Studies into the Identity of the Sites of Insulin-Stimulated Insulin Receptor Serine Phosphorylation. Characterization of Synthetic Peptide Substrates for the Insulin-Stimulated Insulin Receptor Serine Kinase

Biochemistry ◽  
1995 ◽  
Vol 34 (29) ◽  
pp. 9488-9499 ◽  
Author(s):  
Wayne G. Carter ◽  
Kojo A. Asamoah ◽  
Graham J. Sale
Keyword(s):  
Insulin Receptor ◽  
Synthetic Peptide ◽  
Serine Phosphorylation ◽  
Serine Kinase ◽  
Peptide Substrates

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).

Characterization of Synthetic Peptide Substrates for p34cdc2 Protein Kinase

1991 ◽  
Vol 45 (4) ◽  
pp. 391-400 ◽  
Author(s):  
Daniel R. Marshak ◽  
Mark T. Vandenberg ◽  
Young Seuk Bae ◽  
Ii Je Yu
Keyword(s):  
Protein Kinase ◽  
Synthetic Peptide ◽  
Peptide Substrates

Purification and Characterization of an Insulin-Stimulated Insulin Receptor Serine Kinase†

Biochemistry ◽  
1996 ◽  
Vol 35 (45) ◽  
pp. 14340-14351 ◽  
Author(s):  
Wayne G. Carter ◽  
Alexandra C. Sullivan ◽  
Kojo A. Asamoah ◽  
Graham J. Sale
Keyword(s):  
Insulin Receptor ◽  
Purification And Characterization ◽  
Serine Kinase

scholarly journals Phosphatidylinositol 3′-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase

1998 ◽  
Vol 335 (2) ◽  
pp. 397-404 ◽  
Author(s):  
Keith A. CENGEL ◽  
Rosanne E. KASON ◽  
Gregory G. FREUND
Keyword(s):  
Insulin Receptor ◽  
Immune Complexes ◽  
Kinase Activity ◽  
Sh2 Domain ◽  
Serine Phosphorylation ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Serine Kinase ◽  
Insulin Receptor Substrate 1 ◽  
Pas Kinase

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3´-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase–IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate.

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 Characterization of Endoplasmic Reticulum-Localized UDP-d-Galactose: Hydroxyproline O-Galactosyltransferase Using Synthetic Peptide Substrates in Arabidopsis

2009 ◽  
Vol 152 (1) ◽  
pp. 332-340 ◽  
Author(s):  
Takuji Oka ◽  
Fumie Saito ◽  
Yoh-ichi Shimma ◽  
Takehiko Yoko-o ◽  
Yoshiyuki Nomura ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Synthetic Peptide ◽  
Peptide Substrates

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 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.

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