Biomedical subjects

Y Kaburagi

Publications and source records attributed to Y Kaburagi.

At least 37 records · Page 2Linked to original sources

PubMed1997

Four mutant alleles of the insulin receptor gene associated with genetic syndromes of extreme insulin resistance.

We identified four novel mutant alleles of the insulin receptor gene in three patients with genetic syndromes associated with insulin resistance. Two mutant alleles of the insulin receptor gene were identified in a patient with the Rabson-Mendenhall syndrome who was a compound heterozygote for a mutation at the 3'-splice acceptor site of intron 4 (AG-->GG), the first mutation causing an aberrant splicing at this locus, and a deletion of eight base pairs in exon 12. The second patient with leprechaunism was also a compound heterozygote for a deletion of one base pair in exon 19 and a mutation, Thr910-->Met, which causes impaired receptor processing. Interestingly, the third patient with type A syndrome was a simple heterozygote for the identical one base pair deletion. The fact that the same one base pair deletion links to type A in a simple heterozygote and to leprechaunism in a compound heterozygote appears consistent with the hypothesis that the severity of mutations will determine the phenotype.

Alternative Splicing↗

PubMed1996

Annexin II is a novel player in insulin signal transduction. Possible association between annexin II phosphorylation and insulin receptor internalization.

Annexin II is a Ca2+-, phospholipid-, and actin- binding protein that was implicated in the regulation of vesicular traffic and endosome fusion. It is a known substrate for protein kinases including the platelet-derived growth factor receptor, src protein-tyrosine kinase, and protein kinase C. In the present study we investigated the possible involvement of annexin II in insulin signal transduction. Phosphorylation of annexin II in response to insulin treatment of intact Chinese hamster ovary (CHO)-T cells was detected by 5 min and reached maximal levels after a 2-3-h incubation with the hormone. However, unlike other receptor substrates, annexin II failed to undergo insulin-induced Tyr phosphorylation under conditions where receptor internalization was inhibited. This was evident in CHO cells, overexpressing the insulin receptor, in which internalization was inhibited either by tyrosine kinase inhibitors or by lowering the temperature to 4 degrees C, and in CHO cells overexpressing various insulin receptor mutants in which normal internalization was impaired. Hence, Tyr phosphorylation of annexin II could be part of the internalization and sorting mechanism of the insulin receptor.

Androstadienes↗

PubMed1996

Mutant of insulin receptor substrate-1 incapable of activating phosphatidylinositol 3-kinase did not mediate insulin-stimulated maturation of Xenopus laevis oocytes.

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Hypoxia and hypoxia/reoxygenation activate Src family tyrosine kinases and p21ras in cultured rat cardiac myocytes.

We previously reported that both hypoxia and hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly and sequentially activate mitogen-activated protein kinase kinase kinase (MAPKKK) activity of Raf-1. This was followed by the sequential activation of MAP kinase kinase (MAPKK). MAP kinases (p42mopk and p44mopk), and S6 kinase (p90rsk). In this study, we demonstrated that both hypoxia and hypoxia/ reoxygenation caused rapid activation of Src family tyrosine kinases, p60c-src and p59c-fyn, which are upstream mediators of MAP kinase activation. This was followed by the activation of p21ras. Because Src family tyrosine kinases are known to be cell-surface-associated kinases and upstream regulators of p21ras, these results strongly suggested that activation of Src family tyrosine kinases plays a key role in triggering intracellular signaling cascades in cardiac myocytes in response to hypoxia and hypoxia/reoxygenation.

Animals↗

PubMed1996

Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice.

We and others recently generated mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene and demonstrated that they exhibited growth retardation and had resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signalling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study, we investigated the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis were significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis was both wortmannin sensitive and insensitive in wild-type and IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation were not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) was roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20 to 30% of that of IRS-1 in the muscles. In conclusion, (i) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (ii) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles; and (iii) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice).

Adaptor Proteins, Signal Transducing↗

PubMed1996

Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.

Amino Acid Sequence↗

PubMed1996

Signal transduction mechanism of insulin and insulin-like growth factor-1.

