponding antibodies from defatted cell lysates and then immunoblotted (IB) with antiphosphotyrosine or anti-IRS-1 antibodies (pp125FAK only). Phosphorimages of three to four independent experiments were quantitatively evaluated as fold stimulation (mean ± standard deviation). Basal tyrosine phosphorylation and association are set at 1 in each case.

Interaction of pp125^FAK and pp59^Lyn in insulin-mimetic signaling and action in adipocytes. It is generally assumed that pp125^FAK activates Src-class kinases by docking of the phosphorylated tyrosine 397 of pp125^FAK to the SH2 domain of the Src-class kinase, thereby preventing its carboxy-terminal phosphotyrosine residue from binding to the SH2 domain, which is assumed to keep the kinase in the inactive state (10, 47, 52). The involvement of pp125^FAK on pp59^Lyn regulation during PIG signaling was studied by electroporation of rat adipocytes with excess of functional Src docking site peptide (encompassing residues 385 to 405, including Y397 from the amino acid sequence of human pp125^FAK) or nonfunctional Src docking site peptide (with F397 replacing Y397) or an unrelated peptide (encompassing residues 368 to 388 from pp125^FAK) prior to treatment with PIG 41 (Fig. 6). The immune complex kinase assay with pp59^Lyn revealed that the Src docking site peptide Y397 diminished PIG 41-induced tyrosine phosphorylation of pp59^Lyn, IRS-1, and enolase to 15 to 45% of that observed with the mutant peptide F397 or the unrelated peptide. Substrate phosphorylation by pp59^Lyn was more susceptible to inhibition by the Src docking site peptide Y397 than autophosphorylation (Fig. 6). In conclusion, the functional Src docking site peptide functions as a potent inhibitor for pp59^Lyn activation by PIG, most likely by preventing the interaction between pp125^FAK and pp59^Lyn.

View larger version (50K): [in this window] [in a new window]   FIG. 6.   Effect of pp125FAK-Src docking site peptide on PIG-induced activation of pp59Lyn. Isolated rat adipocytes were electroporated with control peptide, mutant Src docking site peptide F397, or wild-type Src docking site peptide Y397 (25 µg/ml) and then incubated in the absence or presence of increasing concentrations of PIG 41. pp59Lyn was immunoprecipitated (IP) with anti-pp59Lyn antibody from defatted cell lysates and then subjected to an immune complex kinase assay for autophosphorylation (by incubation with [32P]ATP and subsequent SDS-PAGE of the total incubation mixture) or substrate phosphorylation (by incubation with unlabeled ATP and either enolase or recombinant IRS-1 and subsequent immunoblotting of the total incubation mixture with antiphosphotyrosine antibody). Phosphorimages of a typical experiment are shown (for pp59Lyn and IRS-1 [left section]) repeated three times with similar results. The basal pp59Lyn autophosphorylation in the presence of the mutant Src docking site peptide F397 was comparable to that for the control peptide (which is shown) only. Phosphorimages of enolase tyrosine phosphorylation were quantitatively evaluated as fold stimulation from four independent experiments (mean ± standard deviation [right section]) with basal values set at 1 in each case.

View larger version (52K): [in this window] [in a new window]   FIG. 7.   Effect of pp125FAK-Src docking site peptide on PIG-induced IRS-1 tyrosine phosphorylation and glucose transport. Isolated rat adipocytes were electroporated with control peptide, mutant Src docking site peptide F397, or wild-type Src docking site peptide Y397 (25 µg/ml) and then incubated in the absence or presence of increasing concentrations of PIG 41. IRS-1 was immunoprecipitated (IP) with anti-IRS-1 antibody from defatted cell lysates and then immunoblotted (IB) with antiphosphotyrosine antibody. Phosphorimages of a typical experiment are shown (upper section). Other portions of the cells were assayed for 2-deoxyglucose transport. Data from four independent experiments were quantitatively evaluated as fold stimulation (mean ± standard deviation), with basal values set at 1 in each case (lower section).

