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D W Rose

Publications and source records attributed to D W Rose.

53 records · Page 3Linked to original sources
PubMed1996

Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors. Evidence for a role in mitogenic signaling.

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. We identified a human cDNA that is a splice variant of the human GRB10 homolog GRB-IR, which we term GRB10/IR-SV1 (for GRB10/GRB-IR splice variant 1). The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domain and a pleckstrin homology domain. Cloning of a full-length human cDNA revealed a predicted coding sequence that was similar to the mouse GRB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletion. The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such that GRB10/IR-SV1 contains an intact pleckstrin homology domain and a distinct amino terminus. The interaction of GRB10/IR-SV1 with the insulin receptor and the insulin-like growth factor I (IGF-I) receptor is mediated by the SH2 domain, and we show that glutathione S-transferase-SH2 domain fusion proteins interact specifically in vitro with the insulin receptor derived from mammalian cells. The GRB10/IR-SV1 SH2 domain also interacted with an approximately 135-kDa phosphoprotein from unstimulated cell lysates, an interaction that decreased after insulin stimulation. We present evidence that the GRB10/IR-SV1 protein plays a functional role in insulin and IGF-I signaling by showing that microinjection of an SH2 domain fusion protein inhibited insulin- and IGF-I-stimulated mitogenesis in fibroblasts, yet had no effect on mitogenesis induced by epidermal growth factor. Our findings suggest that GRB10/IR-SV1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.

Adipose Tissue↗
PubMed1996

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.

3T3 Cells↗
PubMed1996

A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors.

Nuclear receptors regulate gene expression by direct activation of target genes and inhibition of AP-1. Here we report that, unexpectedly, activation by nuclear receptors requires the actions of CREB-binding protein (CBP) and that inhibition of AP-1 activity is the apparent result of competition for limiting amounts of CBP/p300 in cells. Utilizing distinct domains, CBP directly interacts with the ligand-binding domain of multiple nuclear receptors and with the p160 nuclear receptor coactivators, which upon cloning have proven to be variants of the SRC-1 protein. Because CBP represents a common factor, required in addition to distinct coactivators for function of nuclear receptors, CREB, and AP-1, we suggest that CBP/p300 serves as an integrator of multiple signal transduction pathways within the nucleus.

Animals↗
PubMed1996

Involvement of ErbB2 in the signaling pathway leading to cell cycle progression from a truncated epidermal growth factor receptor lacking the C-terminal autophosphorylation sites.

To investigate the mechanisms underlying the enhanced mitogenic activity of the truncated epidermal growth factor receptor (EGFR) lacking the C-terminal autophosphorylation sites (Delta973-EGFR), we studied the intracellular signaling pathways in NR6 cells expressing human wild type EGFR and Delta973-EGFR. Microinjection of dominant/negative p21ras(N17) completely inhibited EGF-induced DNA synthesis in both cell types. EGF stimulated Shc phosphorylation as well as the formation of wild type EGFR.Shc complexes. In contrast, EGF stimulated Shc phosphorylation without formation of Delta973-EGFR.Shc complexes. Tyrosine-phosphorylated Shc formed complexes with Grb2.Sos, and microinjection of anti-Shc antibody and Shc-SH2 GST fusion protein inhibited EGF stimulation of DNA synthesis in both cell lines. EGF markedly increased ErbB2 tyrosine phosphorylation in wild type EGFR cells. In Delta973-EGFR cells, ErbB2 was tyrosine phosphorylated in the basal state and EGFR stimulated further phosphorylation of ErbB2. In addition to ErbB2, additional proteins were tyrosine phosphorylated in Delta973-EGFR cells, mostly in the molecular mass range of 120 170 kDa. Taken together with our findings indicating coupling of ErbB2 to Shc, these data suggest the importance of an alternative signaling pathway in Delta973-EGFR cells mediated by the formation of heterodimeric structures between the truncated EGFR and ErbB2, followed by coupling through Shc to Grb2.Sos and the p21ras pathway, ultimately leading to mitogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Phosphatidylinositol 3-kinase is necessary and sufficient for insulin-stimulated stress fiber breakdown.

