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Insulin-induced activation of phosphoinositide 3-kinase in Fao cells

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Summary

Phosphoinositide 3-kinase (PI3-kinase) plays a crucial role in insulin signal transduction. We studied the molecular mechanism of the insulin-induced activation of PI3-kinase in rat hepatoma Fao cells using an antibody against the 110-kDa catalytic subunit (p110) and two against the 85-kDa regulatory subunit (p85α). PI3-kinase activity increased 1.6-fold in anti-p85 immunoprecipitates after insulin stimulation, whereas it did not increase when cell lysates were first immunoprecipitated with anti-phosphotyrosine or anti-insulin receptor substrate-1 (IRS-1), then with anti-p85, suggesting that the PI3-kinase which associates with tyrosyl phosphoproteins including IRS-1 is responsible for the increase in kinase activity. The activated PI3-kinase molecules constituted 4–6% of the total PI3-kinase, and their specific activity was 11–14 times higher than that of the basal state. Anti-p110 recognized the catalytically active form of p110, and immunoprecipitated p110 only after exposure to insulin. Hence, the epitope of anti-p110, P200-C215, seems to be included in the portion of p110, the conformation of which is changed by insulin stimulation. We conclude that, in response to insulin stimulation, only a small fraction of p85 in the PI3-kinase pool associates with tyrosyl phosphoproteins including IRS-1, and that the specific activity of p110 is increased presumably through a conformational change including the P200-C215 region.

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Abbreviations

PI3-kinase:

Phosphoinositide 3-kinase

p85:

85-kDa subunit of PI3-kinase

p110:

110-kDa subunit of PI3-kinase

IRS-1:

insulin receptor substrate-1

SH2:

src homology 2

SH3:

src homology 3

BCR:

breakpoint cluster region

PMSF:

phenylmethylsulphonyl fluoride

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid

PIP:

phosphatidylinositol phosphate

TLC:

thin layer chromatography

IP (in figures):

immunoprecipitation with the indicated antibody

TBS:

Tris-buffered saline

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Hayashi, T., Okamoto, M., Yoshimasa, Y. et al. Insulin-induced activation of phosphoinositide 3-kinase in Fao cells. Diabetologia 39, 515–522 (1996). https://doi.org/10.1007/BF00403297

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  • DOI: https://doi.org/10.1007/BF00403297

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