The Structural Determinants of PH Domain-Mediated Regulation of Akt Revealed by Segmental Labeling
- Harvard Medical School, Brigham and Women's Hospital, United States
- Harvard Medical School, Dana-Farber Cancer Institute, United States
- Harvard Medical School, United States
Abstract
Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties.
Data availability
Source data: Enzyme kinetics, fluorescence binding data, western blots, SDSPAGE gels have been deposited in Dryad: http://dx.doi:10.5061/dryad.0p2ngf1xg
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The Structural Determinants of PH Domain-Mediated Regulation of Akt Revealed by Segmental LabelingDryad Digital Repository, doi:10.5061/dryad.0p2ngf1xg.
Article and author information
Author details
Funding
National Cancer Institute (CA74305)
- Philip A Cole
Claudia Adams Barr Program in Innovative Cancer Research
- Haribabu Arthanari
Austrian Science Fund (Schroedinger Fellowship)
- Andras Boeszoermenyi
American Heart Association (19POST34380800)
- Andras Boeszoermenyi
National Institutes of Health (EB002026)
- Haribabu Arthanari
Kwanjeong Educational Foundation (pre-doctoral fellowship)
- Hwan Bae
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Chu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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The Structural Determinants of PH Domain-Mediated Regulation of Akt Revealed by Segmental Labeling
eLife 9:e59151.
https://doi.org/10.7554/eLife.59151
Further reading
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- Biochemistry and Chemical Biology
In healthy cells, cyclin D1 is expressed during the G1 phase of the cell cycle, where it activates CDK4 and CDK6. Its dysregulation is a well-established oncogenic driver in numerous human cancers. The cancer-related function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G-Two and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unrecognized substrate of cyclin D1–CDK4/6 in tumor cells overexpressing cyclin D1 during G1 and subsequent phases. The phosphorylation of GTSE1 mediated by cyclin D1–CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 across all cell cycle phases. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1’s role in cell cycle control and oncogenesis beyond RB phosphorylation.
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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
