The structural basis of the multi-step allosteric activation of Aurora B kinase
- University of Oslo, Norway
- University of Pennsylvania, United States
Abstract
Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. The mass spectrometry proteomics data are available through the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD038935.
Article and author information
Author details
Funding
Norges Forskningsråd (187615)
- Dario Segura-Peña
- Oda Hovet
- Hemanga Gogoi
- Stine Malene Hansen Wøien
- Nikolina Sekulic
Norges Forskningsråd (262695)
- Oda Hovet
- Manuel Carrer
- Michele Cascella
Norwegian Supercomputing Program (NN4654K)
- Oda Hovet
- Manuel Carrer
- Michele Cascella
Norges Forskningsråd (325528)
- Nikolina Sekulic
National Institute of General Medical Sciences (R35-GM130302)
- Jennine Dawicki-McKenna
- Ben E Black
National Institute of General Medical Sciences (GM108360)
- Jennine Dawicki-McKenna
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Segura-Peña 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 basis of the multi-step allosteric activation of Aurora B kinase
eLife 12:e85328.
https://doi.org/10.7554/eLife.85328
Further reading
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- Biochemistry and Chemical Biology
The Parkinson’s disease (PD)-linked protein Leucine-Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis–Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has, similar to other Roco protein family members, a KM value of LRRK2 that lies within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. In contrast, the most common PD variant G2019S, located in the kinase domain, showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G domain. Autophosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM and the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer–dimer equilibrium. The LRRK2 T1343A variant has a similar increased kinase activity in cells compared to G2019S and the double mutant T1343A/G2019S has no further increased activity, suggesting that T1343 is crucial for the negative feedback in the LRRK2 signaling cascade. Together, our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase-dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.
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