Mutation of tyrosine 960 within the insulin receptor juxtamembrane domain impairs glucose transport but does not inhibit ligand-mediated phosphorylation of insulin receptor substrate-2 in 3T3-L1 adipocytes.

CSF-1 is equipotent to insulin in its ability to stimulate 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes expressing the colony stimulating factor-1 receptor/insulin receptor chimera (CSF1R/IR). However, CSF-1-stimulated glucose uptake and glycogen synthesis is reduced by 50% in comparison to insulin in 3T3-L1 cells expressing a CSF1R/IR mutated at Tyr960 (CSF1R/IRA960). CSF-1-treated adipocytes expressing the CSF1R/IRA960 were impaired in their ability to phosphorylate insulin receptor substrate 1 (IRS-1) but not in their ability to phosphorylate IRS-2. Immunoprecipitation of IRS proteins followed by Western blotting revealed that the intact CSF1R/IR co-precipitates with IRS-2 from CSF-1-treated cells. In contrast, the CSF1R/IRA960 co-precipitates poorly with IRS-2. These observations suggest that Tyr960 is important for interaction of the insulin receptor cytoplasmic domain with IRS-2, but it is not essential to the ability of the insulin receptor tyrosine kinase to use IRS-2 as a substrate. These observations also suggest that in 3T3-L1 adipocytes, tyrosine phosphorylation of IRS-2 by the insulin receptor tyrosine kinase is not sufficient for maximal stimulation of receptor-regulated glucose transport or glycogen synthesis.

Phosphorylation of the kinase suppressor of ras by associated kinases.

The kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. The physiological role of mammalian KSR is not known. We examined the mechanisms regulating the phosphorylation of this putative kinase in mammalian cells. Wild-type mouse KSR and a mutated KSR protein predicted to create a kinase-dead protein are phosphorylated identically in intact cells and in the immune complex. Phosphopeptide sequencing identified 10 in vivo phosphorylation sites in KSR, all of which reside in the 539 noncatalytic amino terminal amino acids. Expression of the amino terminal portion of KSR alone demonstrated that it was phosphorylated in the intact cell and in an immune complex in a manner indistinguishable from that of intact KSR. These data demonstrate that the kinase domain of KSR is irrelevant to its phosphorylation state and suggest that the phosphorylation of KSR and its association with a distinct set of kinases may affect intracellular signaling.

Kinase suppressor of Ras inhibits the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase by growth factors, activated Ras, and Ras effectors.

Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. We examined the effect of mammalian KSR expression on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibited the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrated that the KSR kinase domain was necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibited cell transformation by activated RasVal-12 but had no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development.

CSF-1 receptor/insulin receptor chimera permits CSF-1-dependent differentiation of 3T3-L1 preadipocytes.

A chimeric growth factor receptor (CSF1R/IR) was constructed by splicing cDNA sequences encoding the extracellular ligand binding domain of the human colony stimulating factor-1 (CSF-1) receptor to sequences encoding the transmembrane and cytoplasmic domains of the human insulin receptor. The addition of CSF-1 to cells transfected with the CSF1R/IR chimera cDNA stimulated the tyrosine phosphorylation of a protein that was immunoprecipitated by an antibody directed against the carboxyl terminus of the insulin receptor. Phosphopeptide maps of the 32P-labeled CSF1R/IR protein revealed the same pattern of phosphorylation observed in 32P-labeled insulin receptor beta subunits. CSF-1 stimulated the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and Shc in cells expressing the CSF1R/IR chimera. Lipid accumulation and the expression of a differentiation-specific marker demonstrated that 3T3-L1 preadipocytes undergo CSF-1-dependent differentiation when transfected with the CSF1R/IR chimera cDNA but not when transfected with the expression vector alone. A 12-amino acid deletion within the juxtamembrane region of the CSF1R/IR (CSF1R/IRDelta960) blocked CSF-1-stimulated phosphorylation of IRS-1 and Shc but did not inhibit CSF-1-mediated differentiation of 3T3-L1 preadipocytes. These observations indicate that adipocyte differentiation can be initiated by intracellular pathways that do not require tyrosine phosphorylation of IRS-1 or Shc.

