Insulin receptor autophosphorylation. I. Autophosphorylation kinetics of the native receptor and its cytoplasmic kinase domain

Biochemistry ◽  
1993 ◽  
Vol 32 (22) ◽  
pp. 5766-5772 ◽  
Author(s):  
R. A. Kohanski
Keyword(s):  
Insulin Receptor ◽  
Kinase Domain ◽  
Cytoplasmic Kinase Domain ◽  
Kinetics Of

NIDDM associated with mutation in tyrosine kinase domain of insulin receptor gene

Diabetes ◽  
1992 ◽  
Vol 41 (4) ◽  
pp. 521-526 ◽  
Author(s):  
S. Cocozza ◽  
A. Porcellini ◽  
G. Riccardi ◽  
A. Monticelli ◽  
G. Condorelli ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Gene ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Insulin Receptor Gene

scholarly journals Impairment of the liver insulin receptor autoactivation cascade at full-term pregnancy in the rat

1995 ◽  
Vol 311 (2) ◽  
pp. 523-529 ◽  
Author(s):  
C Martinez ◽  
J C Molero ◽  
P Ruiz ◽  
A Del Arco ◽  
A Andres ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Structural Changes ◽  
Insulin Receptors ◽  
Kinase Domain ◽  
Full Term ◽  
Beta Subunits ◽  
Pregnant Rats ◽  
Functional Changes ◽  
Domain Truncation ◽  
Alternative Sites

Partially purified liver insulin receptors from full-term pregnant rats show decreased autophosphorylation rates if compared with receptors from virgins. We studied the molecular mechanism of this phenomenon, looking at possible structural and functional changes of several domains. The ATP-binding domain seems to be unaltered in receptors from pregnant rats since Km for ATP was similar to that observed in virgins. In contrast, the Vmax. is decreased some 45%, suggesting changes in the kinase domain. Truncation of a fragment of 10 kDa from the C-terminal tail does not normalize the kinase activity in receptors from pregnant rats, suggesting that this domain is not involved in the inhibitory regulation. Treatment with alkaline phosphatase increases the [32P]Pi incorporation into receptors from pregnant rats; however, the autophosphorylation remains lower than that observed in virgin rats. Tryptic phosphopeptide maps of phosphorylated receptors show that the same phosphopeptides are present in receptors from virgin and pregnant rats. However, the progression through the autoactivation cascade in the kinase domain is impaired in receptors from pregnant rats. Differences in the cleavage by trypsin at the two alternative sites in the kinase domain were observed, indicating possible structural changes in receptors from pregnant rats that could be related to the impairment of the autoactivation cascade. Integrity of the alpha- and beta-subunits, as well as differential expression of the two receptor isotypes, were shown to be unaltered. We conclude that (1) the decreased autophosphorylation rate of the liver insulin receptor from pregnant rats is associated with the impairment of its autoactivation cascade, probably as a consequence of the basal Ser/Thr phosphorylation; and (2) the inhibition of the autoactivation cascade does not account for the overall inhibition of autophosphorylation observed in receptors from pregnant rats.

scholarly journals Alternative splicing, gene localization, and binding of SH2-B to the insulin receptor kinase domain

1999 ◽  
Vol 10 (12) ◽  
pp. 1160-1167 ◽  
Author(s):  
Keats Nelms ◽  
Thomas J. O'Neill ◽  
Shiqing Li ◽  
Stevan R. Hubbard ◽  
Thomas A. Gustafson ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Insulin Receptor ◽  
Kinase Domain ◽  
Gene Localization ◽  
Receptor Kinase ◽  
Insulin Receptor Kinase

scholarly journals Caveolin-1 Interacts with the Insulin Receptor and Can Differentially Modulate Insulin Signaling in Transfected Cos-7 Cells and Rat Adipose Cells

1999 ◽  
Vol 13 (12) ◽  
pp. 2013-2024 ◽  
Author(s):  
Fredrik H. Nystrom ◽  
Hui Chen ◽  
Li-Na Cong ◽  
Yunhua Li ◽  
Michael J. Quon
Keyword(s):  
Insulin Receptor ◽  
Insulin Signaling ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Binding Motif ◽  
Intact Cells ◽  
Caveolin 1 ◽  
Mitogen Activated Protein ◽  
Adipose Cells

Abstract Caveolae may function as microdomains for signaling that help to determine specific biological actions mediated by the insulin receptor (IR). Caveolin-1, a major component of caveolae, contains a scaffolding domain (SD) that binds to a caveolin-1 binding motif in the kinase domain of the IR in vitro. To investigate the potential role of caveolin-1 in insulin signaling we overexpressed wild-type (Cav-WT) or mutant (Cav-Mut; F92A/V94A in SD) caveolin-1 in either Cos-7 cells cotransfected with IR or rat adipose cells (low and high levels of endogenous caveolin-1, respectively). Cav-WT coimmunoprecipitated with the IR to a much greater extent than Cav-Mut, suggesting that the SD is important for interactions between caveolin-1 and the IR in intact cells. We also constructed several IR mutants with a disrupted caveolin-1 binding motif and found that these mutants were poorly expressed and did not undergo autophosphorylation. Interestingly, overexpression of Cav-WT in Cos-7 cells significantly enhanced insulin-stimulated phosphorylation of Elk-1 (a mitogen-activated protein kinase-dependent pathway) while overexpression of Cav-Mut was without effect. In contrast, in adipose cells, overexpression of either Cav-WT or Cav-Mut did not affect insulin-stimulated phosphorylation of a cotransfected ERK2 (but did significantly inhibit basal phosphorylation of ERK2). Furthermore, we also observed a small inhibition of insulin-stimulated translocation of GLUT4 when either Cav-WT or Cav-Mut was overexpressed in adipose cells. Thus, interaction of caveolin-1 with IRs may differentially modulate insulin signaling to enhance insulin action in Cos-7 cells but inhibit insulin’s effects in adipose cells.

scholarly journals Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation

1989 ◽  
Vol 260 (3) ◽  
pp. 749-756 ◽  
Author(s):  
V Baron ◽  
N Gautier ◽  
N Rochet ◽  
R Ballotti ◽  
B Rossi ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Insulin Receptor Tyrosine Kinase ◽  
Substrate Phosphorylation ◽  
Exogenous Substrates

Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.

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