Insulin Stimulates Phosphatidylinositol 3-Kinase Activity in Rat Neuronal Primary Cultures

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

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Enhanced Phosphatidylinositol 3-Kinase Activity and High Phosphorylation State of Its Downstream Signalling Molecules Mediated by Ret with the MEN 2B Mutation

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1999.1186 ◽

1999 ◽

Vol 262 (1) ◽

pp. 68-75 ◽

Cited By ~ 56

Author(s):

Hideki Murakami ◽

Toshihide Iwashita ◽

Naoya Asai ◽

Yohei Shimono ◽

Yosuke Iwata ◽

...

Keyword(s):

Kinase Activity ◽

Phosphatidylinositol 3 Kinase ◽

Phosphorylation State ◽

Signalling Molecules ◽

Men 2B ◽

Phosphatidylinositol 3

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Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.279.2.e266 ◽

2000 ◽

Vol 279 (2) ◽

pp. E266-E274 ◽

Cited By ~ 9

Author(s):

Paul G. Drake ◽

Alejandro Balbis ◽

Jiong Wu ◽

John J. M. Bergeron ◽

Barry I. Posner

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Insulin Receptor ◽

Rat Liver ◽

Intracellular Signaling ◽

Kinase Activity ◽

Glycogen Synthase ◽

Metabolic Effects ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK β-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

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Insulin-Dependent Phosphatidylinositol 3′-Kinase Activity Co-precipitates with Insulin Receptor in Human Circulating Mononuclear Cells

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1995.1494 ◽

1995 ◽

Vol 209 (1) ◽

pp. 234-241 ◽

Cited By ~ 3

Author(s):

E. Renard ◽

F. Grigorescu ◽

C. Lavabre ◽

C.R. Kahn

Keyword(s):

Insulin Receptor ◽

Kinase Activity ◽

Mononuclear Cells ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Dependent ◽

Phosphatidylinositol 3

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Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology

Journal of Cell Science ◽

10.1242/jcs.108.12.3745 ◽

1995 ◽

Vol 108 (12) ◽

pp. 3745-3756 ◽

Cited By ~ 2

Author(s):

K. Takegawa ◽

D.B. DeWald ◽

S.D. Emr

Keyword(s):

Amino Acid ◽

Schizosaccharomyces Pombe ◽

Mammalian Cells ◽

Membrane Fraction ◽

Kinase Activity ◽

Temperature Sensitive ◽

Wild Type ◽

Phosphatidylinositol 3 Kinase ◽

Acid Protein ◽

Phosphatidylinositol 3

We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S.pombe strains are temperature-sensitive for growth and the mutant cells contain enlarged vacuoles. Furthermore, while wild-type strains exhibit substantial levels of phosphatidylinositol 3-kinase activity, this activity is not detected in the vps34 delta strain. S.pombe Vps34p-specific antiserum detects a single protein in cells of -90 kDa that fractionates almost exclusively with the crude membrane fraction. Phosphatidylinositol 3-kinase activity also is localized mainly in the membrane fraction of wild-type cells. Immunoisolated Vps34p specifically phosphorylates phosphatidylinositol on the D-3 position of the inositol ring to yield phosphatidylinositol(3)phosphate. but does not utilize phosphatidylinositol(4)phosphate or phosphatidylinositol(4,5)bisphosphate as substrates. In addition, when compared to the mammalian p110 phosphatidylinositol 3-kinase, S. pombe Vps34p is relatively insensitive to the inhibitors wortmannin and LY294002. Together, these results indicate that S. pombe Vps34 is more similar to the phosphatidylinositol-specific 3-kinase, Vps34p from S. cerevisiae, and is distinct from the p110/p85 and G protein-coupled phosphatidylinositol 3-kinases from mammalian cells. These data are discussed in relation to the possible role of Vps34p in vesicle-mediated protein sorting to the S. pombe vacuole.

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