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Identification of Tyrosine Phosphatases That Dephosphorylate the Insulin Receptor

A BRUTE FORCE APPROACH BASED ON “SUBSTRATE-TRAPPING” MUTANTS*
Open AccessPublished:March 31, 2000DOI:https://doi.org/10.1074/jbc.275.13.9792
      Many pharmacologically important receptors, including all cytokine receptors, signal via tyrosine (auto)phosphorylation, followed by resetting to their original state through the action of protein tyrosine phosphatases (PTPs). Establishing the specificity of PTPs for receptor substrates is critical both for understanding how signaling is regulated and for the development of specific PTP inhibitors that act as ligand mimetics. We have set up a systematic approach for finding PTPs that are specific for a receptor and have validated this approach with the insulin receptor kinase. We have tested nearly all known human PTPs (45) in a membrane binding assay, using “substrate-trapping” PTP mutants. These results, combined with secondary dephosphorylation tests, confirm and extend earlier findings that PTP-1b and T-cell PTP are physiological enzymes for the insulin receptor kinase. We demonstrate that this approach can rapidly reduce the number of PTPs that have a particular receptor or other phosphoprotein as their substrate.
      PTP
      protein-tyrosine phosphatase
      GST
      glutathioneS-transferase
      IRK
      insulin receptor kinase
      PCR
      polymerase chain reaction
      PBS
      phosphate-buffered saline
      TC-PTP
      T-cell PTP
      pNPP
      para-nitrophenylphosphate
      Many cellular receptors signal via tyrosine phosphorylation (
      • Fantl W.J.
      • Johnson D.E.
      • Williams L.T.
      ,
      • Kazlauskas A.
      ). The tyrosine kinases required for this activity are often recruited upon ligand binding, as in the Jak-Stat pathways utilized by cytokine receptors (growth hormone, interleukin-10, leptin, leukemia inhibitory factor, tumor necrosis factor, and interferon receptors). Alternatively, receptors themselves have kinase activity, like epidermal growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and insulin receptors. In either case, the receptors are returned to their original state through the activity of protein-tyrosine phosphatases (PTPs).1 Although nearly 50 distinct mammalian PTPs have now been identified (
      • Hooft van Huijsduijnen R.
      ), assignment of PTPs to receptors and other tyrosine phosphorylated substrates has been slow (
      • Tonks N.K.
      • Neel B.G.
      ,
      • Neel B.G.
      • Tonks N.K.
      ). In theory, a pharmacological inhibitor of the PTP that is specific for a receptor would act as a receptor agonist. This concept is well illustrated in type II diabetes, where generic PTP inhibitors such as vanadium salts have been known for over a hundred years to restore insulin sensitivity (cited in Ref.
      • Morinville A.
      • Maysinger D.
      • Shaver A.
      ). In addition, a PTP knock-out mouse was recently shown to have increased insulin sensitivity (
      • Elchebly M.
      • Payette P.
      • Michaliszyn E.
      • Cromlish W.
      • Collins S.
      • Loy A.L.
      • Normandin D.
      • Cheng A.
      • Himms-Hagen J.
      • Chan C.C.
      • Ramachandran C.
      • Gresser M.J.
      • Tremblay M.L.
      • Kennedy B.P.
      ). Because it is easier to develop drugs that block an enzyme than it is to find ligand agonists and because many of the ligands involved (growth hormone, leptin, insulin, and β-interferon) have therapeutic value, there is considerable interest in identifying specific PTPs for these receptors.
      We have undertaken a systematic approach to PTP substrate identification that is based on “substrate-trapping” mutants of PTPs (
      • Neel B.G.
      • Tonks N.K.
      ,
      • Flint A.J.
      • Tiganis T.
      • Barford D.
      • Tonks N.K.
      ,
      • Kishimoto T.K.
      • Kahn J.
      • Migaki G.
      • Mainolfi E.
      • Shirley F.
      • Ingraham R.
      • Rothlein R.
      ,
      • LaMontagne Jr., K.R.
      • Flint A.J.
      • Franza Jr., B.R.
      • Pandergast A.M.
