Defective internalization of insulin and its receptor in cells expressing mutated insulin receptors lacking kinase activity.

The effects of high glucose on insulin-receptor tyrosine kinase activity and gene expression were investigated in 3T3-HIR cells. Cells incubated for 48 h in the presence of 25 mM glucose showed a 5-fold increase in the amount of insulin receptors per cell, receptor autophosphorylation and phosphorylation of the exogenous substrate poly(Glu/Tyr) compared with cells grown in the absence of glucose but in the presence of 25 mM fructose. These effects were associated with a 4-fold stimulation in steady-state levels of insulin-receptor mRNA. Significant cellular glucose utilization and lactate production were observed in the presence of high glucose in the culture medium, indicating a functional glycolytic pathway in glucose-treated cells, but not in cells treated with fructose. Such a differential response to hexoses favours the hypothesis of a carbohydrate regulation via a glycolytic intermediate. This was further supported by a similar glucose-induced increase in mRNA levels of the enzyme glyceraldehyde-3-phosphate dehydrogenase. To test the hypothesis that the stimulatory effect of glucose on amount of insulin receptors and phosphorylation state could result from post-transcriptional modifications, cells exposed to glucose were incubated with actinomycin D, a potent inhibitor of gene transcription. In cells challenged with high glucose plus inhibitor, insulin-receptor mRNA half-life was increased from 1 to 3 h, indicating that posttranscriptional mechanisms are involved in these processes of glucose regulation. Inhibition of protein synthesis by cycloheximide induced an overexpression of insulin-receptor mRNA levels in the presence of glucose, suggesting that labile repressor protein(s) could be implicated in the effects of glucose. We conclude that (1) long-term culture with high glucose increases the amount of insulin receptors and their tyrosine kinase activity and (2) the glucose-induced increase in insulin-receptor mRNA levels can be accounted for, at least in part, by posttranscriptional events.

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Red blood cell age, pyruvate kinase activity, and insulin receptors. Evidence that monocytes and RBCs may behave differently

Diabetes ◽

10.2337/diabetes.32.11.1017 ◽

1983 ◽

Vol 32 (11) ◽

pp. 1017-1022 ◽

Cited By ~ 5

Author(s):

A. Camagna ◽

R. De Pirro ◽

L. Tardella ◽

L. Rossetti ◽

R. Lauro ◽

...

Keyword(s):

Blood Cell ◽

Pyruvate Kinase ◽

Red Blood Cell ◽

Kinase Activity ◽

Insulin Receptors ◽

Pyruvate Kinase Activity ◽

Cell Age

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Red Blood Cell Age, Pyruvate Kinase Activity, and Insulin Receptors: Evidence that Monocytes and RBCs May Behave Differently

Diabetes ◽

10.2337/diab.32.11.1017 ◽

1983 ◽

Vol 32 (11) ◽

pp. 1017-1022 ◽

Cited By ~ 5

Author(s):

A. Camagna ◽

R. D. Pirro ◽

L. Tardella ◽

L. Rossetti ◽

R. Lauro ◽

...

Keyword(s):

Blood Cell ◽

Pyruvate Kinase ◽

Red Blood Cell ◽

Kinase Activity ◽

Insulin Receptors ◽

Pyruvate Kinase Activity ◽

Cell Age

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Insulin receptors prepared with iodoacetamide show enhanced autophosphorylation and receptor kinase activity.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)89608-2 ◽

1991 ◽

Vol 266 (11) ◽

pp. 7051-7057

Author(s):

C H Li ◽

M L Moule ◽

C C Yip

Keyword(s):

Kinase Activity ◽

Insulin Receptors ◽

Receptor Kinase

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Insulin activates p21Ras and guanine nucleotide releasing factor in cells expressing wild type and mutant insulin receptors.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)80685-1 ◽

1993 ◽

Vol 268 (27) ◽

pp. 19998-20001

Author(s):

B Draznin ◽

L Chang ◽

J.W. Leitner ◽

Y Takata ◽

J.M. Olefsky

Keyword(s):

Insulin Receptors ◽

Guanine Nucleotide ◽

Wild Type ◽

Mutant Insulin ◽

In Cells

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Activation of c-Src in cells bearing v-Crk and its suppression by Csk

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4706-4713.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4706-4713

Author(s):

H Sabe ◽

M Okada ◽

H Nakagawa ◽

H Hanafusa

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Src Kinase ◽

Cellular Protein ◽

Morphological Transformation ◽

Protein Tyrosine ◽

In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

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The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain

Journal of Cell Science ◽

10.1242/jcs.105.2.519 ◽

1993 ◽

Vol 105 (2) ◽

pp. 519-528

Author(s):

F. Boschelli ◽

S.M. Uptain ◽

J.J. Lightbody

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Cell Cycle Control ◽

Sh2 Domain ◽

Wild Type ◽

Type V ◽

Cdc28 Kinase ◽

In Cells

The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.