View larger version (63K): [in this window] [in a new window]   FIG. 8.   Effect of pp125FAK regulatory loop peptide on PIG-induced tyrosine phosphorylation of pp125FAK and associated IRS-1. Isolated rat adipocytes were electroporated with regulatory loop peptide Y576/577 or control peptide F576/577 (50 µg/ml) and then incubated in the absence or presence of increasing concentrations of PIG 41. pp125FAK was immunoprecipitated (IP) with anti-pp125FAK antibody from defatted cell lysates and then either immunoblotted (IB) with antiphosphotyrosine or anti-pp125FAK antibodies or reimmunoprecipitated with anti-IRS-1 antibody as indicated. Immunoprecipitated IRS-1 was immunoblotted with antiphosphotyrosine or anti-IRS-1 antibodies. Phosphorimages of a typical experiment are shown (left section) repeated three times with similar results. Quantitative evaluation is given as fold stimulation (mean ± standard deviation [right section]). Basal tyrosine phosphorylation is set at 1 in each case.

View larger version (41K): [in this window] [in a new window]   FIG. 9.   Time course of PIG-induced tyrosine phosphorylation of pp125FAK, pp59Lyn, and IRS-1. Isolated rat adipocytes, nonadherent 3T3-L1 adipocytes, and adherent 3T3-L1 adipocytes were stimulated in the presence of 3 µM PIG 41 for various periods of time. pp125FAK, pp59Lyn, and IRS-1 were immunoprecipitated (IP) with corresponding antibodies from defatted cell lysates and then immunoblotted (IB) with antiphosphotyrosine antibody. Phosphorimages of three to five independent experiments were quantitatively evaluated as percentages of the control (mean ± standard deviation). Maximal tyrosine phosphorylation is set at 100% for each time course.

Integrin engagement in PIG signaling and action in adipocytes. pp125^FAK can be activated by the integrin heterodimeric transmembrane cell surface receptors that mediate interactions between the cell surface and the extracellular matrix (29, 45). The presence of ₁-integrin in rat adipocytes has been demonstrated previously (14). Therefore, we studied the effect of clustering of this integrin on IRS-1 tyrosine phosphorylation by PIG 41. ₁-Integrin clustering can be provoked by incubating cells with activating anti-₁-integrin monoclonal antibody in the presence of the extracellular matrix protein fibronectin, which correlates with the activation of the integrin signaling pathway and enhancement of tyrosine phosphorylation of certain proteins (e.g., pp125^FAK) in a variety of cell types (25, 32), including isolated rat adipocytes (14).

View larger version (61K): [in this window] [in a new window]   FIG. 10.   Effect of integrin clustering on stimulation of IRS-1 tyrosine phosphorylation by PIG 41. Isolated rat adipocytes or nonadherent 3T3-L1 adipocytes were treated in the absence or presence of anti-1-integrin antibody plus fibronectin or anti-3-integrin antibody plus poly-L-lysine in the absence or presence of 0.5 mM GRGDSP or GRADSP peptide and then stimulated (5 min) with increasing concentrations of PIG 41. IRS-1 and pp59Lyn were immunoprecipitated (IP) with corresponding antibodies from defatted rat adipocyte and 3T3-L1 adipocyte lysates, respectively, and then immunoblotted (IB) with antiphosphotyrosine antibody. Phosphorimages from a typical experiment repeated three times with similar results are shown (left section) or were quantitatively evaluated from five independent experiments, respectively, as fold stimulation (mean ± standard deviation [right section]) with basal values set at 1 in each case.

View larger version (39K): [in this window] [in a new window]   FIG. 11.   Effect of integrin clustering on glucose transport activation by PIG 41. Isolated rat adipocytes, nonadherent 3T3-L1 adipocytes, or adherent 3T3-L1 adipocytes were treated in the absence or presence of anti-1-integrin antibody plus fibronectin or anti-3-integrin antibody plus poly-L-lysine and then incubated in the absence or presence of increasing concentrations of PIG 41. 2-Deoxyglucose transport was assayed and quantitatively evaluated from four independent experiments as fold stimulation (mean ± standard deviation) with basal values set at 1 in each case.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In mammals, there is a clear functional distinction between insulin receptors and other members of the protein tyrosine kinase family. Whereas insulin receptors regulate metabolic pathwayse.g., the translocation of the glucose transporter isoform 4 (GLUT4) from tubulovesicular structures of the trans-Golgi network to the plasma membrane in muscle and adipose tissue (9)all other receptor or non-receptor tyrosine kinases so far identified appear to control cell growth and differentiation. Consequently, the question arises of whether this specificity reflects a strict heterogeneity of the intracellular signaling pathways or whether it is the expression and the selectivity of the kinases alone that determine cellular responses (43). If the latter possibility were true, one would expect that other tyrosine kinases could mimic the metabolic effects of insulin in cells equipped with an insulin-sensitive GLUT4 translocation apparatus and concomitantly expressing the respective tyrosine kinase. This assumption has been verified in some cases, but not in others (17). Many signaling components and processes are shared by both insulin and growth factors in insulin-sensitive cells, yet their actions differ. For instance, the PDGF receptor tyrosine kinase manages to phosphorylate IRS-1 at tyrosine residues in 3T3-L1 adipocytes, which, however, does not correlate with increased glucose transport (18, 73). There are numerous examples of positive cross talk from receptors other than the insulin receptor feeding into the IRS-1-PI 3K pathway, including the G protein-coupled receptors for angiotensin and endothelin (26, 65, 66, 74); the cytokine receptors for interferons, interleukins, and tumor necrosis factor alpha (1, 69, 70, 75); the growth factor receptors for PDGF and IGF-1 (17, 41, 50); and transmembrane cell adhesion molecules, such as the integrins (54, 68). However, only in rare cases have the tyrosine kinases directly feeding into the IRS-1-PI 3K pathway and their mode of regulation been elucidated.