      • Tonks N.K.
      ,
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ,
      • Garton A.J.
      • Flint A.J.
      • Tonks N.K.
      ,
      • Pannifer A.D.B.
      • Flint A.J.
      • Tonks N.K.
      • Barford D.
      ,
      • Cote J.F.
      • Charest A.
      • Wagner J.
      • Tremblay M.L.
      ,
      • Taddei N.
      • Chiarugi P.
      • Cirri P.
      • Fiaschi T.
      • Stefani M.
      • Camici G.
      • Raugei G.
      • Ramponi G.
      ,
      • Timms J.F.
      • Carlberg K.
      • Gu H.
      • Chen H.
      • Kamatkar S.
      • Nadler M.J.
      • Rohrschneider L.R.
      • Neel B.G.
      ). The most effective of these mutants have a Asp → Ala mutation about 30 amino acids N-terminal of the catalytic HCSAG motif (
      • Hooft van Huijsduijnen R.
      ,
      • Neel B.G.
      • Tonks N.K.
      ,
      • Taddei N.
      • Chiarugi P.
      • Cirri P.
      • Fiaschi T.
      • Stefani M.
      • Camici G.
      • Raugei G.
      • Ramponi G.
      ). These mutants, like their wild type counterparts, form an enzyme-substrate intermediate involving the conserved PTP cysteine and the tyrosine phosphate of the substrate but are consequently unable to release the dephosphorylated substrate. This approach has been used previously to identify intracellular substrates for PTPs (
      • LaMontagne Jr., K.R.
      • Flint A.J.
      • Franza Jr., B.R.
      • Pandergast A.M.
      • Tonks N.K.
      ,
      • Garton A.J.
      • Flint A.J.
      • Tonks N.K.
      ,
      • Timms J.F.
      • Carlberg K.
      • Gu H.
      • Chen H.
      • Kamatkar S.
      • Nadler M.J.
      • Rohrschneider L.R.
      • Neel B.G.
      ). We have cloned the catalytic domains of the (nearly) full set of known human PTPs and expressed them either as wild type or as trapping mutants in glutathione S-transferase (GST) fusion proteins in bacteria. We have used these PTPs to test their affinity for the autophosphorylated insulin receptor kinase (IRK). Positive PTPs were validated in secondary assays for dephosphorylation of full-length IRK and of an IRK phosphopeptide that contained the tyrosines that are autophosphorylated in the IRK.

      DISCUSSION

      The accumulation of new genomic and cDNA sequences is rapidly outpacing the rate at which functions are assigned to genes. It is therefore imperative to devise new, systematic approaches to fit gene products functionally in metabolic and regulatory pathways. PTPs are ideally suited to such an approach. The family is manageable in size, and PTP catalytic domains are easily and unambiguously picked up in computer-assisted similarity searches. In the “PTP screen” described here we have chosen to focus on the catalytic domains of PTPs, because these domains are thought to be sufficient for substrate specificity (
      • Jia Z.
      • Barford D.
      • Flint A.J.
      • Tonks N.K.
      ), like kinase domains (
      • Songyang Z.
      • Carraway III, K.L.
      • Eck M.J.
      • Harrison S.C.
      • Feldman R.A.
      • Mohammadi M.
      • Schlessinger J.
      • Hubbard S.R.
      • Smith D.P.
      • Eng C.
      • Lorenzo M.J.
      • Ponder B.A.J.
      • Mayer B.J.
      • Cantley L.C.
      ). On the substrate side we have selected the insulin receptor kinase, because it autophosphorylates and may therefore be considered as a physiological PTP substrate. In addition, IRK dephosphorylation has been intensively studied in the past, and those earlier results could serve as a yardstick for our approach. The major candidate PTPs previously implicated in IRK dephosphorylation are PTP-1b and LAR (
      • Li P.M.
      • Zhang W.R.
      • Goldstein B.J.
      ,
      • Ahmad F.
      • Considine R.V.
      • Goldstein B.J.
      ,
      • Norris K.
      • Norris F.
      • Kono D.H.
      • Vestergaard H.