Insulin and insulin-like growth factor-1 (IGF-1) are two structurally related hormones which produce similar biological activities such as metabolic and growth promoting actions. Their receptors, insulin and IGF-1 receptors, also share similarities in both structure and functions such as tyrosine-specific protein kinase. We identified insulin receptor substrate-1 (IRS-1) as a common substrate for insulin and IGF-1 receptor tyrosine kinases. We generated IRS-1 knockout mice and showed that IRS-1 plays a physiological role in signal transduction and biological actions of insulin and IGF-1. We also identified pp190 (IRS-2) as an alternative substrate for IRS-1.

Animals↗

PubMed1996

Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1 and identification of insulin receptor substrate-2.

To clarify the physiological roles of insulin receptor substrate-1 (IRS-1) in vivo, we made mice with a targeted disruption of the IRS-1 gene locus. Mice homozygous for targeted disruption of the IRS-1 gene were born alive but were retarded in embryonal and postnatal growth. They also had resistance to the glucose-lowering effects of insulin, insulin-like growth factor-1 (IGF-1) and factor-2 (IGF-2). These data suggest the existence of both IRS-1-dependent and IRS-1-independent pathways for signal transduction of insulin and IGFs. Moreover, we identified tyrosine phosphorylation of a 190-kDa protein (pp 190) as a novel substrate (IRS-2) for insulin receptor kinase in livers of IRS-1 deficient mice which can bind both P13-kinase and Ash/Grb2.

Animals↗

PubMed1996

Antibody production to heat shock proteins with Mr 65 kD (HSP65) in cutaneous inflammation: a possible relation to focal infection.

In order to correlate the immunomodulatory roles of homologous heat shock proteins with Mr 65 kD (HSP65) to skin diseases, antibody level to recombinant-HSP65 of Mycobacterium leprae was quantified with enzyme-linked immunosorbent assay (ELISA) in the sera of patients. In psoriasis, an insignificant increase was observed in anti-HSP65 IgG (0.111 +/- 0.053, mean +/- SD in 0D492 nm, n = 22), compared with a normal group (0.080 +/- 0.032, n = 9). However, psoriasis of acute guttate-type (PGA), which is often induced after tonsillar infection, showed a significant increase (0.178 +/- 0.032 n = 4, p <0.001), but psoriasis vulgaris did not (PV) (0.101 +/- 0.053, n = 12), nor generalized psoriasis pustulosa (PP) (0.087 +/- 0.025, n = 6). Similarly, patients with palmoplantar pustulosis (PPP) with tonsillar or periodontal infection showed significantly high anti-H5P65 IgG (0.230 +/- 0.065, n = 7, p <0.0001), compared with only a mild increase in PPP without suspected infectious foci (0.139 +/- 0.066, n = 13, p <0.05). Possible staphylococcal infection in the oral cavity was suggested by an additional ELISA assay to staphylococcal antigen: anti-staphylokinase IgG showed a significant increase in PPP with infectious foci (0.110 +/- 0.028 n = 3, p <0.01) compared with the normal group (0.039 +/- 0.014), while PPP without them showed only a mild change (0.060 +/- 0.017, n = 6, p <0.05). We assume that immunoreaction to H5P65 may be involved in psoriatic skin inflammation associated with focal infection.

Antibody Formation↗

PubMed1995

Upstream mechanisms of glycogen synthase activation by insulin and insulin-like growth factor-I. Glycogen synthase activation is antagonized by wortmannin or LY294002 but not by rapamycin or by inhibiting p21ras.

This study was undertaken to define intracellular signaling pathways upstream to glycogen synthase activation. First, we examined the role of the two pathways of insulin signaling, Ras-dependent and wortmannin/LY294002-sensitive, in glycogen synthase activation. Although negative dominant Ras (Ras17N) induction in PC12 cells markedly decreased activities of mitogen-activated protein kinase (MAP) and pp90 S6 kinase in response to insulin or insulin-like growth factor I (IGF-I), activation of glycogen synthase by these agents was unaffected by negative dominant Ras induction. In contrast, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, antagonized glycogen synthase activation in response to insulin or IGF-I. Next, we examined the contribution of pp70 S6 kinase, one of the wortmannin/LY294002-sensitive signaling molecules on glycogen synthase activation. Immunosuppressant rapamycin completely blocked activation of pp70 S6 kinase by insulin or IGF-I, but rapamycin alone or in combination with induction of negative dominant Ras failed to antagonize glycogen synthase activation by these hormones. These data suggest that 1) activation of Ras-MAP kinase is not necessary for stimulation of glycogen synthase and 2) activation of wortmannin/LY294002-sensitive pathway, independent of pp70 S6 kinase, plays a key role in glycogen synthase regulation in PC12 cells.

