Identification and analysis of PH domain‐containing targets of phosphatidylinositol 3‐kinase using a novel in vivo assay in yeast
Open accessAbstract
Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.
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PI(3,4,5)P3 and its derivative PI(3,4)P2 interact with the lipid-binding PH domain in a range of protein effectors, resulting in their recruitment to membrane-signalling complexes and/or modulation of their activity.
“PI(3,4,5)P3 and PI(3,4)P2 interact with the lipid-binding PH-domain in a range of protein effectors, resulting in their recruitment to membrane-signalling complexes and/or modulation of their activity [78–81].”
From Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane (2020) · cites this paper as [78]
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Research works printed but not linked: 6
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- Colicelli,J., Birchmeier,C., Michaeli,T., O'Neill,K., Riggs,M. and Wigler,M. (1989) Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase. Proc. Natl Acad. Sci. USA, 86, 3599-3603.
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Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane
Katan, M., & Cockcroft, S. (2020) · Essays in Biochemistry · doi:10.1042/ebc20200041 · Cited by 176 · see citing works
Park, W. S., Do Heo, W., Whalen, J. H., O'Rourke, N. A., Bryan, H. M., Meyer, T., & Teruel, M. N. (2008) · Molecular Cell · doi:10.1016/j.molcel.2008.04.008 · Cited by 167 · see citing works
- Authors
- Isakoff, S. J., Cardozo, T., Andreev, J., Li, Z., Ferguson, K. M., Abagyan, R., Lemmon, M. A., Aronheim, A., & Skolnik, E. Y.
- Affiliations
- NYU Langone Health (US)
- University of Pennsylvania (US)
- Technion – Israel Institute of Technology (IL)
- Corresponding author
- Skolnik, Edward Y.
- ORCID
- Venue
- The EMBO Journal
- Published
- 1998-09-15
- Volume
- 17
- Issue
- 18
- Pages
- 5374-5387
- ISSN
- 0261-4189 (ISSN-L)
- 1460-2075
- PMC
- PMC1170863
- OpenAlex
- W2141206157
- Cited by
- 324
- References
- 0
- Citations by year
- 2026: 1 · 2025: 5 · 2024: 1 · 2023: 3 · 2022: 5 · 2021: 4 · 2020: 4 · 2019: 9 · 2018: 6 · 2017: 5 · 2016: 3 · 2015: 11 · 2014: 7 · 2013: 8 · 2012: 12
- FWCI
- 9.09
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- The EMBO Journal (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- American Diabetes Association
- Damon Runyon Cancer Research Foundation
- National Institute of Diabetes and Digestive and Kidney Diseases
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Keywords
- phosphatidylinositol 3-kinase, pleckstrin homology domain, PtdIns(3,4)P2, phosphatidylinositol 3,4,5-trisphosphate, Saccharomyces cerevisiae, Akt, Bruton's tyrosine kinase, consensus sequence, Gab1, DOS, myosin X, phosphoinositide signaling
- MeSH terms
- Amino Acid Sequence, Blood Proteins, Cell Membrane, Fungal Proteins, Models, Molecular, Mutation, Phosphoproteins, Protein Binding, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Second Messenger Systems, Consensus Sequence, Conserved Sequence, Sequence Homology, Amino Acid, Phosphatidylinositol Phosphates, ras Proteins, Phosphatidylinositol 3-Kinases
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