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Positive and Negative Regulation of Phosphoinositide 3-Kinase-Dependent Signaling Pathways by Three Different Gene Products of the p85α Regulatory Subunit

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.8035-8046.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 8035-8046 ◽

Cited By ~ 109

Author(s):

Kohjiro Ueki ◽

Petra Algenstaedt ◽

Franck Mauvais-Jarvis ◽

C. Ronald Kahn

Keyword(s):

Glucose Transport ◽

Kinase Activity ◽

Glycogen Synthesis ◽

Catalytic Subunit ◽

Regulatory Subunit ◽

Regulatory Subunits ◽

Irs Proteins ◽

Terminal Structure ◽

Stimulation Of ◽

In Cells

ABSTRACT Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimulation of glucose transport and glycogen synthesis. The gene for the p85α regulatory subunit yields three splicing variants, p85α, AS53/p55α, and p50α. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal region of 304, 34, and 6 amino acids, respectively. To determine if these regulatory subunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditions, we expressed each regulatory subunit in fully differentiated L6 myotubes using adenovirus-mediated gene transfer with or without coexpression of the p110α catalytic subunit. PI 3-kinase activity associated with p50α was greater than that associated with p85α or AS53. Increasing the level of p85α or AS53, but not p50α, inhibited both phosphotyrosine-associated and p110-associated PI 3-kinase activities. Expression of a p85α mutant lacking the p110-binding site (Δp85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated activity. Insulin stimulation of two kinases downstream from PI-3 kinase, Akt and p70 S6 kinase (p70S6K), was decreased in cells expressing p85α or AS53 but not in cells expressing p50α. Similar inhibition of PI 3-kinase, Akt, and p70S6K was observed, even when p110α was coexpressed with p85α or AS53. Expression of p110α alone dramatically increased glucose transport but decreased glycogen synthase activity. This effect was reduced when p110α was coexpressed with any of the three regulatory subunits. Thus, the three different isoforms of regulatory subunit can relay the signal from IRS proteins to the p110 catalytic subunit with different efficiencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform. These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the catalytic subunit or interaction with additional signaling molecules.

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Insulin Action in Cells Expressing Truncated or Kinase-Defective Insulin Receptors: Dissection of Multiple Hormone-Signaling Pathways

Diabetes Care ◽

10.2337/diacare.13.3.302 ◽

1990 ◽

Vol 13 (3) ◽

pp. 302-316 ◽

Cited By ~ 12

Author(s):

D. A. McClain

Keyword(s):

Signaling Pathways ◽

Insulin Action ◽

Insulin Receptors ◽

Hormone Signaling ◽

In Cells

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Tyrosine kinase activity of liver insulin receptor is inhibited in rats at term gestation

Biochemical Journal ◽

10.1042/bj2630267 ◽

1989 ◽

Vol 263 (1) ◽

pp. 267-272 ◽

Cited By ~ 9

Author(s):

C Martínez ◽

P Ruiz ◽

A Andrés ◽

J Satrústegui ◽

J M Carrascosa

Keyword(s):

Insulin Receptor ◽

Kinase Activity ◽

Insulin Signalling ◽

Insulin Receptors ◽

Insulin Binding ◽

Late Gestation ◽

Receptor Kinase ◽

Pregnant Rats ◽

Insulin Resistant ◽

32P Incorporation

Late gestation is associated with insulin resistance in rats and humans. It has been reported that rats at term gestation show active hepatic gluconeogenesis and glycogenolysis, and diminished lipogenesis, despite normal or mildly elevated plasma insulin concentrations, indicating a state of resistance to the hormone action. Since autophosphorylation of the insulin receptor has been reported to play a key role in the hormone signal transduction, we have partially purified plasma-membrane liver insulin receptors from virgin and 22-day-pregnant rats and studied their binding and kinase activities. (1) Insulin binding to partially purified receptors does not appear to be influenced by gestation, as indicated by the observed KD and Bmax. values. (2) The rate of autophosphorylation and the maximal 32P incorporation into the receptor beta-subunit from pregnant rats at saturating concentrations of insulin are markedly decreased with respect to the corresponding values for virgin rats. (3) The diminished autophosphorylation rate was due to a decreased responsiveness of the kinase activity to the action of insulin. (4) Phosphorylation of the exogenous substrates casein and poly(Glu80Tyr20) by insulin-receptor kinase was also less when receptors from pregnant rats were used. These results show the existence of an impairment at the receptor kinase level of the insulin signalling mechanism that might be related to the insulin-resistant state characteristic of term gestation in rats.

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