In the present study, we identified the non-receptor tyrosine kinases, pp125^FAK and pp59^Lyn, and ₁-integrin as components of the PIG-dependent signaling pathway upstream of IRS-1, which upon direct interaction of the kinases induces insulin-independent stimulation of glucose transport in adipocytes. This conclusion is based on the following findings. (i) The insulin-mimetic metabolic activity of structurally different PIG compounds correlates well with their ability to induce tyrosine phosphorylation of pp125^FAK (Fig. 4), its substrate paxillin (Fig. 4), and IRS-1 (Fig. 3B), as well as autophosphorylation of pp59^Lyn (Fig. 3A). (ii) Introduction into isolated adipocytes of antibodies which potently inhibit pp59^Lyn (Fig. 2) and pp125^FAK kinase activities (Table 4) as well as of the functional Src docking site and regulatory loop peptides derived from pp125^FAK (Fig. 7 and 8) drastically impairs PIG-dependent IRS-1 tyrosine phosphorylation and glucose transport: the former peptide by direct interference with downstream signaling to pp59^Lyn (Fig. 6), the latter by direct inhibition of full activation of pp125^FAK (Fig. 7). (iii) The time courses for PIG-dependent activation of pp125^FAK and pp59^Lyn are similar in shape to that for tyrosine phosphorylation of IRS-1, peaking in sequential order (Fig. 8). (iv) The PIG-dependent tyrosine phosphorylation of pp125^FAK and of pp125^FAK-associated IRS-1 is more pronounced in nonadherent than in adherent 3T3-L1 adipocytes (Fig. 4B and 5). (v) ₁-Integrin clustering blocks PIG-dependent pp59^Lyn autophosphorylation, IRS-1 tyrosine phosphorylation, and glucose transport (Fig. 10 and 11).

These results are compatible with the following model: PIG compounds trigger activation of pp125^FAK, which is antagonized by integrin engagement. Phosphotyrosine 397 of activated and autophosphorylated pp125^FAK docks to the SH2 domain of pp59^Lyn, which is thereby activated. pp59^Lyn phosphorylates pp125^FAK at the tyrosines 576 and 577 within its regulatory loop, which is a prerequisite for tyrosine phosphorylation of IRS-1 associated with pp125^FAK. Thus, pp125^FAK may act as a common signaling platform for pp59^Lyn and IRS-1, from which the metabolic signal (e.g., to the glucose transport system) originates.

Apparently the mechanisms for pp125^FAK activation operating during cell adhesion-integrin clustering and PIG action differ fundamentally. The inhibition of PIG signaling in response to simultaneous integrin engagement may be explained by some conformational or allosteric interference at pp125^FAK directly or at components located upstream, including the integrins. Integrins interact with extracellular matrix proteins, such as fibronectin and vitronectin. So far we have not obtained any experimental evidence for binding of a radiolabeled PIG derivative to recombinant ₁-integrin in vitro by using either a binding assay or immunoprecipitation with anti-₁-antibody (N. Hanekop and G. Müller, unpublished data). However, during studies of inactivation and reconstitution of isolated rat adipocytes for PIG action, we previously identified an N-ethylmaleimide-, trypsin-, and salt-sensitive component in the plasma membrane, which is required for PIG-induced tyrosine phosphorylation of IRS-1 and glucose transport activation (40). This 115-kDa polypeptide specifically interacts with PIG 41 (M. Gerl, M. Quint, and G. Müller, unpublished data) and presumably acts as a PIG receptor which may transmit the PIG signal across the plasma membrane or along the cell surface to ₁-integrin by a mechanism which we are currently investigating (37, 38). This molecular mode of PIG action differs strikingly from the classical view of transport of PIG-like molecules across the plasma membrane into the cytosol, where they act as allosteric activators or inhibitors of insulin-regulated enzymes of glucose metabolism (21). We were unable to measure specific uptake of a radiolabeled PIG derivative into isolated rat adipocytes (C. Piossek and G. Müller, unpublished data), arguing against a direct intracellular site of PIG action.

