Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

Kathleen H Holt; Ann Louise Olson; W Scott Moye-Rowley; Jeffrey E Pessin

doi:10.1128/mcb.14.1.42-49.1994

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Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

Journal article

Open access

Peer reviewed

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

Kathleen H Holt, Ann Louise Olson, W Scott Moye-Rowley and Jeffrey E Pessin

Molecular and cellular biology, Vol.14(1), pp.42-49

01/1994

DOI: 10.1128/mcb.14.1.42-49.1994

PMCID: PMC358354

PMID: 8264609

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https://europepmc.org/articles/pmc358354View

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Abstract

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.

Gene Expression

Amino Acid Sequence

beta-Galactosidase - genetics

beta-Galactosidase - metabolism

DNA, Fungal - genetics

Enzyme Activation

Genes, Fungal

Genes, Reporter

Molecular Sequence Data

Phosphatidylinositol 3-Kinases

Phosphotransferases (Alcohol Group Acceptor) - chemistry

Phosphotransferases (Alcohol Group Acceptor) - genetics

Phosphotransferases (Alcohol Group Acceptor) - metabolism

Protein Conformation

Recombinant Fusion Proteins - genetics

Recombinant Fusion Proteins - metabolism

Saccharomyces cerevisiae - enzymology

Saccharomyces cerevisiae - genetics

Sequence Deletion

Details

Title: Subtitle: Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits
Creators: Kathleen H Holt - Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242
Ann Louise Olson
W Scott Moye-Rowley
Jeffrey E Pessin
Resource Type: Journal article
Publication Details: Molecular and cellular biology, Vol.14(1), pp.42-49
DOI: 10.1128/mcb.14.1.42-49.1994
PMID: 8264609
PMCID: PMC358354
ISSN: 0270-7306
eISSN: 1098-5549
Grant note: DK33823 / NIDDK NIH HHS DK25925 / NIDDK NIH HHS
Language: English
Date published: 01/1994
Academic Unit: Molecular Physiology and Biophysics; Internal Medicine
Record Identifier: 9984297504802771

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