scholarly journals Studies on an insulin-stimulated insulin receptor serine kinase activity: separation of the kinase activity from the insulin receptor and its reconstitution back to the insulin receptor

1995 ◽  
Vol 308 (3) ◽  
pp. 915-922 ◽  
Author(s):  
K A Asamoah ◽  
P G P Atkinson ◽  
W G Carter ◽  
G J Sale
Keyword(s):  
Myelin Basic Protein ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Basic Protein ◽  
Serine Phosphorylation ◽  
Serine Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Mitogen Activated Protein

In cells insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Recently we have achieved co-purification of the insulin receptor with insulin-stimulated insulin receptor serine kinase activity. We now show that the co-purified serine kinase activity can be removed by NaCl washing and reconstituted by adding back the NaCl eluate. Reconstitution enabled higher serine phosphorylation than achieved with the co-purified preparation. Myelin basic protein was discovered to be a potent substrate for insulin-stimulated serine phosphorylation by the co-purified preparation, with the activity responsible having similar properties to the serine kinase activity towards the receptor. Myelin basic protein was also phosphorylated on serine by the NaCl eluate. Myelin basic protein phosphorylated by the co-purified preparation or the NaCl eluate gave the same set of phosphoserine peptides. The major myelin basic protein serine kinase activity in the NaCl eluate co-purified exactly on Mono Q with the activity that restored insulin-stimulated insulin receptor serine phosphorylation. These results provide strong evidence for the true separation of the serine kinase from the insulin receptor and the distinctiveness of the serine kinase activity from the insulin receptor tyrosine kinase and mitogen-activated protein kinases. The procedures developed for the isolation of the serine kinase and the establishment of an effective in vitro substrate should allow purification of the kinase. The protocols also provide flexible systems for identifying the functions of the insulin-stimulated serine phosphorylations and the respective kinase(s).

scholarly journals Evidence that a novel serine kinase catalyses phosphorylation of the insulin receptor in an insulin-dependent and tyrosine kinase-dependent manner

1988 ◽  
Vol 256 (3) ◽  
pp. 903-909 ◽  
Author(s):  
D M Smith ◽  
G J Sale
Keyword(s):  
Cyclic Amp ◽  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Cyclic Gmp ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Serine Phosphorylation ◽  
Serine Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Insulin Dependent

Insulin receptor was co-purified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. By using this ‘in vitro’ system, the mechanism of activation of the serine kinase by insulin was explored. Peptide 1150, histone, poly(Glu-Tyr), eliminating Mn2+ (Mg2+ only), treatment at 37 degrees C (1 h), N-ethylmaleimide, phosphate, beta-glycerol phosphate and anti-phosphotyrosine antibody all inhibited insulin-receptor tyrosine kinase activity and the ability of insulin to stimulate phosphorylation of the insulin receptor on serine. Additionally, direct stimulation of the receptor tyrosine kinase by vanadate increased serine phosphorylation of the insulin receptor. Insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the insulin receptor. The activity of the insulin-sensitive receptor serine kinase was not augmented by cyclic AMP, cyclic GMP, Ca2+, Ca2+ + calmodulin, Ca2+ + phosphatidylserine + diolein or spermine, or inhibited appreciably by heparin. Additionally, the serine kinase phosphorylated casein or phosvitin poorly and was active with Mn2+. This indicates that it is distinct from Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, cyclic AMP- and cyclic GMP-dependent protein kinases, casein kinases I and II and insulin-activated ribosomal S6 kinase. Taken together, these data indicate that a novel species of serine kinase catalyses the insulin-dependent phosphorylation of the insulin receptor and that activation of this receptor serine kinase by insulin requires an active insulin-receptor tyrosine kinase.

scholarly journals Two systems in vitro that show insulin-stimulated serine kinase activity towards the insulin receptor

1988 ◽  
Vol 250 (2) ◽  
pp. 509-519 ◽  
Author(s):  
D M Smith ◽  
M J King ◽  
G J Sale
Keyword(s):  
Insulin Receptor ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Kinase Activity ◽  
Serine Phosphorylation ◽  
Dependent Manner ◽  
Serine Kinase ◽  
Two Systems ◽  
32P Incorporation

Two systems in vitro are described that show insulin-stimulated phosphorylation of the insulin receptor on serine residues. In the first system, insulin receptor was purified partially from Fao rat hepatoma cells by direct solubilization of the cells in Triton X-100 and chromatography on wheat-germ-agglutinin-agarose. Phosphorylation of these preparations with [gamma-32P]ATP in the presence or absence of insulin resulted in 32P incorporation exclusively into phosphotyrosine residues. Serine kinase activity towards the insulin receptor was reconstituted by adding extracts of Fao cells. Prior exposure of the cells to insulin stimulated serine kinase activity towards the insulin receptor in extracts 7.2-fold. A receptor serine kinase activity enhanced by treatment of cells with cyclic AMP analogues was also retained in the reconstituted system. In the second system, insulin receptor and insulin-sensitive serine kinase activity towards the insulin receptor were co-purified from human placenta. The protocol involved preparation of membranes, before solubilization and chromatography on wheat-germ-agglutinin-agarose, by using gentle procedures designed not to disrupt a potentially labile association between the insulin receptor and the serine kinase. Serine kinase activity in these preparations towards the insulin receptor was stimulated up to 10-fold by insulin, and the stoicheiometry of serine phosphorylation was estimated to be approx 0.8 mol/mol of insulin receptor for phosphorylations performed in the presence of insulin. Thus a preparation of insulin receptor is described for the first time that is phosphorylated to high stoicheiometry on serine in an insulin-dependent manner. Conditions that facilitate recovery and assay of serine kinase activity are defined and discussed. These systems provide a basis for characterizing the nature of the insulin-sensitive serine kinase that phosphorylates the insulin receptor, and defining its role in insulin action and control of receptor function.

