Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2

Thank you for sending your work entitled “Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2” for consideration at eLife. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom, Philip Cole is a member of our Board of Reviewing Editors.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

The manuscript by Zorba et al is a multifaceted study aimed at clarifying the roles of autophosphorylation and TPX2 mechanisms of activation of the Aurora A kinase (AAK) as well as the molecularity of autophosphorylation of AAK. Several key findings include:

1) The stimulation by AAK autophosphorylation of Thr288 (by 100-fold) is roughly matched by TPX2 stimulation of unphosphorylated AAK (by 40-fold)

2) The domain swapped dimer of the unphosphorylated AAK in the presence of TPX2 represents a possible interaction of intermolecular autohosphorylation.

3) The solution phase assays especially with a dead mutant as substrate demonstrate the kinetic competence of intermolecular autophosphorylation.

Additional bolstering data include molecular modeling showing how the disordered loop in monomer II of AAK could be positioned for phosphoryl transfer in the active site of monomer I of AAK, analytical ultracentrifugation and SAXS showing the presence of a minor population of solution phase dimer in AAK, and ITC and enzymatic experiments demonstrating that aa1-25 of TPX2 are necessary and sufficient for high affinity binding (ca 2 uM) and activation. The careful use of Abs and mass spec really help to provide quantitative information about the state of Thr288 phosphorylation in the various enzyme forms. Importantly, the results here fill gaps in the literature about AAK regulation and contradict those from a recent study from Dodson et al reporting that AAK autophosphorylation is intramolecular. Zorba et al point out several limitations in Dodson et al that can account for the differing conclusions. Overall, Zorba et al is carefully executed, well-written, and makes a set of significant findings that should interest a broad readership. We ask the authors to address the issues below.

1) A missing piece that if added would significantly enhance this study relates to the structural basis of AAK autophosphorylation. The domain swapped AAK dimer shows surfaces of interaction in the C-lobes that one assumes are important for intermolecular autophosphorylation if the authors' model is correct. It would be very useful to mutagenize these contacting residues to ascertain their contributions to the kinetics of AAK autophosphorylation. A hypothesis to be tested would be that once autophosphorylation is completed (even if slower in such contact mutants), activity of such AAK mutants with peptide substrates would be near normal.

2) The data in Figure 5B at 1 µM AAK suggests a half-life of at least 400 seconds. This would predict a half-life of at least 4000 seconds (67 minutes) at 100 nM. These slow rates invite concerns about the physiologic significance of autophosphorylation as an activation mechanism of AAK rather than its activation by an upstream kinase such as PAK1 or CDK11. On the other hand, it appears that the assays were carried out at 25°C, which could slow things down. Moreover, the local effective concentration of AAK may be higher than its total cellular concentration. We hope that the authors can provide a perspective on whether the preponderance of existing evidence supports that AAK autophosphorylation is relevant to AAK's normal cellular function.