Expression of the insulin receptor with a recombinant vaccinia virus. Biochemical evidence that the insulin receptor has intrinsic serine kinase activity.

We have previously reported the tight association of a serine kinase activity with the human insulin receptor (Lewis, R. E., Wu, G. P., MacDonald, R. G., and Czech, M. P. (1990) J. Biol. Chem. 265, 947-954). We tested the possibility that the associated serine kinase activity was intrinsic to the receptor catalytic domain. The ratio of phosphoserine to phosphotyrosine on insulin receptors phosphorylated in vitro was used as an index of the associated serine kinase activity. Phosphorylation and phosphoamino acid analysis of insulin proreceptors revealed associated serine kinase activity early in receptor synthesis. Insulin receptors were expressed in HeLa cells using a recombinant vaccinia virus. The ratio of phosphoserine to phosphotyrosine on insulin receptors expressed by the recombinant vaccinia virus was determined relative to endogenous insulin receptors in cells treated with alpha-amanitin to block host cell mRNA synthesis. alpha-Amanitin treatment had no effect on the ratio of phosphoserine to phosphotyrosine on insulin receptors expressed from the recombinant virus even though they were present in a 4000-fold excess above endogenous receptors. We conclude that the serine kinase activity associated with the insulin receptor is intrinsic to the receptor catalytic domain. Receptor-catalyzed autophosphorylation of serine may play an important role in modulating insulin receptor signaling.

Phorbol ester stimulates phosphorylation on serine 1327 of the human insulin receptor.

Phorbol esters stimulate the phosphorylation of the insulin receptor on discrete serine and threonine residues in intact cells. Phosphorylation of the insulin receptor cytoplasmic domain on serine, threonine, and tyrosine residues regulates receptor tyrosine kinase activity and signaling. In these studies, we demonstrate that phorbol ester treatment of intact COS-1 cells transiently expressing the human insulin receptor stimulates phosphorylation of serine 1327 within the carboxyl-terminal tail of the insulin receptor beta subunit. Phosphopeptide maps of wild-type (Ser1327) and mutant (Ala1327) human insulin receptors revealed the absence of a single phosphopeptide in the Ala1327 receptors when compared with wild-type receptors from phorbol ester-treated cells. Phosphoamino acid analysis revealed phosphoserine within the phosphopeptide from wild-type receptors that is absent in the Ala1327 receptor. The synthetic peptide 1327S (KRSYEEHIPYTHMNGGKK) corresponding to amino acids 1325-1342 of the human insulin receptor is phosphorylated on serine by protein kinase C. After digestion with trypsin, the phosphorylated synthetic peptide comigrated with the serine-phosphorylated peptide isolated from wild-type insulin receptors that was absent from the Ala1327 mutant. Ser1327 is proximal to autophosphorylation sites Tyr1328 and Tyr1334. The potential effects of serine phosphorylation at position 1327 on subsequent phosphorylation of these tyrosines by the insulin receptor kinase were examined using synthetic peptides. The chemically modified peptide 1327S(P) was synthesized with the stoichiometric addition of phosphate to the side chain hydroxyl of a serine corresponding to position 1327 of the insulin receptor. Kinetic analysis revealed that the addition of phosphate to the serine improved substrate recognition by the insulin receptor tyrosine kinase almost 2-fold. The average Km was 1.44 mM for the peptide 1327S(P) versus 2.64 mM for peptide 1327S. However, when compared with the unphosphorylated control peptide, 1327S, the serine-phosphorylated peptide 1327S(P) also reduced the Vmax of the insulin receptor tyrosine kinase 53%. Radiosequence analysis revealed that the chemical addition of phosphate to the serine in peptide 1327S(P) inhibited insulin receptor-catalyzed phosphorylation of the tyrosine on 1327S(P) corresponding to Tyr1334 but not of the tyrosine corresponding to Tyr1328. These data suggest that the juxtaposition of a serine phosphorylation site adjacent to receptor tyrosine phosphorylation sites provides the potential for regulation of insulin receptor autophosphorylation and signaling through its carboxyl-terminal tail.

