Structural and Biochemical Evidence for an Autoinhibitory Role for Tyrosine 984 in the Juxtamembrane Region of the Insulin Receptor

Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of α-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in k cat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus. Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of α-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in k cat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus. The insulin receptor is a member of the receptor tyrosine kinase (RTK) 1The abbreviations used are: RTK, receptor tyrosine kinase; IRS, insulin receptor substrate; IGF1, insulin-like growth factor-1; IRR, insulin receptor-related receptor; IRK, tyrosine kinase domain of the insulin receptor; HEK, human embryonic kidney. family of cell surface receptors. Unlike the majority of RTKs, which are monomeric in the absence of ligand, the insulin receptor is a disulfide-linked heterotetramer comprising two extracellular α subunits and two membrane-spanning β subunits (1Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.C. Tsubokawa M. Mason A. Seeburg P.H. Grunfeld C. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1526) Google Scholar, 2Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz F. Kan Y.W. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (978) Google Scholar). Upon insulin binding to the α subunits, the insulin receptor undergoes a poorly characterized structural rearrangement that facilitates autophosphorylation of specific tyrosine residues in the cytoplasmic portion of the β subunits. Tyrosine autophosphorylation stimulates receptor catalytic (tyrosine kinase) activity (3Rosen O.M. Herrera R. Olowe Y. Petruzzelli L.M. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3237-3240Crossref PubMed Scopus (306) Google Scholar) and creates recruitment sites for downstream signaling molecules such as the insulin receptor substrate (IRS) proteins (4White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (625) Google Scholar) and APS (5Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). Three tyrosine residues in the cytoplasmic juxtamembrane region of the insulin receptor, Tyr-965, Tyr-972, and Tyr-984, are conserved to various extents in the insulin receptor subfamily of RTKs (Fig. 1). This subfamily includes the insulin receptor, the insulin-like growth factor-1 (IGF1) receptor, the insulin receptor-related receptor (IRR), and insulin receptor-like proteins in invertebrates such as Daf-2 in Caenorhabditis elegans and DIR in Drosophila melanogaster. Tyr-965, an autophosphorylation site that is not conserved in the invertebrate receptors, appears to be involved in receptor endocytosis (6Rajagopalan M. Neidigh J.L. McClain D.A. J. Biol. Chem. 1991; 266: 23068-23073Abstract Full Text PDF PubMed Google Scholar, 7Backer J.M. Shoelson S.E. Weiss M.A. Hua Q.X. Cheatham R.B. Haring E. Cahill D.C. White M.F. J. Cell Biol. 1992; 118: 831-839Crossref PubMed Scopus (87) Google Scholar). Tyr-972, invariant in the insulin receptor subfamily, is an essential autophosphorylation site (NPXY motif) that recruits IRS proteins (4White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (625) Google Scholar), Shc (8Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar), and Stat-5b (9Chen J. Sadowski H.B. Kohanski R.A. Wang L.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2295-2300Crossref PubMed Scopus (113) Google Scholar) via a phosphotyrosinebinding domain in these adapter proteins. Tyr-984 is also invariant in this subfamily, yet its role in insulin receptor signaling has not been determined. Biochemical studies indicate that Tyr-984 is not, to any appreciable extent, a site of autophosphorylation (10Kohanski R.A. Biochemistry. 