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M Kasuga

Publications and source records attributed to M Kasuga.

At least 19 recordsLinked to original sources
PubMed1996

Protein-free cell culture on an artificial substrate with covalently immobilized insulin.

Insulin was immobilized on a surface-hydrolyzed poly(methyl methacrylate) film. Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins. Small amounts of immobilized insulin (1-10% of the required amount of free insulin) were sufficient to stimulate cell proliferation. In addition, the maximal mitogenic effect of immobilized insulin was greater than that of free insulin. Immobilized insulin activated the insulin receptor and downstream signaling proteins, and this activation persisted for longer periods than that obtained with free insulin, probably explaining the greater mitogenic effect of the immobilized insulin. Finally the immobilized-insulin film was usable repeatedly without marked loss of activity.

Animals↗
PubMed1996

Subcellular trafficking kinetics of GLU4 mutated at the N- and C-terminal.

The glucose transporter isoform, GLUT4, has been expressed in Chinese hamster clones and its subcellular trafficking has been determined following labelling at the cell surface with the impermeant bis-mannose photolabel, 2-N-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos -4-yloxy)-2-propylamine (ATM-BMPA). ATM-BMPA-tagged GLUT4 leaves the cell surface rapidly and equilibrates to give an internal/surface distribution ratio of approx. 3.5 after 60 min. GLUT4 in which the N-terminal phenylalanine-5 and glutamine-6 are mutated to alanine-N-(FQ-AA) and in which the C-terminal leucine-489 and -490 are mutated to alanine C-(LL-AA) have low internal/surface ratios of 0.64 and 1.24 respectively. If all cell-surface transporters are able to recycle, as would be the case for a two-pool recycling model with a single intracellular pool, then analysis suggests that the wild-type GLUT4 distribution ratio is dependent on endocytosis and exocytosis rate constants of 0.074 and 0.023 min(-1). These values are similar, but not identical, to those found for GLUT4 trafficking in adipocytes. The distribution of the N-(FQ-AA) transporter appears to be due to a decrease in endocytosis with reduced intracellular retention, while the distribution of the C-(LL_AA) transporter appears to be mainly due to poor intracellular retention. These results are also considered in terms of a consecutive intracellular pool model in which GLUT4 targeting domains alter the distribution between recycling endosomes and a slowly recycling compartment. In this case the more rapid apparent exocytosis of the mutated GLUT4 is due to their failure to reach a slowly recycling compartment with a consequent return to the plasma membrane by default. It is suggested that overexpression of transporters increases the proportion that are recycled in this way. Wortmannin is shown to decrease glucose transport activity and cell-surface photolabelled transporters in a manner consistent with an inhibition of transporter recycling. Studies on the rate of loss of transport activity and ATB-BMPA-tagged transporter in wortmannin-treated cells confirm that the N-(FQ-AA) mutant is endocytosed more slowly than the wild-type GLUT4. Taken together, these results suggest that the mutation at either the N- or the C-terminal domain can reduce movement to a slowly recycling intracellular compartment but that neither domain alone is entirely sufficient to produce wild-type GLUT4 trafficking behaviour.

Amino Acid Sequence↗
PubMed1996

Cleavage of vesicle-associated membrane protein (VAMP)-2 and cellubrevin on GLUT4-containing vesicles inhibits the translocation of GLUT4 in 3T3-L1 adipocytes.

We have identified VAMP isoforms, VAMP-2 and cellubrevin, on GLUT4-containing vesicle membranes isolated from 3T3-Ll adipocytes. These proteins translocate from a low density microsomal fraction to the plasma membrane upon insulin stimulation in a fashion similar to GLUT4. VAMP-1 was not detected in this low density microsomal fraction nor on purified GLUT4-containing vesicles. In streptolysin-O permeabilized 3T3-L1 adipocytes, both VAMP-2 and cellubrevin were cleaved with botulinum neurotoxin isoform B, BoNTx/B. In addition, BoNTx/B partially inhibited insulin-stimulated GLUT4 translocation and glucose transport activity. We conclude that the synaptobrevin isoforms are important components of the insulin-dependent translocation of GLUT4 to the cell surface in adipocytes.

3T3 Cells↗
PubMed1996

Interaction of Shc with adaptor protein adaptins.

