Biomedical subjects

M P Czech

Publications and source records attributed to M P Czech.

At least 37 records · Page 2Linked to original sources

PubMed1998

Stimulation of IRS-1-associated phosphatidylinositol 3-kinase and Akt/protein kinase B but not glucose transport by beta1-integrin signaling in rat adipocytes.

The signal transduction pathway by which insulin stimulates glucose transport is not understood, but a role for complexes of insulin receptor substrate (IRS) proteins and phosphatidylinositol (PI) 3-kinase as well as for Akt/protein kinase B (PKB) has been proposed. Here, we present evidence suggesting that formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation are insufficient to stimulate glucose transport in rat adipocytes. Cross-linking of beta1-integrin on the surface of rat adipocytes by anti-beta1-integrin antibody and fibronectin was found to cause greater IRS-1 tyrosine phosphorylation, IRS-1-associated PI 3-kinase activity, and Akt/PKB activation, detected by anti-serine 473 antibody, than did 1 nM insulin. Clustering of beta1-integrin also significantly potentiated stimulation of insulin receptor and IRS-1 tyrosine phosphorylation, IRS-associated PI 3-kinase activity, and Akt/PKB activation caused by submaximal concentrations of insulin. In contrast, beta1-integrin clustering caused neither a change in deoxyglucose transport nor an effect on the ability of insulin to stimulate deoxyglucose uptake at any concentration along the entire dose-response relationship range. The data suggest that (i) beta1-integrins can engage tyrosine kinase signaling pathways in isolated fat cells, potentially regulating fat cell functions and (ii) either formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation is not necessary for regulation of glucose transport in fat cells or an additional signaling pathway is required.

Adipocytes↗

PubMed1998

Interaction of insulin receptor substrate-1 with the sigma3A subunit of the adaptor protein complex-3 in cultured adipocytes.

Signaling through the insulin receptor tyrosine kinase involves its autophosphorylation in response to insulin and the subsequent tyrosine phosphorylation of substrate proteins such as insulin receptor substrate-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membrane-bound, and this localization may be important in targeting downstream signaling elements that mediate insulin action. Since IRS-1 localization to membranes may occur through its association with specific membrane proteins, a 3T3-F442A adipocyte cDNA expression library was screened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors. A cDNA clone that encodes sigma3A, a small subunit of the AP-3 adaptor protein complex, was demonstrated to bind IRS-1 utilizing this cloning strategy. The specific interaction between IRS-1 and sigma3A was further verified by in vitro binding studies employing baculovirus-expressed IRS-1 and a glutathione S-transferase (GST)-sigma3A fusion protein. IRS-1 and sigma3A were found to co-fractionate in a detergent-resistant population of low density membranes isolated from basal 3T3-L1 adipocytes. Importantly, the addition of exogenous purified GST-sigma3A to low density membranes caused the release of virtually all of the IRS-1 bound to these membranes, while GST alone had no effect. These results are consistent with the hypothesis that sigma3A serves as an IRS-1 receptor that may dictate the subcellular localization and the signaling functions of IRS-1.

3T3 Cells↗

PubMed1998

Cross-talk between insulin receptor and integrin alpha5 beta1 signaling pathways.

The ligation and clustering of cell surface alphabeta heterodimeric integrins enhances cell adhesion and initiates signaling pathways that regulate such processes as cell spreading, migration, differentiation, proliferation and apoptosis. Here we show that insulin treatment of Chinese hamster ovary cells expressing insulin receptors (CHO-T) markedly promotes cell adhesion onto a fibronectin matrix, but not onto bovine serum albumin or poly-lysine. Incubation of cells with a GRGDSP peptide that specifically binds integrins (but not the nonspecific GRADSP peptide) abolishes this insulin effect, as does the potent phosphoinositide 3-kinase (PI 3-kinase) inhibitor wortmannin. Moreover, a specific blocking monoclonal anti-alpha5beta1 integrin antibody, PB-1, blocks insulin-stimulated cell adhesion onto fibronectin. Conversely, activating alpha5beta1 integrins on CHO-T cells by adherence onto fibronectin markedly potentiates the action of insulin to enhance insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation. Activation of alpha5beta1 integrin also markedly potentiates the recruitment of p85-associated PI 3-kinase activity to IRS-1 in response to submaximal levels of insulin in CHO-T cells. These data indicate that insulin potently activates integrin alpha5beta1 mediated CHO-T cell adhesion, while integrin alpha5beta1 signaling in turn enhances insulin receptor kinase activity and formation of complexes containing IRS-1 and PI 3-kinase. These findings raise the hypothesis that insulin receptor and alpha5beta1 integrin signaling act synergistically to enhance cell adhesion.