1993; 32: 5773-5780Crossref PubMed Scopus (43) Google Scholar, 11Favelyukis S. Till J.H. Hubbard S.R. Miller W.T. Nat. Struct. Biol. 2001; 8: 1058-1063Crossref PubMed Scopus (281) Google Scholar). Previous crystallographic studies on the tyrosine kinase domain of the insulin receptor (IRK) have elucidated the mechanism by which trans-autophosphorylation of tyrosines in the kinase activation loop stimulates catalytic activity (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (962) Google Scholar, 13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (787) Google Scholar). These studies were performed using a cytoplasmic domain construct comprising the core tyrosine kinase domain (residues 989–1283) and the juxtamembrane region proximal to the kinase domain (residues 978–988). This original construct included two amino acid substitutions in the kinase-proximal juxtamembrane region, Cys-981 → Ser and Tyr-984 → Phe. Cys-981, which is not conserved even among mammalian insulin receptors, was substituted with serine to prevent formation of disulfide-linked dimers in vitro. At the time of the initial structural studies, it was unclear whether Tyr-984 was an autophosphorylation site, and to avoid potential autophosphorylation heterogeneity, this tyrosine was substituted with phenylalanine. Use of the Tyr-984 → Phe IRK protein (IRKY984F) in past structural studies, and the disordered state of Phe-984 therein, has left obscure the function of this conserved tyrosine. To elucidate the role of Tyr-984 in insulin receptor function, we have determined a crystal structure of unphosphorylated IRK containing wild-type Tyr-984. Although crystals of wild-type IRK were obtained, the N-terminal kinase lobe in this crystal form is poorly ordered, and the position of Tyr-984 could not be ascertained. Crystals of a mutant IRK in which conserved Asp-1132 in the catalytic loop was substituted with asparagine (IRKD1132N) diffract to high resolution, and a structure of this protein has been determined at 1.9 Å resolution. In this crystal structure, Tyr-984 is involved in several hydrophobic and hydrogen-bonding interactions with residues in α-helix C (αC) and in the five-stranded β sheet in the N-terminal lobe of the kinase. These interactions suggest that Tyr-984 serves a structural role in the kinase activation mechanism. Comparison of the in vitro steady-state kinetics properties of wild-type IRK and IRKY984F shows that the wild-type enzyme has a 4-fold lower k cat than IRKY984F. Moreover, full-length insulin receptors bearing a Tyr-984 → Ala mutation are hyperphosphorylated in transiently transfected mammalian cells, either in the basal (without insulin) state or upon insulin stimulation, consistent with an autoinhibitory role for Tyr-984. These data, together with previous structural studies of activated IRK (13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (787) Google Scholar), suggest a novel strategy for designing small molecule activators of the insulin receptor with potential application to noninsulin-dependent diabetes mellitus. IRK Protein Production—Site-directed mutagenesis was performed using the QuikChange system (Stratagene). Mutant IRK cDNAs were subcloned into the baculovirus vector pFastBac (Invitrogen). All of the constructs were verified by DNA sequencing. Wild-type and mutant IRK proteins (residues 978–1283) were purified from baculovirus-infected insect cells as described by Hubbard et al. (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (962) Google Scholar). Tris-phosphorylated IRK proteins were produced as described by Hubbard (13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (787) Google Scholar). Crystallographic Studies—Crystals of IRKD1132N were grown at 4 °C by vapor diffusion in hanging drops containing 1.5 μl of protein stock solution (15 mg/ml IRKD1132N, 50 mm Tris-HCl, pH 7.5, and 170 mm NaCl) plus 1.5 μl of reservoir buffer (20% polyethylene glycol 8000, 100 mm Tris-HCl, pH 7.5, 30 mm NaCl, and 5 mm dithiothreitol). The crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 57.47 Å, b = 69.48 Å, and c = 88.81 Å when frozen. There is one molecule in the asymmetric unit, and the solvent content is 51%. The crystals were transferred stepwise (in ethylene glycol content) into a cryo-solvent consisting of 20% polyethylene glycol 8000, 100 mm Tris-HCl, pH 7.5, 200 mm NaCl, and 15% ethylene glycol. Data from a single crystal were collected at Beamline at the and were using and W. 1997; PubMed Scopus Google Scholar). the unit cell of the IRKD1132N crystals was to the unit cell of crystals of an → Ala mutant IRK J.H. M. Kohanski R.A. Hubbard S.R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) Data was used as the for and of the IRKD1132N and were with J. M. L.M. Biol. 1998; PubMed Scopus Google Scholar), and was performed with T.A. M. A. 1991; PubMed Scopus Google Scholar). Steady-state kinetics for wild-type IRK and both the unphosphorylated and were using a M.A. Biochemistry. 