The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of approximately 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.

Adaptor Protein Complex 2↗
PubMed1996

Molecular cloning of p125Nap1, a protein that associates with an SH3 domain of Nck.

Binding proteins to the Src homology 3 (SH3) domains of Nck were screened by the use of glutathione S-transferase fusion proteins. Two proteins of 140 and 125 kDa were detected, both of which associated preferentially with the first SH3 domain of Nck. The 125-kDa protein, designated as Nap1 for Nck-associated protein 1, was purified and the corresponding rat cDNA was isolated. The predicted amino acid sequence revealed that p125Nap1 does not contain any known functional motif but shows sequence homology to Hem family gene. Using specific antibodies, p125Nap1 was shown to associate with Nck both in vitro and in intact cells. Further characterization of p125Nap1 may clarify the protein-protein interaction in the downstream signaling of Nck.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells.

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (approximately 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti(-)[insulin receptor substrate 1 (IRS1)] and anti-alpha-p85 antibodies show the same level of insulin-stimulated activity, approximately 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of alpha-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

3T3 Cells↗
PubMed1995

Induction of Fas-mediated apoptosis in p53-transfected human colon carcinoma cells.

To investigate the biological function of p53 in colon carcinoma cells, a wild-type p53 expression plasmid under the control of the human cytomegalovirus promoter was stably transfected into the human colon adenocarcinoma cell line WiDr, which carries a mutation of the p53 gene at codon 273. Exogenous wild-type p53 transcripts were detected at various expression levels in 8 of 117 G418-resistant clones. The growth rates of the wild-type p53+ clones in culture did not change significantly. The efficiency of colony formation in soft agar, however, was completely suppressed in two wild-type p53+ clones. This is the first to demonstrate the feasibility of stable transfection of the wild-type p53 gene under the control of non-inducible promoter in human colon cancer cells. The major alteration found was that wild-type p53+ cells which were incubated with anti-Fas IgM showed marked cytolysis with preferential over-expression of wild-type p53 accompanied by overexpression of a cyclin-dependent kinase inhibitor, WAF1, whereas the endogenous mutant p53 retained its expression level. The findings suggest that a Fas-initiated pathway is incidentally linked to a p53-dependent apoptotic pathway through the reconstituted wild-type p53 gene in WiDr cells. This model should help elucidating the additional role of the p53 tumor suppressor gene and the mechanism of apoptosis in colon carcinoma cells.

Adenocarcinoma↗
PubMed1995

Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity.

C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.

Affinity Labels↗
PubMed1995

Localization and subcellular distribution of SH-PTP2, a protein-tyrosine phosphatase with Src homology-2 domains, in rat brain.

SH-PTP2 is a protein-tyrosine phosphatase with src homology-2 (SH2) domains and shown to be highly expressed in the rat brain. Light microscopic immunohistochemistry showed that specific immunoreactivity was widely distributed, most abundant in neurophil, weak in neuronal somata, and absent from white matter. By electron microscopic immunohistochemistry, intense labeling is observed on synapses and concentrated in the pre- and post-synaptic plasma membranes. In subcellular fractionation analysis of brain, SH-PTP2 was mainly observed in the particulate fraction, particularly in myelin and synaptosomes. SH-PTP2 was further recovered in the synaptic plasma membrane. SH-PTP2 was extracted from brain membrane with a detergent such as Triton X-100 or Nonidet P-40 but not with 1 M NaCl. Furthermore, SH-PTP2 was coimmunoprecipitated with a 100 kDa tyrosine-phosphorylated membrane protein, which may couple SH-PTP2 to brain membranes. These results suggest that SH-PTP2 associates with synaptic membranes and may play a role in the synaptic communications in the brain.

Animals↗
PubMed1995

Ras-independent and wortmannin-sensitive activation of glycogen synthase by insulin in Chinese hamster ovary cells.