Amino Acid Sequence↗

PubMed1995

The role of the NPXY motif in the insulin receptor in tyrosine phosphorylation of insulin receptor substrate-1 and Shc.

The insulin receptor phosphorylates insulin receptor substrate-1 (IRS-1) and Shc on tyrosine residues, both of which associate with the protein-abundant Src homology/growth factor receptor-bound protein 2(ASH/GRB2) leading to p21ras activation. Juxtamembrane Tyr960 of the insulin receptor required for tyrosine phosphorylation of both IRS-1 and Shc is contained in the NPXY motif, which is also present in other tyrosine kinase receptors and oncogene products. In this study, the role of this motif in insulin's signaling was examined in Chinese hamster ovary cells expressing insulin receptors with mutations in this motif. All alterations in Tyr960 examined decreased tyrosine phosphorylation of both IRS-1 and Shc to a similar extent. The replacements of Asn957 and the deletion of NPE impaired tyrosine phosphorylation of Shc and IRS-1, although tyrosine phosphorylation of Shc was more severely affected than that of IRS-1. The amount of ASH/GRB2 bound to IRS-1 and Shc in vitro and in vivo was also decreased in these cells. These data suggest that the NPXY motif in the insulin receptor is important for tyrosine phosphorylation of both IRS-1 and Shc as well as subsequent signaling.

Amino Acid Sequence↗

PubMed1994

Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1.

Insulin receptor substrate-1 (IRS-1) is the major substrate of insulin receptor and IGF-1 receptor tyrosine kinases; it has an apparent relative molecular mass of 160-190,000 (M(r), 160-190K) on SDS polyacrylamide gel. Tyrosine-phosphorylated IRS-1 binds the 85K subunit of phosphatidylinositol 3-kinase which may be involved in the translocation of glucose transporters and the abundant src homology protein (ASH)/Grb2 which may be involved in activation of p21ras and MAP kinase cascade. IRS-1 also has binding sites for Syp and Nck and other src homology 2 (SH2) signalling molecules. To clarify the physiological roles of IRS-1 in vivo, we made mice with a targeted disruption of the IRS-1 gene locus. Mice homozygous for targeted disruption of the IRS-1 gene were born alive but were retarded in embryonal and postnatal growth. They also had resistance to the glucose-lowering effects of insulin, IGF-1 and IGF-2. These data suggest the existence of both IRS-1-dependent and IRS-1-independent pathways for signal transduction of insulin and IGFs.

Animals↗

PubMed1994

Signal transduction pathways from insulin receptors to Ras. Analysis by mutant insulin receptors.

We have examined the involvement of insulin receptor (IR) substrate-1 (IRS-1) and/or Shc in the upstream of Ras activation in insulin signaling using Chinese hamster ovary (CHO) cell lines overexpressing wild-type (CHO-IR) cells) or mutant insulin receptors. In CHO-IR cells, insulin rapidly phosphorylated IRS-1 and Shc at tyrosine residues and stimulated the formation of the active GTP-bound Ras (Ras.GTP). In contrast, a CHO cell line overexpressing the kinase-negative mutant insulin receptor substituting Arg1018 for Lys1018 was unable to tyrosine-phosphorylate IRS-1 and Shc and failed to activate Ras in response to insulin. A CHO cell line overexpressing the mutant insulin receptor, substituting Ala960 for Tyr960 and which was known to exhibit impaired tyrosine phosphorylation of IRS-1 and biological effects evoked by insulin, showed severely impaired insulin-dependent tyrosine phosphorylation of Shc and moderately impaired activation of Ras. Another cell line overexpressing the mutant insulin receptor, lacking 82 amino acids of the C terminus of beta-subunit and which was recently reported to retain normal insulin-dependent tyrosine phosphorylation of IRS-1, showed slightly impaired Ras activation at 10(-7) M insulin with severely reduced tyrosine phosphorylation of Shc protein. Furthermore, insulin did not induce the association of tyrosine-phosphorylated IRS-1 and Shc in CHO-IR cells. These results suggest that Shc and IRS-1 lie in the separate signaling pathways and that the tyrosine phosphorylation of IRS-1 with or without some low level of Shc phosphorylation may be enough to stimulate the submaximal accumulation of Ras.GTP complex and may need synergistically the higher level of tyrosine phosphorylation of Shc to induce the full activation of Ras in insulin signaling.

