MAP kinases and mTOR mediate insulin-induced phosphorylation of insulin receptor substrate-1 on serine residues 307, 612 and 632.
OBJECTIVE
Insulin-induced IRS-1 serine phosphorylation could be physiologically important to regulate insulin action. In a hyperinsulinaemic state such as obesity or Type 2 diabetes, this phosphorylation could be modified and exacerbate insulin resistance. We aimed at identifying serine residues in IRS-1 phosphorylated in response to insulin stimulation and at determining the involved kinases.


METHODS
3T3-L1 adipocytes, muscle and adipose tissue of mice were subjected to Western Blot analysis with phosphospecific antibodies to identify phosphorylation sites in IRS-1 following insulin treatment. Pharmacological inhibitors were used to determine the serine kinases involved in this phosphorylation.


RESULTS
In 3T3-L1 adipocytes, insulin promoted the phosphorylation of serine 307, 612 and 632 with Serine(612/632) more rapidly phosphorylated than Serine(307). Insulin-induced phosphorylation of Serine(307) was dependent on the activation of a PI 3-kinase/mTOR pathway. The phosphorylation of Serine(612/632) required the activation of the MAP kinase pathway following short-term insulin stimulation and activation of the PI 3-kinase/mTOR pathway following prolonged insulin stimulation. Phosphorylation of Serine(307) and Serine(632) occurred in vivo in skeletal muscle and white adipose tissue of mice injected with insulin and was dependent on the activation of mTOR. Moreover, inhibition of mTOR led to a persistent PI 3-kinase activation by insulin.


CONCLUSIONS
Insulin-induced IRS-1 serine phosphorylation is a complex process involving different sites and kinases. This complexity could be physiologically important to accurately regulate insulin signalling. Abnormal phosphorylation of these serine residues in hyperinsulinaemic state could participate in the down-regulation of insulin signalling.
Gual
P
P
INSERM U568 and IFR50, Faculté de Médecine, Nice Cedex 02, France.
Grémeaux
T
T
Gonzalez
T
T
Le Marchand-Brustel
Y
Y
Tanti
J-F
JF
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D007328:Insulin / Q000494:pharmacology* / Q000502:physiology; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases
D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D010768:Phosphoserine; D011494:Protein Kinases; D058570:TOR Serine-Threonine Kinases; C526295:Irs1 protein, mouse; C546843:mTOR protein, mouse