      • Pedersen O.
      • Theofilopoulos A.N.
      • Moller N.P.
      ). Of these, PTP-1b is well established, because a mouse mutated in PTP-1b displayed highly increased insulin sensitivity (
      • Elchebly M.
      • Payette P.
      • Michaliszyn E.
      • Cromlish W.
      • Collins S.
      • Loy A.L.
      • Normandin D.
      • Cheng A.
      • Himms-Hagen J.
      • Chan C.C.
      • Ramachandran C.
      • Gresser M.J.
      • Tremblay M.L.
      • Kennedy B.P.
      ). By contrast, LAR is expressed only at low levels in insulin-responsive tissues (
      • Norris K.
      • Norris F.
      • Kono D.H.
      • Vestergaard H.
      • Pedersen O.
      • Theofilopoulos A.N.
      • Moller N.P.
      ), and LAR knock-out phenotypes have been ambiguous (
      • Ren J.M.
      • Li P.M.
      • Zhang W.R.
      • Sweet L.J.
      • Cline G.
      • Shulman G.I.
      • Livingston J.N.
      • Goldstein B.J.
      ,
      • Schaapveld R.Q.
      • Schepens J.T.
      • Robinson G.W.
      • Attema J.
      • Oerlemans F.T.
      • Fransen J.A.
      • Streuli M.
      • Wieringa B.
      • Hennighausen L.
      • Hendriks W.J.
      ).
      What we have found in our survey is that PTP specificity increased with substrates that were more physiologically relevant. Thus, nearly all PTPs hydrolyzed pNPP, a tyrosine-phosphate mimetic; a subset dephosphorylated an insulin-like peptide and only four (PTP-1b, TC-PTP, PTP-γ, and Sap1) trapped the entire intracellular IRK. Our results confirm that mutant PTP substrate trapping closely mimics physiological dephosphorylation. The binding on filters was inhibited by pervanadate and depended on the phosphatase being mutated and the substrate being phosphorylated. IRK substrate as bound on the nitrocellulose membrane had (at least partially) maintained correct folding, because incubation of nonphosphorylated IRK immobilized on a membrane in suitable ATP-containing buffer resulted in autophosphorylation (data not shown). The membrane binding assay also appeared to be robust in the sense that the set of PTPs testing positive did not depend on incubation time, as in the dephosphorylation assay (Fig. 2). Surprisingly, GST-PTP-γ was reproducibly positive in the membrane binding assay, yet the wild type protein failed to efficiently dephosphorylate IRK. The wild type GST-PTP-γ protein preparation had activity on pNPP and peptide substrates (Tables I and II). A possible explanation for this discrepancy is that three tyrosines are involved in IRK autophosphorylation. It is possible that PTP-γ recognizes and dephosphorylates only one or two of these. That might explain why complete dephosphorylation of the IRK is slow, whereas release of free phosphate from the IRK-peptide is efficient. Among the three PTPs that are positive in all three assays, PTP-1b and TC-PTP are structurally very similar (
      • Hooft van Huijsduijnen R.
      ). TC-PTP is abundantly and widely expressed, but homozygous mice mutated in this gene die soon after birth (
      • You-Ten K.E.
      • Muise E.S.
      • Itie A.
      • Michaliszyn E.
      • Wagner J.
      • Jothy S.
      • Lapp W.S.
      • Tremblay M.L.
      ), so that the function of this PTP is as yet unresolved. Sap1 has very limited tissue expression that does not coincide with IRK expression (
      • Seo Y.
      • Matozaki T.
      • Tsuda M.
      • Hayashi Y.
      • Itoh H.
      • Kasuga M.
      ,
      • Matozaki T.
      • Suzuki T.
      • Uchida T.
      • Inazawa J.
      • Ariyama T.
      • Matsuda K.
      • Horita K.
      • Noguchi H.
      • Mizuno H.
      • Sakamoto C.
      • Kasuga M.