1995; PubMed Scopus Google Scholar, M. F. M. J. Miller W.T. Nature. 1997; PubMed Scopus Google Scholar). All of the were at 30 °C in μl of buffer containing 100 mm Tris-HCl, pH 7.5, mm mm mg/ml and of either or 5 mm or mm and mg/ml of either or mg/ml and mm or mm were determined by data to the The of the initial measurements and of of the enzyme J.M. Ellis L. Biochem. 1992; PubMed Scopus Google Scholar). of for enzyme of with were data of Wild-type and Tyr-984 → Ala in mammalian vector the full-length insulin receptor was by R. in the insulin receptor were into the vector which the cytoplasmic domain of the receptor Kohanski R.A. Biochemistry. 1997; PubMed Scopus Google Scholar), using the QuikChange system (Stratagene). The was into as a as described by et al. M. E. Kohanski R.A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). Wild-type and mutant were purified using the cells were in and in with on the of μl of and of DNA were used to to the by the The cells were into two and grown for in with and The cells were for at °C in containing to insulin stimulation. insulin was with mm to various and to the cells for at The cells were on and with The cells were by the to of 200 μl of buffer mm pH 7.5, 200 mm NaCl, mm and mm and on for The cell were collected and by at for at 4 in the were on containing wild-type and mutant insulin receptors were on the to the proteins were transferred to that were in in mm pH 7.5, 100 mm NaCl, and and the kinase domain of the insulin receptor by R. were used to insulin receptor phosphorylation and protein was used as the was used for The phosphorylation content of insulin receptors was determined by the on a using for of crystal structure of IRKD1132N in its unphosphorylated state was determined by replacement and at 1.9 Å resolution. c in the of Tyr-984 is in and a of the structure appears in The data and are in data and in is for the = 100 in is for the includes protein and = 100 and c are the and structure was determined from of the protein The in is for the = 100 includes protein and = 100 and c are the and structure was determined from of the protein in a In the original crystal structure of unphosphorylated IRK (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (962) Google Scholar), the activation loop the between the and of the and in the activation loop as a to conserved Asp-1132 and in the catalytic This activation loop which the site, is of a M. E. Kohanski R.A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). In the IRKD1132N structure, the of asparagine for Asp-1132 in the catalytic loop between Asp-1132 and to a in which the activation loop is from the site and disordered (Fig. IRKD1132N has catalytic of the role of Asp-1132 in the mechanism M. E. Kohanski R.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of IRKD1132N is to the structure of IRK → in which of by in the activation loop is for the activation loop from to J.H. M. Kohanski R.A. Hubbard S.R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The in the IRKD1132N crystal structure is in with solution studies that the of the site in this and other IRK M. E. Kohanski R.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the juxtamembrane region proximal to the kinase domain (residues in the IRKD1132N structure from in IRK In the IRKD1132N structure, this in the N-terminal kinase lobe and Tyr-984 is in a hydrophobic at the between the five-stranded β sheet and in the N-terminal lobe (Fig. In to hydrophobic between Tyr-984 and and the group of Tyr-984 is to invariant in the insulin receptor is also to invariant In a of conserved residues in the insulin receptor subfamily is in the between the juxtamembrane region and the kinase Tyr-984, and (Fig. In the IRKD1132N structure, (αC) in with juxtamembrane two with the of and one with the of (Fig. and This of the juxtamembrane region is by between the of (αC) and of in wild-type and in the juxtamembrane is conserved in insulin and receptors and is a in In the crystal of the receptor kinase domain in its unphosphorylated S. M. L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) or state A. A. W. R. