Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

Decision letter after peer review:

Thank you for submitting your article "Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Volker Dötsch as Reviewing Editor and Reviewer #3, and the evaluation has been overseen by Philip Cole as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Stefan Knapp (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. All reviewers agreed that the experiments reported interesting results on the regulation of PKA activity by local concentration. The main criticism focuses on the use of overexpression and the not reported relative concentrations of PKA and PKAR-IIβ.

Essential revisions:

1) Information on endogenous levels of PKA and of the regulatory subunit should be provided to show that at least overall levels are within the physiological range. A IHC comparison would be most useful as PKA concentration could be probed at different cellular locations in the presence and absence of PKA pathway stimulation.

2) More information on the use of overexpression or stable expressing cell lines is needed in the description of the experiments and figure legends.

3) A mathematical model that describes the quantitative behavior of the system would be very useful.Reviewer #1:

This is a very thorough and important study demonstrating quantitative control signaling through changes in the abundance and localization of a regulatory kinase subunit. The authors use live imaging experiments in microfluidic devices to reveal nonmonotonic dependence of PKA activity on the level of its regulatory subunit and provide evidence that it translates into corresponding changes of cell polarization and cell migration. The presentation is very clear and the results are convincing. Given the quantitative nature of the results, the authors could try to summarize their observations using a mathematical model. This would make the discussion even more convincing.

Reviewer #2:

The authors developed a FRET based sensor system that allows for the positioning of PKA at the plasma membrane in the absence of direct pathway stimulation. The system consists of a rapamycin-inducible dimerization domain which was achieved by linking the binding partner of rapamycin, FKBP, to fluorescent tagged PKA regulatory subunit PKAR-IIβ (construct PKAR-FKBP-FP) and a second fluorescent protein linked to the membrane-targeting sequence of Lyn kinase (Lyn-FRB). This assay system was used to shed light on the question of how localization affects PKA signalling outputs including complex phenotypic responses such a polarity and cell migration. The study also addresses the role of the regulatory subunit which on the one hand is a potent inhibitor of PKA activity but it also targets PKA to cellular locations, a prerequisite for PKA dependent signalling, thus functional also as a local "activator". The authors indeed found evidence of this paradoxical activating role of the regulatory subunit, which was transient in some cell types. An innovative microfluidic device controlled rapamycin gradients revealing that cell polarity can be tuned by the slope of PKA-R gradient.

The developed inducible FRET system provides interesting insights into the role of PKA location and the regulatory subunit. However, the system relies on ectopic expression only and it is not clear how the expression level compares with endogenous PKA levels. The main cell system (HeLa) is not the most obvious choice of cell lines for these studies as these cells are genetically highly compromised.

As the authors nicely show that PKA activity is highly dependent on PKAR-IIβ concentration, the relative levels of this regulatory subunit should be compared to levels in a PKA relevant cellular system. It is not clear if transient transfections or stable cell lines were used. It would have been helpful to add this information to the figure legends.

Reviewer #3:

LaCroix et al., investigate the catalytic activity of the protein kinase PKA in the context of the translocation of the regulatory subunit PKA-R to the plasma membrane, For this forced translocation they use the rapamycin / FBBP system with one component tethered to the regulatory subunit and the other to a plasma membrane anchor. They observe a rapid translocation of the regulatory unit to the plasma membrane as well as a translocation of the catalytic subunit of the kinase. Probing the activity of the kinase they observe a complex behavior with the activity first increasing and then decreasing. This behavior also depends on the rapamycin concentration used with lower concentrations – translating into a slower translocation of the regulatory subunit to the plasma membrane – resulting in a slower but sustained increase in catalytic activity. They also demonstrate the effect by measuring cell migration using microfluidic devices and rapamycin gradients.

The interpretation of the authors is that for the full activity a dimer of the catalytic subunit must be bound to the dimeric regulatory unit which then gets activated at the plasma membrane by binding to cyclic AMP. If the concentration of the regulatory unit at the membrane increases the catalytic subunits will progressively bind to so far un-complexed regulatory subunits which will – after an initial increase in activity – decrease the activity again. This interpretation is logical. A limitation, however, is that the authors do not provide measures of the (relative) cellular concentrations of the catalytic and the regulatory subunits. Since they use a lentiviral or transient transfection system it remains unclear if the observed effect is relevant at all under basal expression conditions or are created by the overexpression of one of the components.