Activation of glycogen synthase is one of the major metabolic events triggered by exposure of cells to insulin. The molecular mechanism by which insulin activates glycogen synthase was investigated. The possible role of Ras and mitogen-activated protein kinase cascade was investigated with a stable cell line, CHO-IR-C/S 46, that overexpresses insulin receptors and a catalytically inactive SH-PTP 2 protein phosphatase and in which insulin does not induce the formation of the Ras-GTP complex or the subsequently activation of the mitogen-activated protein kinase cascade. Insulin activated glycogen synthase in this cell line to a similar extent as in parental CHO-IR cells. The importance of heteromeric phosphoinositide (PI) 3-kinase in insulin activation of glycogen synthase was examined in a stable cell line, CHO-IR/delta p85, that overexpresses insulin receptors and a dominant negative mutant (delta p85) of the 85-kDa subunit of PI 3-kinase that lacks the binding site for the catalytic 110-kDa subunit. Insulin-dependent activation of PI-3 kinase and glucose transport, but not the formation of the Ras-GTP complex, are markedly attenuated in this cell line. In CHO-IR/delta p85 cells, insulin activated glycogen synthase to a similar extent as in parental CHO-IR cells. The failure of overproduction of the mutant (delta p85) protein to inhibit insulin activation of glycogen synthase was also confirmed by transient expression in Rat 1 cells with the use of a recombinant vaccinia virus. However, wortmannin abolished insulin activation of glycogen synthase in all cell lines. These data suggest that existence of a Ras-independent and wortmannin-sensitive pathway for activation of glycogen synthase by insulin.

Androstadienes↗
PubMed1995

EGF-induced activation of 70-kDa S6 kinase in CHO cells expressing human EGF receptors.

We investigated epidermal growth factor (EGF)-induced activation of 85-kDa/110-kDa phosphatidylinositol (PI)-3-kinase and 70-kDa S6 kinase in Chinese hamster ovary cells expressing the human EGF receptor. EGF-induced activation of p70 S6 kinase was comparable to that induced by insulin, whereas that of PI-3-kinase in anti-phosphotyrosine immunoprecipitates was very small compared with insulin. Wortmannin, a p85/p110 PI-3-kinase inhibitor, inhibited EGF-induced activation of p70 S6 kinase in a dose-dependent manner. Given that several proteins homologous to catalytic subunit of p85/p110 PI-3-kinase have been identified and that wortmannin inhibits distinct form of PI-3-kinase, the present results suggest that wortmannin-sensitive kinases that resemble catalytic subunit of p85/p110 PI-3-kinase may participate in the signaling pathway from EGF receptors to p70 S6 kinase.

Androstadienes↗
PubMed1995

Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and fat cells by inducing the redistribution of a specific glucose transporter, GLUT4, from intracellular vesicles to the cell surface. Phosphoinositide (PI) 3-kinase has been implicated as a key intermediate in insulin-stimulated glucose transport by studies that have examined the effects of wortmannin and LY294002, which are thought to be specific inhibitors of this enzyme. However, the specificity of these compounds for PI 3-kinase has recently been questioned. Epidermal growth factor, which activates mitogen-activated protein kinase in mouse 3T3-L1 adipocytes, has now been shown to have no effect on PI 3-kinase activity or GLUT4 translocation in these cells. Furthermore, microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase, which lacks a binding site for the catalytic 110-kDa subunit, inhibited GLUT4 translocation induced by insulin in 3T3-L1 adipocytes; microinjection of the wild-type protein had no effect. These observations indicate that PI 3-kinase is necessary for insulin-induced GLUT4 translocation and glucose transport in adipocytes.

3T3 Cells↗
PubMed1995

Phosphoinositide 3-kinase as an upstream regulator of the small GTP-binding protein Rac in the insulin signaling of membrane ruffling.

Membrane ruffling and the closely linked response of fluid-phase pinocytosis were investigated in Chinese hamster ovary cells that stably overexpress the human insulin receptor and a mutant 85-kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) that lacks a binding site for the catalytic 110-kDa subunit of this enzyme. Both membrane ruffling and pinocytosis induced by insulin were markedly impaired in these cells. Microinjection of Rac, a Ras-related small GTP-binding protein, induced membrane ruffling in human epidermoid carcinoma KB cells, and this effect of Rac was not blocked by coinjection of delta p85 or by exposure of cells to wortmannin, a specific PI 3-kinase inhibitor. These results suggest that PI 3-kinase is essential not only for insulin-stimulated membrane ruffling but also for pinocytosis, and that PI 3-kinase possibly functions upstream of Rac in the signal transduction pathway.

Animals↗