Affinity chromatography of catalytic autoantibody to vasoactive intestinal peptide.

An autoantibody in human IgG that hydrolyzes vasoactive intestinal peptide (VIP) was identified. The IgG did not hydrolyze VIP unless an unidentified inhibitor was removed by dialysis. The VIP antibody was fractionated by affinity chromatography on immobilized VIP by using IgG without VIP-hydrolytic activity as the starting material. The affinity-purified antibody catalyzed the hydrolysis of VIP (nominal kcat/Km: 1.1 X 10(6) M-1 min-1). The values of Km for the affinity-purified antibody preparation and unfractionated IgG (110 nM and 112 nM) suggested relatively tight antibody-VIP binding. A comparison of the reverse phase HPLC profiles of antibody-treated [Tyr10-125I]VIP with that of synthetic [125I]VIP(1-16) suggested that unfractionated IgG and the affinity-purified antibody cleaved the same peptide bond in VIP (Gln16-Met17).

Site specificity of a catalytic vasoactive intestinal peptide antibody. An inhibitory vasoactive intestinal peptide subsequence distant from the scissile peptide bond.

Ten fragments of vasoactive intestinal peptide (VIP) were tested for reactivity with a human catalytic autoantibody that cleaves full-length VIP(1-28) at the Gln16-Met17 peptide bond. A large COOH-terminal subsequence, VIP(15-28), was bound by the autoantibody with high affinity (Ki 1.25 nM), suggesting that it is the antibody binding epitope. VIP(22-28), a short subsequence distant from the scissile bond, inhibited the binding (Ki 242 microM) and hydrolysis (Ki 260 microM) of full-length VIP by the catalytic autoantibody in a competitive fashion. The autoantibody did not show detectable binding of short VIP subsequences that encompass the scissile bond (VIP(15-21), VIP(11-17), and VIP(13-20]. These data show that residues 22-28, located four amino acids distant from the scissile bond, contribute in recognition of VIP by the catalytic autoantibody.

Characterization of autoantibodies to vasoactive intestinal peptide in asthma.

Vasoactive intestinal peptide (VIP) is a potent relaxant of the airway smooth muscle. In this study, VIP-binding autoantibodies were observed in the plasma of 18% asthma patients and 16% healthy subjects. Immunoprecipitation studies and chromatography on DEAE-cellulose and immobilized protein G indicated that the plasma VIP-binding activity was largely due to IgG antibodies. Saturation analysis of VIP binding by the plasmas suggested the presence of one or two classes of autoantibodies, distinguished by their apparent equilibrium affinity constants (Ka). The autoantibodies from asthma patients exhibited a larger VIP-binding affinity compared to those from healthy subjects (Ka 7.8 x 10(9) M-1 and 0.13 x 10(9) M-1, respectively; P less than 0.005). The antibodies were specific for VIP, judged by their poor reaction with peptides bearing partial sequence homology with VIP (peptide histidine isoleucine, growth hormone releasing factor and secretin). IgG prepared from the plasma of an antibody-positive asthma patient inhibited the saturable binding of 125I-VIP by receptors in guinea pig lung membranes (by 39-59%; P less than 0.001). These observations are consistent with a role for the VIP autoantibodies in the airway hyperresponsiveness of asthma.

Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody.

Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.

Receptor binding of guinea pig and pig vasoactive intestinal peptides by rat lung.

Guinea pig vasoactive intestinal peptide (gpVIP) differs from other mammalian VIPs in four of its 28 amino acid residues. In the present study, the gpVIP displaced 125I-labelled pig VIP (pVIP) binding by rat lung membranes with 7.7-fold lower potency than pVIP. Degradation of gpVIP by rat lung membranes, assessed by radioimmunoassay and h.p.l.c., was 1.9-fold greater than that of pVIP. This difference in degradation of the two peptides was not large enough to account for the lower receptor-binding potency of gpVIP. The amino acid residues that distinguish pVIP from gpVIP are likely to contribute to the interaction of VIP with receptors and peptide hydrolases in lung membranes.