Animals↗

PubMed1994

[The role of phosphorylation cascade in insulin action].

Insulin induces a wide variety of growth and metabolic response in many cell types. Insulin initiates its biological effects by activation of tyrosine kinase in the beta-subunit and phosphorylates several proteins, such as insulin receptor substrate-1 (IRS-1), Shc. thereby activating phosphatidyl inositol 3-kinase activity, and ras activity. MAP kinase cascade activated by ras, 70kDaS6 kinase lying downstream of PI3-kinase, and the regulation of glycogen synthase have been discussed.

Animals↗

PubMed1993

Normal insulin receptor substrate-1 phosphorylation in autophosphorylation-defective truncated insulin receptor. Evidence that phosphorylation of substrates might be sufficient for certain biological effects evoked by insulin.

A mutant human insulin receptor that lacked the 82 amino acids of the COOH terminus of the beta-subunit (del82) was studied. Both the wild type insulin receptor (HIR) and the mutant receptor were expressed in Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation and tyrosine kinase activities toward exogenous substrates of solubilized and partially purified del82 were severely impaired. When CHO cells transfected with del82 (CHO-del82) were stimulated with insulin, autophosphorylation was decreased to a great extent compared with cells expressing HIR (CHO-HIR). Nevertheless, tyrosine phosphorylation of an endogenous substrate, pp185, and insulin receptor substrate-1 (IRS-1) in CHO-del82 was comparable with that in CHO-HIR. Insulin-stimulated activation of phosphatidylinositol 3-kinase activity in CHO-del82 was also equivalent to that in CHO-HIR. Moreover, CHO-del82 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake and thymidine incorporation into DNA. Insulin-induced internalization in CHO-del82 was decreased by 46% as compared with that in CHO-HIR. These data suggest that: 1) the COOH-terminal domain of the insulin receptor may play an inhibitory role in the phosphorylation of pp185 and IRS-1; and 2) phosphorylation of substrates such as pp185 and IRS-1, rather than autophosphorylation of the receptor per se, correlates better with certain biological effects that were mediated by insulin, suggesting that phosphorylation of the substrates might be sufficient for transducing signals downstream.

Animals↗

PubMed1993

Site-directed mutagenesis of the juxtamembrane domain of the human insulin receptor.

We have studied the functions of the juxtamembrane domain (941-989) of the human insulin receptor by site-directed mutagenesis. Tyrosine phosphorylation of pp185 was impaired in Chinese hamster ovary cells expressing the receptors with the alteration of Tyr960, but not of Tyr953 or Tyr972, to Phe (CHO-Y960F cells) as compared with cells expressing the normal receptors. In CHO-Y960F cells, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the activation of phosphatidylinositol 3-kinase in the anti-phosphotyrosine and anti-IRS-1 immunoprecipitates, the activation of mitogen-activated protein (MAP) kinase, and biological actions were also impaired. In addition, although the deletion of residues 954-965 severely impaired insulin internalization, the deletion of NPXY (957-960), the internalization signal of the low density lipoprotein receptor, did not affect internalization. Moreover, neither the deletions around Tyr953 nor the alterations of the tyrosines (953, 960, or 972) significantly reduced internalization. These data suggest that: 1) Tyr960 is important for the recognition of pp185/IRS-1, the association of phosphatidylinositol 3-kinase with pp185/IRS-1, and the activation of MAP kinase; 2) MAP kinase may lie downstream of pp185/IRS-1 in insulin's signal transduction; and 3) the juxtamembrane domain, but not NPXY or individual tyrosines, is important for insulin internalization.

Amino Acid Sequence↗

PubMed1993

Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2.