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), the tyrosine to Tyr-984, is in the hydrophobic as for Tyr-984 in the IRKD1132N is to conserved in in the receptor kinase in the IRKD1132N structure, Tyr-984 is to conserved This in the of in the receptor kinase could be a of the in the insulin receptor the receptor to Tyr-984 (Fig. 1). In the unphosphorylated IRKY984F structure (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (962) Google Scholar), the proximal juxtamembrane region is in the as in the IRKD1132N structure, the of Phe-984 is to solvent and and the hydrophobic between and the β sheet is with In this juxtamembrane conserved and are poorly ordered, and between and conserved is not The of the proximal juxtamembrane region in the activated IRK structure (13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (787) Google Scholar) is from the in the unphosphorylated IRKD1132N and IRKY984F In the activated the juxtamembrane is from the β (Fig. This juxtamembrane is to that in the structure of the form of the receptor kinase domain S. Till J.H. Hubbard S.R. Miller W.T. Nat. Struct. Biol. 2001; 8: 1058-1063Crossref PubMed Scopus (281) Google Scholar), which wild-type tyrosine at and in a space These structural indicate that the phosphorylation state of the activation loop the of the proximal juxtamembrane Steady-state of whether the interactions between the proximal juxtamembrane region, Tyr-984, and residues in the N-terminal kinase lobe catalytic the steady-state kinetics and were determined for the purified soluble kinase wild-type IRK and IRKY984F. measurements were performed using a M.A. Biochemistry. 1995; PubMed Scopus Google Scholar, M. F. M. J. Miller W.T. Nature. 1997; PubMed Scopus Google Scholar) on both the unphosphorylated and of the The data in demonstrate that is than IRKY984F in the basal The k cat for is lower than for with in the for and substrate and the k cat and are The in k cat upon phosphorylation of and IRKY984F are and by a in kinetics abbreviations are: unphosphorylated wild-type unphosphorylated Tyr-984 → Phe wild-type and Tyr-984 → Phe in a abbreviations are: unphosphorylated wild-type unphosphorylated Tyr-984 → Phe wild-type and Tyr-984 → Phe of Wild-type and Tyr-984 → Ala in from in vitro kinetics studies that Tyr-984 catalytic activity in the soluble kinase domain, we whether full-length insulin receptors bearing the Tyr-984 → Ala are hyperphosphorylated in cells were transiently transfected with either wild-type or Tyr-984 → Ala insulin receptors, and the of tyrosine phosphorylation of the receptors to and following with insulin was were performed and consistent The data in 4 demonstrate that tyrosine phosphorylation levels in the Tyr-984 → Ala insulin receptors are elevated the levels in wild-type receptors, both in the basal state and upon insulin stimulation. The of phosphorylation in the mutant receptors is in the absence of with an = increase of The of of mutant wild-type receptors as the of insulin was yet was at 100 insulin (Fig. At the time of the structural studies of IRK (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (962) Google Scholar), it was not whether Tyr-984, an invariant tyrosine in the juxtamembrane region of insulin receptor subfamily was a site of and an construct was in which Tyr-984 was by phenylalanine. To elucidate the role of conserved Tyr-984 in insulin receptor function, we and purified an IRK protein from baculovirus-infected insect cells containing tyrosine at initial in vitro it was that wild-type IRK autophosphorylation at a than IRKY984F. To the catalytic properties of wild-type IRK and we for enzyme the steady-state kinetics and substrate and k The results of these indicate that k cat is 4-fold lower for wild-type IRK than for IRKY984F in the basal To the mechanism by which wild-type IRK is catalytically in the basal state to we determined the crystal structure of unphosphorylated IRK containing Tyr-984. In this structure, the juxtamembrane region proximal to the kinase domain (residues in the N-terminal kinase with Tyr-984 in a hydrophobic between and the β sheet in the N-terminal lobe (Fig. The interactions of Tyr-984 in the β and a of the in the unphosphorylated and IRK suggest the structural mechanism by which Tyr-984 catalytic activity in the basal of the of IRK activation loop autophosphorylation and is the of the N-terminal lobe the lobe to (13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (787) Google Scholar). In to an of the N-terminal the lobe (Fig. This the β of facilitates formation of a catalytically important between conserved and conserved in the activated IRK structure, the proximal juxtamembrane region is from the β and These indicate in to the activation the proximal juxtamembrane by Tyr-984, from its catalytically In protein is a structural in the of catalytic in kinase is not to form the conserved acid J. J. Nature. 