scholarly journals The effect of fasting on the activation in vivo of the insulin receptor kinase

1990 ◽  
Vol 265 (3) ◽  
pp. 887-890 ◽  
Author(s):  
I Contreras ◽  
G L Dohm ◽  
S Abdallah ◽  
J A Wells ◽  
N Mooney ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Serum Glucose ◽  
Tyrosine Kinase Activity ◽  
Insulin Receptor Tyrosine Kinase ◽  
And Control

Fasting causes insulin resistance in liver and fat, and increases insulin sensitivity in muscle. We studied the response in vitro and in vivo to insulin of the insulin receptor tyrosine kinase in muscle and liver from 72 h fasted and control rats. Insulin was injected intraperitoneally together with glucose, and blood and tissue samples were obtained 0, 5, 15 and 30 min later. Basal serum glucose and insulin levels were significantly higher in control than in fasting rats. Serum glucose rose to approximately 300 mg/dl at 5 min and then progressively declined without hypoglycaemia. Receptors were prepared from whole tissue by wheat germ lectin affinity chromatography. 125I-insulin binding to purified receptors was increased by fasting in both muscle (18%) and liver (50%). In untreated fasting and control animals, muscle and liver insulin receptor tyrosine kinase activity was stimulated to similar levels by insulin added in vitro. With only insulin treatment in vivo, muscle receptor tyrosine kinase behaved similarly in fasting and control animals with maximal activation at 15 min post injection. In liver, insulin in vivo stimulated receptor tyrosine kinase activity maximally at 5 min post injection in both fasting and control, but in fasting animals the treatment in vivo caused a significantly larger and more prolonged activation of the enzymic activity, possibly due to a decrease in the rate of dephosphorylation and deactivation of the beta subunits.

scholarly journals Insulin receptor substrate 1 is phosphorylated by the serine kinase activity of phosphatidylinositol 3-kinase

1994 ◽  
Vol 304 (1) ◽  
pp. 17-21 ◽  
Author(s):  
J F Tanti ◽  
T Grémeaux ◽  
E Van Obberghen ◽  
Y Le Marchand-Brustel
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Serine Phosphorylation ◽  
Kinase Assay ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Serine Kinase ◽  
Phosphatidylinositol 3

Insulin receptor substrate (IRS) 1, which is tyrosine phosphorylated in response to insulin, presents multiple serine/threonine phosphorylation sites. To search for a serine kinase activity towards IRS 1, immunoprecipitates from basal or stimulated 3T3-L1 adipocytes were used in an in vitro kinase assay. When IRS 1 was isolated from insulin-treated cells, serine phosphorylation of IRS 1 occurred, which we attribute to the kinase activity of the phosphatidylinositol 3-kinase (PI3-kinase). Importantly, in an in vitro reconstitution assay, an excess of the PI3-kinase subunit prevents this phosphorylation. Together, our results suggest that following insulin stimulation, PI3-kinase associates with IRS 1, allowing for its serine phosphorylation. This phosphorylation event could play a role in the modulation of insulin signalling.

scholarly journals Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase

1991 ◽  
Vol 280 (3) ◽  
pp. 769-775 ◽  
Author(s):  
H Hayashi ◽  
N Miyake ◽  
F Kanai ◽  
F Shibasaki ◽  
T Takenawa ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Phosphorylated Form ◽  
Phosphatidylinositol 3

Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in the anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. This enzyme may therefore be a mediator of insulin signal transduction [Endemann, Yonezawa & Roth (1990) J. Biol. Chem. 265, 396-400; Ruderman, Kapeller, White & Cantley (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415]. At least two questions remain to be elucidated. Firstly, does the insulin receptor tyrosine kinase phosphorylate phosphatidylinositol 3-kinase directly, or does it phosphorylate a protein associated with the 3-kinase? Second, if the enzyme is a direct substrate for the insulin receptor tyrosine kinase, does tyrosine phosphorylation of phosphatidylinositol 3-kinase by the kinase alter the specific enzyme activity, or does the amount of the tyrosine-phosphorylated form of the phosphatidylinositol 3-kinase increase, with no change in the specific activity? We report here evidence that the 85 kDa subunit of highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor in vitro, but not by a mutant insulin receptor which lacks tyrosine kinase activity. We found that an increase in enzyme activity was detected in response to insulin not only in the anti-phosphotyrosine immunoprecipitates of the cytosol, but also in the cytosolic fraction before immunoprecipitation. In addition, we partially separated the tyrosine-phosphorylated form from the unphosphorylated form of the enzyme, by using a f.p.l.c. Mono Q column. The insulin-stimulated phosphatidylinositol 3-kinase activity was mainly detected in the fraction containing almost all of the tyrosine-phosphorylated form. This result suggests that tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase may increase the specific activity of the former enzyme in vivo.

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