Insulin activates the ras proto-oncogene product p21ras (Ras) by stimulating conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9015-9019) is composed of one Src homology (SH)2 and two SH3 domains and highly homologous to the Caenorhabditis elegans protein sem-5 that couples a tyrosine kinase to a Ras protein. We have studied an interaction of ASH with insulin-stimulated tyrosine-phosphorylated proteins in Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR cells). In an anti-ASH (alpha ASH) immunoprecipitates, we detected a 170-kDa phosphoprotein that was recognized by an anti-phosphotyrosine antibody and an anti-insulin receptor substrate 1 antibody (alpha IRS-1) from the insulin-stimulated [32P]orthophosphate-labeled CHO-HIR cells. We failed to detect the tyrosine phosphorylation of the protein ASH. These data suggested that insulin stimulates IRS-1.ASH complex formation in intact cells. Incubation of an ASH fusion protein with the lysates of insulin-stimulated CHO-HIR cells revealed that the fusion protein of ASH was able to bind the tyrosine-phosphorylated 170-kDa protein that was recognized by alpha IRS-1. We also demonstrated that fusion protein of ASH was able to bind the fusion protein of tyrosine-phosphorylated IRS-1 fragments, suggesting that ASH is able to bind tyrosine-phosphorylated IRS-1 directly. These data suggest that IRS-1.ASH complex formation may play a role in coupling the insulin receptor kinase to a Ras signaling pathway. Furthermore, we observed an insulin-stimulated phosphatidylinositol (PI) 3-kinase activity in alpha ASH immunoprecipitates, suggesting the formation of an ASH.IRS-1.PI 3-kinase complex. This complex formation was detected as early as 10 s after insulin stimulation in intact CHO-HIR cells. This is the first report that supports the notion that IRS-1 binds several signal transducing molecules containing SH2 domains, thus serves as an SH2 docking protein that transduces insulin's signal multidirectionally.

Adaptor Proteins, Signal Transducing↗

PubMed1992

Enhanced insulin-induced mitogenesis and mitogen-activated protein kinase activities in mutant insulin receptors with substitution of two COOH-terminal tyrosine autophosphorylation sites by phenylalanine.

We have studied the function of a mutant human insulin receptor in which two COOH-terminal autophosphorylation sites (Tyr-1316 and -1322) were replaced by phenylalanine (F/Y COOH-terminal 2 tyrosines (CT2)). In addition, we have also constructed a mutant receptor in which Lys-1018 in the ATP-binding site was changed to arginine (R/K 1018). Both the wild type insulin receptor (HIR) and the mutant receptors were expressed in Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation of solubilized and partially purified F/Y CT2 was decreased by approximately 30% compared with the HIR. Tyrosine kinase activities of F/Y CT2 and HIR toward exogenous substrates were almost equal. When CHO cells transfected with F/Y CT2 (CHO-F/Y CT2) were stimulated with insulin, autophosphorylation of the beta-subunit of the insulin receptor and the phosphorylation of an endogenous substrate (pp185) in the intact cell were normal compared with cells expressing HIR (CHO-HIR). CHO-F/Y CT2 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake. However, the dose-response curve of insulin-stimulated thymidine incorporation in CHO-F/Y CT2 was shifted to the left (approximately 5-7-fold) compared with that in CHO-HIR. There was no significant difference in insulin-like growth factor 1-stimulated thymidine incorporation between CHO-F/Y CT2 and CHO-HIR. Furthermore, the dose-response curve of insulin-stimulated kinase activity toward myelin basic protein in CHO-F/Y CT2 was also shifted to the left (approximately 5-fold) compared with that in CHO-HIR. Kinase assays in myelin basic protein-containing gels revealed that both species of MAP kinases (M(r) 44,000, 42,000) were more sensitive to activation by insulin in CHO-F/Y CT2 than in CHO-HIR. This observation was confirmed in immune complex kinase assays toward microtubule-associated protein 2 (MAP2) using specific antibodies against mitogen-activated protein (MAP) kinase. R/K 1018 mutant insulin receptors showed an absence of insulin-stimulated kinase activity and CHO cells transfected with R/K 1018 (CHO-R/K 1018) failed to enhance 2-deoxyglucose uptake or thymidine incorporation in response to insulin. In addition, R/K 1018 kinase-defective insulin receptors were unable to mediate insulin-stimulated MAP kinase activation. These data suggest that: 1) tyrosine kinase activity of the insulin receptor is required for activation of insulin-stimulated MAP kinases and 2) phosphorylation of COOH-terminal tyrosine residues may play an inhibitory role in mitogenic signaling through regulation of MAP kinases.

Animals↗