      ); its involvement in IRK regulation is therefore doubtful. Interestingly, the IRK phosphopeptide that we have used is from a short sequence that is perfectly shared between IRK and insulin-like growth factor 1 receptor. It is therefore likely that among the set of PTPs that had tested positive in Table II are some that (also) have autophosphorylated insulin-like growth factor 1 receptor as their substrate.
      Overall, we conclude that the combination of our membrane binding assay and activity tests, as applied to a large panel of PTPs, is a reliable first step in identifying potential substrates. The resulting small PTP set has then to be further evaluated in terms of intracellular localization of the PTPs (
      • Mauro L.J.
      • Dixon J.E.
      ), their tissue distribution, the effect of overexpression, and, ideally, gene inhibition using antisense or knock-out approaches. Shortening the list of PTP candidates before initiating these elaborate studies by a systematic approach as presented in this work should significantly shorten the path to PTP substrate assignments.

      REFERENCES

        • Fantl W.J.
        • Johnson D.E.
        • Williams L.T.
        Annu. Rev. Biochem. 1993; 62: 453-481
        • Kazlauskas A.
        Curr. Opin. Genet. Dev. 1994; 4: 5-14
        • Hooft van Huijsduijnen R.
        Gene (Amst.). 1998; 225: 1-8
        • Tonks N.K.
        • Neel B.G.
        Cell. 1996; 87: 365-368
        • Neel B.G.
        • Tonks N.K.
        Curr. Opin. Cell Biol. 1997; 9: 193-204
        • Morinville A.
        • Maysinger D.
        • Shaver A.
        Trends Pharmacol. Sci. 1998; 19: 452-460
        • Elchebly M.
        • Payette P.
        • Michaliszyn E.
        • Cromlish W.
        • Collins S.
        • Loy A.L.
        • Normandin D.
        • Cheng A.
        • Himms-Hagen J.
        • Chan C.C.
        • Ramachandran C.
        • Gresser M.J.
        • Tremblay M.L.
        • Kennedy B.P.
        Science. 1999; 283: 1544-1548
        • Flint A.J.
        • Tiganis T.
        • Barford D.
        • Tonks N.K.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685
        • Kishimoto T.K.
        • Kahn J.
        • Migaki G.
        • Mainolfi E.
        • Shirley F.
        • Ingraham R.
        • Rothlein R.
        Agents Actions. 1995; 47: 121-134
        • LaMontagne Jr., K.R.
        • Flint A.J.
        • Franza Jr., B.R.
        • Pandergast A.M.
        • Tonks N.K.
        Mol. Cell. Biol. 1998; 18: 2965-2975
        • Tiganis T.
        • Bennett A.M.
        • Ravichandran K.S.
        • Tonks N.K.
        Mol. Cell. Biol. 1998; 18: 1622-1634
        • Garton A.J.
        • Flint A.J.
        • Tonks N.K.
        Mol. Cell. Biol. 1996; 16: 6408-6418
        • Pannifer A.D.B.
        • Flint A.J.
        • Tonks N.K.
        • Barford D.
        J. Biol. Chem. 1998; 273: 10454-10462
        • Cote J.F.
        • Charest A.
        • Wagner J.
        • Tremblay M.L.
        Biochemistry. 1998; 37: 13128-13137
        • Taddei N.
        • Chiarugi P.
        • Cirri P.
        • Fiaschi T.
        • Stefani M.
        • Camici G.
        • Raugei G.
        • Ramponi G.
        FEBS Lett. 1994; 350: 328-332
        • Timms J.F.
        • Carlberg K.
        • Gu H.
        • Chen H.
        • Kamatkar S.
        • Nadler M.J.
        • Rohrschneider L.R.
        • Neel B.G.
        Mol. Cell. Biol. 1998; 18: 3838-3850
        • Kuroda K.
        • Hauser C.
        • Rott R.
        • Klenk H.D.
        • Doerfler W.
        EMBO J. 1986; 5: 1359-1365
        • Herrera R.
        • Lebwohl D.
        • Garcia de Herreros A.
        • Kallen R.G.
        • Rosen O.M.
        J. Biol. Chem. 1988; 263: 5560-5568
        • Zhang Z.Y.
        • Wu L.
        • Chen L.