Androstadienes↗

PubMed1998

Association of acyl-CoA synthetase-1 with GLUT4-containing vesicles.

GLUT4, the glucose transporter present in insulin-sensitive tissues, resides in intracellular vesicular structures and translocates to the cell surface in response to insulin. In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from rat adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 antibody. We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein band, long chain acyl-CoA synthetase-1, specifically present in immunoadsorbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimmune serum. Acyl-CoA synthetase activity detected in GLUT4-enriched vesicles prepared by gradient centrifugation from insulin-treated adipocytes was decreased to about the same extent as GLUT4 protein. Additionally, immunoadsorbed GLUT4 vesicles were found to catalyze palmitoylation of proteins when incubated with labeled palmitate, a pathway that requires palmitate esterification with CoA. These data indicate that the insulin-sensitive membrane compartment that sequesters GLUT4 in fat cells contains long chain acyl-CoA synthetase-1 and its product fatty acyl-CoA, shown previously to be required for budding and fusion in membrane trafficking processes.

Acyl Coenzyme A↗

PubMed1998

Regulation of GRP1-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol 3,4,5-trisphosphate.

Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases. This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that dioctanoyl PtdIns(3,4,5)P3 binds the PH domain of GRP1 with a Kd = 0.5 microM, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns(4,5)P2. Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and a low concentration of sodium cholate. PtdIns(3,4,5)P3-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 microM inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to PtdIns(3,4,5)P3 in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking.

ADP-Ribosylation Factor 1↗

PubMed1998

Direct targets of phosphoinositide 3-kinase products in membrane traffic and signal transduction.

Phosphoinositide 3-kinases are a family of lipid kinases that phosphorylate the 3' position of the inositol ring on phosphatidylinositol and higher-phosphorylated polyphosphoinositides. The multiplicity of cellular functions influenced by the activity of these enzymes has captured the attention of researchers working on two important fields of cell biology--signal transduction and membrane traffic. This review discusses how the recent identification of proteins that interact directly with 3'-polyphosphoinositides has revealed novel potential interconnections between these fundamental cellular processes.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1998

Effects of general receptor for phosphoinositides 1 on insulin and insulin-like growth factor I-induced cytoskeletal rearrangement, glucose transporter-4 translocation, and deoxyribonucleic acid synthesis.

We investigated the effects of general receptor for phosphoinositides-1 (GRP1), a recently cloned protein that binds 3,4,5-phosphatidylinositol [PtdIns(3,4,5)P3] with high affinity, but not PtdIns(3,4)P2 nor PtdIns(3)P, on insulin and insulin-like growth factor I (IGF-I)-induced cytoskeletal rearrangement, glucose transporter-4 (GLUT4) translocation, and DNA synthesis. GRP1 consists of an NH2-terminally located coiled coil domain followed by a Sec7 domain and a COOH-terminal pleckstrin homology (PH) domain that is required for PtdIns binding. We used microinjection of glutathione-S-transferase fusion proteins containing residues 239-399 (PH domain), residues 52-260 (Sec7 domain), residues 5-71 (N-terminal domain), full-length GRP1, and an antibody (AB) raised against full-length GRP1 coupled with immunofluorescent detection of actin filament rearrangement, GLUT4 translocation, and 3'-bromo-5'-deoxyuridine incorporation. Microinjection of these constructs and the AB had no effect on insulin-induced GLUT4 translocation or DNA synthesis. However, microinjection of the GRP1-PH and the GRP1-Sec7 domain as well as the alpha-GRP1-AB significantly inhibited insulin- and IGF-I-stimulated actin rearrangement in an insulin receptor-overexpressing cell line (HIRcB) compared with that in control experiments. Coinjection of GRP1-Sec7 along with constitutively active Rac (Q67L) did not inhibit Rac-induced actin rearrangement. Furthermore, GRP1 is not able to bind and act as a nucleotide exchange factor for the small GTP-binding proteins of the Rho family. As GRP1 acts as a guanine nucleotide exchange factor for ARF6 proteins, we propose a signaling pathway distinct from the small GTP-binding protein Rac, connecting PtdIns(3,4,5)P3 via GRP1 to ARF6, leading to insulin- and IGF-I-induced actin rearrangement.