Rat-1 fibroblasts overexpressing the human insulin receptor undergo rapid actin rearrangement in response to insulin. Breakdown of stress fibers present in quiescent cells is followed by transient membrane ruffling and a return of stress fibers. We investigated the signaling pathways that mediate this insulin-stimulated reorganization of the actin cytoskeleton, which was visualized with rhodamine-phalloidin. Treatment of cells with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin prevented insulin action at the preliminary step of stress fiber breakdown. Cellular microinjection of a polyclonal antibody directed against the p85 subunit of PI3-kinase as well as a purified recombinant p85-SH2 domain protein also inhibited actin reorganization. Transient expression of a constitutively active form of PI3-kinase (p110*) was sufficient to cause both stress fiber breakdown and membrane ruffling in the absence of insulin. Microinjection of a polyclonal anti-Shc antibody or dominant negative N17-Ras protein did not affect actin dynamics, and although constitutively active V12-Ras caused modest cytoskeletal reorganization, this effect was blocked by pretreatment with wortmannin. In summary, activation of PI3-kinase is necessary and sufficient to stimulate actin rearrangement, indicating that PI3-kinase may initiate the only signaling cascade required for insulin to induce cytoskeletal restructuring.

Actins↗
PubMed1995

Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway.

Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Mechanisms of enhanced transmembrane signaling by an insulin receptor lacking a cytoplasmic beta-subunit domain.

We have recently characterized a mutant insulin receptor (HIR delta 978) in which the insulin receptor beta-subunit was truncated at amino acid residue 978. Compared with parental Rat1 cells, the cells expressing the truncated receptor exhibited enhanced sensitivity to insulin's biologic actions. All of these effects are now extended to transcriptional events, since we now show enhanced sensitivity to insulin stimulation of c-fos mRNA expression. These effects were insulin-specific, since insulin-like growth factor-1 stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, and thymidine incorporation into DNA were normal. In addition, the truncated receptor exhibited enhanced sensitivity only in vivo, but not in vitro, since the kinase activity of wheat germ agglutinin-purified receptor preparations was comparable between HIR delta 978 and parental Rat1 insulin receptors. Parental rat endogenous insulin-like growth factor-1 receptors and transfected human insulin receptors form hybrid receptors as well as homologous tetrameric receptors. The normal heterotetrameric receptors possess kinase activity in vivo leading to tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with the p85 regulatory subunit of phosphatidyl inositol 3-kinase. Interestingly, preincubation with human-specific anti-insulin receptor antibody abolished the increased insulin sensitivity in glucose incorporation into glycogen in HIR delta 978 cells. Furthermore, microinjection of anti-IRS-1 antibody into HIR delta 978 cells inhibited insulin stimulation of DNA synthesis. In summary: 1) truncated receptors on the cell surface confer enhanced insulin sensitivity in vivo; 2) the normal heterotetrameric receptors are functionally active and couple to IRS-1 efficiently; and 3) IRS-1 is an important molecule transmitting insulin's biological signals in HIR delta 978 cells.

Animals↗
PubMed1995

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.

Animals↗
PubMed1994

Syp (SH-PTP2) is a positive mediator of growth factor-stimulated mitogenic signal transduction.

Syp (SH-PTP2) was recently identified as a phosphotyrosine phosphatase containing two SH2 domains within its primary structure. In response to appropriate growth factor stimulation, Syp becomes phosphorylated on tyrosine residues and associates with insulin receptor substrate 1 (IRS-1) and/or the corresponding growth factor receptor via its SH2 domains, leading to increased Syp activity. To assess the importance of Syp in mitogenic signaling, we microinjected mammalian fibroblasts with several reagents designed to interfere with Syp SH2/phosphotyrosine interaction in vivo. Insulin-, insulin-like growth factor-1-, and epidermal growth factor-stimulated DNA synthesis, indicated by bromodeoxyuridine (BrdUrd) incorporation, was dramatically decreased following microinjection of a Syp antibody (Ab) (65-85%) or a Syp GST-SH2 fusion protein (approximately 90%) in comparison with cells microinjected with control IgG or glutathione S-transferase (GST), respectively. In addition, microinjection of an IRS-1-derived phosphonopeptide, which inhibits in vitro binding of Syp-SH2 to IRS-1 with an ED50 value of approximately 23 microM, also decreased BrdUrd incorporation in vivo by approximately 50-75%. Microinjection of the Syp Ab, Syp GST-SH2 fusion protein, or the phosphonopeptide had no effect on serum-stimulated BrdUrd incorporation. In conclusion, disruption of Syp function in living cells inhibited cell cycle progression in response to growth factor stimulation, indicating that Syp is a critical positive regulator of mitogenic signal transduction.