        Biochemistry. 1995; 34: 16088-16096
        • Wishart M.J.
        • Dixon J.E.
        Trends Biochem. Sci. 1998; 23: 301-306
        • Mooney R.A.
        • Kulas D.T.
        • Bleyle L.A.
        • Novak J.S.
        Biochem. Biophys. Res. Commun. 1997; 235: 709-712
        • Seely B.L.
        • Staubs P.A.
        • Reichart D.R.
        • Berhanu P.
        • Milarski K.L.
        • Saltiel A.R.
        • Kusari J.
        • Olefsky J.M.
        Diabetes. 1996; 45: 1379-1385
        • Huyer G.
        • Liu S.
        • Kelly J.
        • Moffat J.
        • Payette P.
        • Kennedy B.
        • Tsaprailis G.
        • Gresser M.J.
        • Ramachandran C.
        J. Biol. Chem. 1997; 272: 843-851
        • Bell S.D.
        • Denu J.M.
        • Dixon J.E.
        • Ellington A.D.
        J. Biol. Chem. 1998; 273: 14309-14314
        • Kato H.
        • Faria T.N.
        • Stannard B.
        • Roberts Jr., C.T.
        • LeRoith D.
        Mol. Endocrinol. 1994; 8: 40-50
        • Ng D.H.
        • Harder K.W.
        • Clark-Lewis I.
        • Jirik F.
        • Johnson P.
        J. Immunol. Methods. 1995; 179: 177-185
        • Jia Z.
        • Barford D.
        • Flint A.J.
        • Tonks N.K.
        Science. 1995; 268: 1754-1758
        • Songyang Z.
        • Carraway III, K.L.
        • Eck M.J.
        • Harrison S.C.
        • Feldman R.A.
        • Mohammadi M.
        • Schlessinger J.
        • Hubbard S.R.
        • Smith D.P.
        • Eng C.
        • Lorenzo M.J.
        • Ponder B.A.J.
        • Mayer B.J.
        • Cantley L.C.
        Nature. 1995; 373: 536-539
        • Li P.M.
        • Zhang W.R.
        • Goldstein B.J.
        Cell. Signal. 1996; 8: 467-473
        • Ahmad F.
        • Considine R.V.
        • Goldstein B.J.
        J. Clin. Invest. 1995; 95: 2806-2812
        • Norris K.
        • Norris F.
        • Kono D.H.
        • Vestergaard H.
        • Pedersen O.
        • Theofilopoulos A.N.
        • Moller N.P.
        FEBS Lett. 1997; 415: 243-248
        • Ren J.M.
        • Li P.M.
        • Zhang W.R.
        • Sweet L.J.
        • Cline G.
        • Shulman G.I.
        • Livingston J.N.
        • Goldstein B.J.
        Diabetes. 1998; 47: 493-497
        • Schaapveld R.Q.
        • Schepens J.T.
        • Robinson G.W.
        • Attema J.
        • Oerlemans F.T.
        • Fransen J.A.
        • Streuli M.
        • Wieringa B.
        • Hennighausen L.
        • Hendriks W.J.
        Dev. Biol. 1997; 188: 134-146
        • You-Ten K.E.
        • Muise E.S.
        • Itie A.
        • Michaliszyn E.
        • Wagner J.
        • Jothy S.
        • Lapp W.S.
        • Tremblay M.L.
        J. Exp. Med. 1997; 186: 683-693
        • Seo Y.
        • Matozaki T.
        • Tsuda M.
        • Hayashi Y.
        • Itoh H.
        • Kasuga M.
        Biochem. Biophys. Res. Commun. 1997; 231: 705-711
        • Matozaki T.
        • Suzuki T.
        • Uchida T.
        • Inazawa J.
        • Ariyama T.
        • Matsuda K.
        • Horita K.
        • Noguchi H.
        • Mizuno H.
        • Sakamoto C.
        • Kasuga M.
        J. Biol. Chem. 1994; 269: 2075-2081
        • Mauro L.J.
        • Dixon J.E.
        Trends Biochem. Sci. 1994; 19: 151-155