3T3 Cells↗

PubMed1997

A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains.

Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4, 5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1997

Association of cytosolic Rab4 with GDI isoforms in insulin-sensitive 3T3-L1 adipocytes.

Translocation of an intracellular pool of GLUT4 glucose transporters to the fat and muscle cell surface is thought to involve small GTP-binding proteins such as the Rab4 protein. The cycling of Rab proteins between cytosol and intracellular membranes necessary for their function appears to be regulated by GDP-dissociation inhibitors (GDI), three of which have been cloned thus far. Previous data suggest that Rab4 binds two of these isoforms of GDI (1 and 2) similarly when purified proteins are employed [Shisheva, A., et al. (1994) Mol. Cell. Biol. 14, 3459-3468]. In the present study, we have analyzed the cytosolic Rab4 in complexes with GDI-1 or GDI-2 in intact cells using a coprecipitation technique. We show here that in insulin-sensitive 3T3-L1 adipocytes and other cultured cells, Rab4 simultaneously forms stable cytosolic complexes with both GDI-1 and GDI-2. Acute insulin treatment of the cultured adipocytes significantly increases cytosolic levels of Rab4 which can be quantitatively immunoprecipitated with anti-Rab4 antibodies. Surprisingly, the increased cytosolic Rab4 due to insulin action is predominantly associated with cytosolic GDI-1. The levels of cytosolic Rab4-GDI-2 complexes were virtually unaltered by insulin. Insulin-dependent alterations of Rab4 and GDI-1 phosphorylation were not detected in 32P-labeled 3T3-L1 adipocytes, suggesting another mechanism accounts for the specificity of Rab4 binding to GDI-1. Taken together, these data suggest there is selective formation of Rab4-GDI-1 complexes in response to insulin which plays a role in the action of insulin on membrane trafficking.

3T3 Cells↗

PubMed1997

Signaling by phosphoinositide-3,4,5-trisphosphate through proteins containing pleckstrin and Sec7 homology domains.

Signal transmission by many cell surface receptors results in the activation of phosphoinositide (PI) 3-kinases that phosphorylate the 3' position of polyphosphoinositides. From a screen for mouse proteins that bind phosphoinositides, the protein GRP1was identified. GRP1 binds phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4, 5)P3] through a pleckstrin homology (PH) domain and displays a region of high sequence similarity to the yeast Sec7 protein. The PH domain of the closely related protein cytohesin-1, which, through its Sec7 homology domain, regulates integrin beta2 and catalyzes guanine nucleotide exchange of the small guanine nucleotide-binding protein ARF1, was also found to specifically bind PtdIns(3,4,5)P3. GRP1 and cytohesin-1 appear to connect receptor-activated PI 3-kinase signaling pathways with proteins that mediate biological responses such as cell adhesion and membrane trafficking.

ADP-Ribosylation Factor 1↗

PubMed1997

Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development.

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Regulation of phosphatidylinositol 3,4,5-trisphosphate 5'-phosphatase activity by insulin.

Polyphosphoinositides are thought to be mediators of cellular signaling pathways as well as regulators of cytoskeletal elements and membrane trafficking events. It has recently been demonstrated that a class of phosphatidylinositol (PI) 3,4,5-P3 5'-phosphatases contains SH2 domains and proline-rich regions, which are present in many signaling proteins. We report here that insulin stimulation of Chinese hamster ovary cells (CHO-T) expressing human insulin receptors causes an 8-10-fold increase in PI 3,4,5-P3 5'-phosphatase activity in anti-phosphotyrosine immunoprecipitates of the cell lysates. This insulin-sensitive polyphosphoinositide 5'-phosphatase did not catalyze dephosphorylation of PI 4,5-P2. No change in 5'-phosphatase activity was detected in insulin receptor or IRS-1 immune complexes in response to insulin. However, insulin treatment of CHO-T cells markedly increased the PI 3,4,5-P3 5'-phosphatase activity associated with Shc and Grb2. The insulin-regulated polyphosphoinositide 5'-phosphatase was not immunoreactive with antibody raised against the recently cloned SHIP 5'-phosphatase reported to associate with Shc and Grb2 in B lymphocytes. These data demonstrate that insulin causes formation of complexes containing a PI 3,4,5-P3 5'-phosphatase, and Shc or Grb2, or both, suggesting an important role of this enzyme in insulin signaling.