Amino Acid Sequence↗
PubMed1994

Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor.

Shc proteins contain a single SH2 domain, lack catalytic activity, and are substrates for activated receptors for insulin, insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF). Treatment with these growth factors induced rapid tyrosine phosphorylation of Shc. We investigated the potential role of Shc in mitogenic signaling. Affinity-purified antibodies were microinjected into living Rat1 fibroblasts overexpressing human insulin receptors. Bromodeoxyuridine incorporation into newly synthesized DNA was subsequently studied to assess the importance of Shc. Cellular microinjection of anti-Shc antibody inhibited BrdU incorporation induced by insulin, IGF-1, and EGF, but did not affect cells stimulated by fetal calf serum. Microinjection of an oncogenic p21ras protein (T24) into quiescent cells produced constitutively active mitogenic signaling, and comicroinjection of T24 with the anti-Shc antibody restored insulin and EGF stimulation of DNA synthesis. Immunoprecipitates of Shc from lysates of insulin-stimulated cells removed 70-80% of guanine nucleotide-releasing factor activity. These results indicate that Shc is an important component in a mitogenic signal transduction pathway that is shared by insulin, IGF-1, and EGF. The functional locus of Shc is either upstream of p21ras or lies on a distinct branch of the pathway leading to cell cycle progression.

Animals↗
PubMed1994

Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction.

Insulin treatment of mammalian cells immediately stimulates the tyrosine phosphorylation of a cellular protein of 185 kDa referred to as pp185 or IRS-1 (insulin receptor substrate 1). The potential role of the IRS-1 protein in insulin signaling has been examined by microinjecting affinity-purified antibodies into living cells. Stably transfected Rat-1 fibroblasts, which overexpress the human insulin receptor, were microinjected and subsequently stimulated with insulin or other growth factors. Progression through the cell cycle was monitored by using a single-cell assay, which employs bromodeoxyuridine labeling of DNA and analysis with immunofluorescence microscopy. Microinjection of anti-IRS-1 antibody completely inhibited incorporation of bromodeoxyuridine into the nuclei of cells stimulated with insulin or insulin-like growth factor I but did not affect cells stimulated with serum or a variety of purified growth factors. These studies indicate that IRS-1 is a critical component of the insulin and insulin-like growth factor I signaling pathways, which lead to DNA synthesis and cell growth.

Amino Acid Sequence↗
PubMed1994

Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression.

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.

Animals↗
PubMed1992

Expression of c-fos and AP-1 activity in senescent human fibroblasts is not sufficient for DNA synthesis.

Human fibroblasts have a limited replicative life span when maintained in culture after which they become unresponsive to treatment with mitogens, a phenomenon most commonly called senescence. Experiments indicating that serum does not induce expression of the c-fos proto-oncogene in senescent fibroblasts raised the issue of a potential central role for c-fos in the phenotype of sustained growth arrest. This was directly tested by microinjection of oncogenic c-Ha-ras protein into senescent fibroblasts. While ras injection was found to induce marked nuclear c-fos expression and functional AP-1 transcription activity, this did not lead to DNA synthesis. These results suggest that the senescence phenotype cannot be solely attributed to the absence of c-fos expression and that the proliferative block in these cells is either independent of AP-1 transcriptional activity, downstream of it, or involves multiple molecular mechanisms.

Cell Division↗
PubMed1989

Possible involvement of the 90-kDa heat shock protein in the regulation of protein synthesis.