Cell adhesion, as well as PIG action, leads to tyrosine phosphorylation of IRS-1 to almost the full insulin response in both isolated rat adipocytes and nonadherent 3T3-L1 adipocytes (14; this study). However, only PIG molecules activate glucose transport to up to 90% of the maximal insulin effect (12), whereas direct integrin clustering has no effect (14). This raises the question of how specificity is determined during PIG, insulin, and integrin signaling in adipocytes. Certainly, integrin engagement per se is not sufficient for glucose transport activation in insulin-responsive cells. The desired specificity may be based on (i) additional input(s) from a different signaling cascade which emerges from the putative PIG receptor and bypasses IRS-1 and PI 3K or (ii) different modes or kinetics of targeting or activation of IRS-1 and PI 3K. The involvement of (at least) two signals for glucose transport activation in adipocytes, one derived from the IRS-1-PI 3K pathway, and the other one unknown so far, is reminiscent of insulin action. It has been argued that insulin might differentially activate specific intracellular pools of (various isoforms of) PI 3K (41, 49). Interestingly, a cell-permeable derivative of phosphatidylinositol(3,4,5)trisphosphate, a lipid signaling product of PI 3K, was shown to stimulate glucose uptake in 3T3-L1 adipocytes treated with insulin together with the PI 3K inhibitor wortmannin, but to have no effect alone, confirming that PI 3K activation is required but not sufficient for insulin signaling to the glucose transport system (19). The insulin-mediated dissociation of the recently discovered Synip protein from VAMP2 and Syntaxin 4, the v- and t-SNAREs of the GLUT4 translocation apparatus, which thereby allows VAMP2-Syntaxin 4 interaction and final fusion of GLUT4 vesicles with the plasma membrane, may represent the PI 3K-independent signal for glucose transport activation (31). A recent report with 3T3-L1 adipocytes and L6 myotubes provided further evidence that insulin stimulates two independent signals contributing to stimulation of glucose transport with PI 3K leading to plasma membrane insertion of GLUT4 without stimulating glucose transport and a PI 3K-independent pathway involving p38 mitogen-activated protein kinase, which leads to activation of GLUT4 recruited at the cell surface (61). Apparently PIG signaling fuels both the PI 3K-dependent and PI 3K-independent pathways. Specificity may be achieved by (i) differential expression of the putative PIG receptor and/or its downstream signaling components in adipose or muscle versus non-insulin-responsive tissues and (ii) different time courses and sites for tyrosine phosphorylation of IRS proteins. The residues phosphorylated on IRS-1 in response to integrin clustering and PIG action in adipocytes have not been identified so far. Interestingly, in vitro phosphorylation of recombinant IRS-1 with pp60^Fyn revealed a unique cohort of phosphotyrosine residues in comparison to the insulin receptor (60), arguing for differential phosphorylation of IRS-1 by non-receptor tyrosine kinases.

The physiological relevance of insulin-mimetic PIG signaling in adipocytes remains a matter of speculation so far. Adipocytes do not harbor focal adhesions (55), but they clearly express pp125^FAK and some other components of integrin signaling, including pp59^Lyn (rat adipocytes) and its homolog pp60^Fyn (3T3-L1 adipocytes). Thus, the role of these kinases and their regulation by the PIG signaling cascade may be different in terminally differentiated insulin-responsive cells compared to in dividing cells. The PIG signaling cascade constituted by the PIG receptor, ₁-integrin (as a negative regulator), pp125^FAK, pp59^Lyn, and IRS-1 apparently operates as a positive cross talk to metabolic insulin signaling. We speculate that GPI proteins or PIG molecules derived thereby by lipolytic cleavage in response to certain physiological stimuli, as demonstrated recently in rat adipocytes upon insulin and glucose challenge (33), act as natural ligands for the PIG receptor. These ligands may monitor environmental situations reflecting some aspect or parameter of cell adhesion or migration (such as coordination between proliferation and differentiation and metabolic activity) different from that signaled by the extracellular matrix-integrin interaction.