1993; PubMed Scopus Google Scholar). binding to kinase the β in of kinase even in the absence of activation loop phosphorylation K. E. J. J. Nature. 1995; PubMed Scopus Google Scholar). In the tyrosine kinase interactions between the domain and the a position of to kinase activity W. Nature. 1997; PubMed Scopus Google Scholar, F. J. Nature. 1997; PubMed Scopus Google Scholar). structural between and the activation loop has been for S. A. J. Nat. Struct. Biol. PubMed Scopus Google Scholar). of and rearrangement of the activation loop the interactions that the of in the loop of the structural and data indicate that activation of IRK in the basal state results from of the proximal juxtamembrane region from the β mutation of The wild-type IRK and IRKY984F have catalytic that the of the activation loop on is that of the proximal juxtamembrane To whether by Tyr-984 as for the soluble kinase domain is for maintaining phosphorylation in the full-length insulin receptor, we transiently transfected cells with either wild-type insulin receptors or mutant insulin receptors in which Tyr-984 was by These studies that in the absence of the tyrosine phosphorylation levels of Tyr-984 mutant receptors were significantly elevated to that of wild-type receptors and were elevated at insulin (Fig. The elevated tyrosine phosphorylation levels in the Tyr-984 → Ala mutant were not to a to the wild-type and mutant receptors kinetics not the of the Tyr-984 → Ala in the full-length insulin receptor is than for the → Ala described M. 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Biol. 2001; PubMed Scopus Google Scholar) or cells not a on or phosphorylation is In mutation of Tyr-984 in the soluble kinase domain a small increase in catalytic k results in a increase in the phosphorylation of the full-length receptor in The in the phosphorylation of these two mutant insulin receptors, Tyr-984 → Ala and → in to the position of in the kinase domain, the proximal juxtamembrane region plays a role in the mechanism by which insulin binding to the extracellular α subunits a structural rearrangement in the cytoplasmic β subunits. is that the interactions between the proximal juxtamembrane and in the N-terminal kinase lobe are important for maintaining a of the two kinase that trans-autophosphorylation in the basal of these interactions mutation of the by the extracellular of insulin) on the cytoplasmic The that insulin tyrosine phosphorylation of the Tyr-984 → Ala mutant receptor that in the receptor are other RTKs either tyrosine at the position of Tyr-984 in the insulin receptor or hydrophobic at that or a which function to Tyr-984, at with to growth a tyrosine at the position of Tyr-984, which in the crystal structure M.A. C. 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Y. K. S. K. M. A. M. Y. Y. Y. Y. 1998; PubMed Scopus Google or L.M. Ellis R. Struct. 8: Full Text Full Text PDF Scopus Google Scholar). Moreover, the of receptors in the insulin receptor subfamily receptors in this subfamily to mutation of conserved Tyr-984, as The structural of the autoinhibitory role of Tyr-984 in the basal state kinase a novel strategy for the of small molecule activators of the insulin receptor. to in the β and Tyr-984 the receptor in the absence of insulin and the receptor in the of The specific target site for such a molecule be the β as in the activated IRK structure (13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (787) Google Scholar) (Fig. of insulin receptor autophosphorylation by on the cytoplasmic domain have been Y. F. C. J. J.R. PubMed Scopus Google Scholar, Goldfine I.D. Kohanski R.A. S.R. S. 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