The heme-sensitive eukaryotic initiation factor (eIF)-2 alpha kinase regulates translational activity in reticulocytes by phosphorylation of the smallest subunit of eukaryotic peptide initiation factor 2, eIF-2. Highly purified preparations of the kinase contain an abundant 90-kDa polypeptide which appears to modulate the activity of the enzyme. The physical properties and structural characteristics of the reticulocyte 90-kDa peptide are similar to those of the 90-kDa heat shock protein (hsp 90) from HeLa and other mammalian cells. The reticulocyte and HeLa cell proteins are shown to be immunologically cross-reactive. A direct comparison of the two proteins by one-dimensional peptide mapping of large peptides generated by limited proteolysis and by reversed-phase high performance liquid chromatography analysis of tryptic peptides indicates that they represent the same protein species. Like the 90-kDa reticulocyte protein, HeLa cell hsp 90 causes increased eIF-2 alpha phosphorylation by the heme-sensitive kinase and is a potent inhibitor of protein synthesis in the reticulocyte lysate system. A potential mechanism for the latter inhibition is inferred. These results implicate hsp 90 in the regulation of protein synthesis via its interaction with and perhaps regulation of the heme-sensitive kinase and phosphorylation of eIF-2 alpha.

Animals↗
PubMed1987

The 90-kilodalton peptide of the heme-regulated eIF-2 alpha kinase has sequence similarity with the 90-kilodalton heat shock protein.

Highly purified preparations of the heme-controlled eIF-2 alpha (eukaryotic peptide initiation factor 2 alpha subunit) kinase of rabbit reticulocytes contain an abundant 90-kilodalton (kDa) peptide that is immunologically cross-reactive with spectrin and that modulates the activity of the enzyme [Kudlicki, W., Fullilove, S., Read, R., Kramer, G., & Hardesty, B. (1987) J. Biol. Chem. 262, 9695-9701]. The amino-terminal sequence of the 90-kDa protein has a high degree of similarity with the known amino-terminal sequences of the Drosophila 83-kDa heat shock protein (20 out of 22 residues) and with other related heat shock proteins. The amino acid sequence of a tryptic phosphopeptide isolated by high-performance liquid chromatography from the eIF-2 alpha kinase associated 90-kDa protein after phosphorylation by casein kinase II is shown to be identical with a 14 amino acid segment of the known sequence of the Drosophila 83-kDa heat shock protein. Results of hydrodynamic studies indicate a highly elongated structure for the reticulocyte protein, characteristic of a structural protein. Additional structural similarities between the eukaryotic heat shock proteins, the reticulocyte eIF-2 alpha kinase associated 90-kDa peptide, and spectrin are discussed.

Amino Acid Sequence↗
PubMedDate not supplied

[Value of anamnesis and clinical examination in degenerative impingement syndrome in comparison with surgical findings--a prospective study].

50 patients with a degenerative shoulder-syndrome with impingement are evaluated by questioning and 17 clinical tests. Also taking in account findings of plain x-rays they are classified preoperatively as "rotator cuff tear" or "no tear". Only 4 of the maneuvers are positive in more than 66% of cases. Jobe test and Eppendorf test are especially useful in diagnosing an impingement syndrome. A painful are is found in only 48.9%. In the rupture group 9.7 maneuvers are positive while in the non rupture group it is 7.7 in the mean (not significant). Because no significant differences are noticed between groups for any of the tests a cuff tear can not be ruled out by a single sign. The overall rate of positive tests of both authors is similar (41.1%, 45.8%) but in detail differences are found in 21.2%. Useful (significant, p < 0.05) data for diagnosing a tear are older age (56.1 years vs. 47.7 years. in the non rupture group), previous (minor) trauma and radiological findings on plain films suggesting periarticular degenerative lesions. A calcifying tendinitis is consistent with pure impingement. The correct diagnosis confirmed by operation is made in 90% (sensitivity 91.3%, specificity 88.9%). This can be achieved only by an experienced clinician who takes into account all anamnestic and clinical findings, especially details that cannot be classified as just positive or negative and thus cannot be computerized.

Adult↗