Animals↗

PubMed1996

Role of the Raf/mitogen-activated protein kinase pathway in p21ras desensitization.

Desensitization of p21(ras) after stimulation of cells by growth factors and phorbol 12-myristate 13-acetate (PMA) correlates with hyperphosphorylation of the guanine nucleotide exchange factor Son-of-sevenless (Sos) and its dissociation from the adaptor protein Grb2 (Cherniack, A., Klarlund, J. K., Conway, B. R., and Czech, M. P. (1995) J. Biol. Chem. 270, 1485-1488). To test the role of the Raf/mitogen-activated protein (MAP) kinase pathway, we utilized cells expressing a chimera composed of the catalytic domain of p74Raf-1 and the hormone binding domain of the estradiol receptor (DeltaRaf-1:ER). Estradiol markedly stimulated DeltaRaf-1:ER and the downstream MEK and MAP kinases in these cells as well as Sos phosphorylation. However, the dissociation of Grb2 from Sos observed in response to PMA was not apparent upon DeltaRaf-1:ER activation. Furthermore, stimulation of DeltaRaf-1:ER did not impair GTP loading of p21(ras) in response to platelet-derived growth factor or epidermal growth factor. We conclude that activation of the Raf/MAP kinase pathway alone in these cells is insufficient to cause disassembly of Sos from Grb2 or to interrupt the ability of Sos to catalyze activation of p21(ras).

3T3 Cells↗

PubMed1996

Mouse p170 is a novel phosphatidylinositol 3-kinase containing a C2 domain.

Phosphatidylinositol (PI) 3-kinases catalyze the formation of 3'-phosphoinositides, which appear to promote cellular responses to growth factors and such membrane trafficking events as insulin-stimulated translocation of intracellular glucose transporters. We report here the cloning of a novel PI 3-kinase, p170, from cDNA of insulin-sensitive mouse 3T3-L1 adipocytes. Mouse p170 utilizes PI and to a limited extent PI 4-P as substrates, in contrast to the PI-specific yeast VPS34 homolog PtdIns 3-kinase and the p110 PI 3-kinases, which phosphorylate PI, PI 4-P, and PI 4,5-P2. Mouse p170 is also distinct from PtdIns 3-kinase or the p110 PI 3-kinases in exhibiting a 10-fold lower sensitivity to wortmannin. Unique structural elements of p170 include C-terminal sequences strikingly similar to the phosphoinositide-binding C2 domain of protein kinase C isoforms, synaptotagmins, and other proteins. These features of mouse p170 are shared with a recently cloned Drosophila PI 3-kinase, DmPI3K_68D. Together, these proteins define a new class of PI 3-kinase likely influenced by cellular regulators distinct from those acting upon p110- or VPS34-like PI 3-kinases.

3T3 Cells↗

PubMed1996

Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles.

Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3-kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller-Harrison, R., Morin, M. and Czech, M. (1995) J. Biol. Chem. 270, 24442-24450). We report here that in response to insulin, activated complexes of IRS-1.PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1 adipocytes. The targeting of PI 3-kinase to rat adipocyte GLUT4-containing vesicles using vesicles prepared by sucrose velocity gradient ultracentrifugation was also demonstrated. Insulin treatment caused a 2.3-fold increase in immunoreactive p85 protein in these GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI 3-kinase activity in GLUT4-containing vesicle extracts increased to a similar extent. HPLC analysis of the GLUT4 vesicle-associated PI 3-kinase activity showed insulin-mediated increases in PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 when PI, PI 4-P, and PI 4,5-P2 were used as substrates. Our data demonstrate that insulin directs the association of PI 3-kinase with GLUT4-containing vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis that PI 3-kinase is involved in the insulin-regulated movement of GLUT4 to the plasma membrane.

3T3 Cells↗

PubMed1996

The beta-adrenergic receptor is a substrate for the insulin receptor tyrosine kinase.

G-protein-linked receptors and intrinsic tyrosine-kinase growth receptors represent two prominent modalities in cell signaling. Cross-regulation among members of both receptor superfamilies has been reported, including the counter-regulatory effects of insulin on beta-adrenergic catecholamine action. Cells stimulated by insulin show loss of function and increased phosphotyrosine content of beta 2-adrenergic receptors. Phosphorylation of tyrosyl residues 350/354 of beta 2-adrenergic receptors is obligatory for counter-regulation by insulin (Karoor, V., Baltensperger, K., Paul, H., Czech, M., and Malbon, C. C. (1995) J. Biol. Chem. 270, 25305-25308), suggesting the hypothesis that G-protein-linked receptors themselves may act as substrates for the insulin receptor and other growth factor receptors. This hypothesis was evaluated directly using recombinant human insulin receptor, hamster beta 2-adrenergic receptor, and an vitro reconstitution and phosphorylation assay. Insulin is shown to stimulate insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor. Phosphoamino acid analysis establishes that insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor in vitro is confined to phosphotyrosine. High pressure liquid chromatography and two-dimensional mapping reveal insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor at residues Tyr132/Tyr141, Tyr350/Tyr354, and Tyr364, known sites of phosphorylation in response to insulin in vivo. Insulin-like growth factor-I receptor as well as the insulin receptor displays the capacity to phosphorylate the beta 2-adrenergic receptor in vitro, establishing a new paradigm, i.e. G-protein-linked receptors acting as substrates for intrinsic tyrosine kinase growth factor receptors.

Amino Acid Sequence↗

PubMed1996

The rat T-cell surface protein RT6 is associated with src family tyrosine kinases and generates an activation signal.

RT6 is a glycosyl-phosphatidylinositol-linked surface molecule present on most mature rat T-cells. RT6+ T-cells can prevent the expression of autoimmune diabetes in the BB rat, but the mechanism is unknown. Because cross-linking of other glycosyl-phosphatidylinositol-linked T-cell proteins is known to activate T-cells, we investigated the signaling properties of RT6. Antibody cross-linking of RT6 enhanced expression of the alpha subunit of the interleukin-2 (IL-2) receptor and potentiated the proliferation of rat T-cells cultured in the presence of phorbol ester plus recombinant IL-2 (rIL-2) and/or rIL-4. RT6 was found to coimmunoprecipitate with five tyrosine phosphorylated proteins including p60fyn and p56lck, members of the src tyrosine kinase family. Pretreatment of T-cells with phorbol ester increased the phosphorylation of proteins that coimmunoprecipitated with RT6, altered the electrophoretic mobility of several of these coimmunoprecipitated phosphoproteins, and increased the amount of p60fyn and p56lck coimmunoprecipitated with RT6. These data indicate that RT6-mediated signaling events may prime T-cells to respond to exogenous cytokines, suggesting a possible mechanism by which surface RT6 may influence T-cell function.

ADP Ribose Transferases↗

PubMed1995

Phosphorylation of tyrosyl residues 350/354 of the beta-adrenergic receptor is obligatory for counterregulatory effects of insulin.

Insulin stimulates a loss of function and increased phosphotyrosine content of the beta 2-adrenergic receptor in intact cells, raising the possibility that the beta 2-receptor itself is a substrate for the insulin receptor tyrosine kinase. Phosphorylation of synthetic peptides corresponding to cytoplasmic domains of the beta 2-adrenergic receptor by the insulin receptor in vitro and peptide mapping of the beta 2-adrenergic receptor phosphorylated in vivo in cells stimulated by insulin reveal tyrosyl residues 350/354 and 364 in the cytoplasmic, C-terminal region of the beta 2-adrenergic receptor as primary targets. Mutation of tyrosyl residues 350, 354 (double mutation) to phenylalanine abolishes the ability of insulin to counterregulate beta-agonist stimulation of cyclic AMP accumulation. Phenylalanine substitution of tyrosyl reside 364, in contrast, abolishes beta-adrenergic stimulation itself.

Amino Acid Sequence↗