Using Markov State Models to Develop a Mechanistic Understanding of Protein Kinase A Regulatory Subunit RIα Activation in Response to cAMP Binding

Britton W Boras,Alexandr Kornev,Susan S Taylor,Andrew D Mcculloch

doi:10.1074/jbc.m114.568907

Britton W Boras, Alexandr Kornev + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m114.568907

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Abstract

Protein kinase A (PKA) holoenzyme consists of two catalytic (C) subunits and a regulatory (R) subunit dimer (R(2)C(2)). The kinase is activated by the binding of cAMPs to the two cyclic nucleotide binding domains (CBDs), A and B, on each R-subunit. Despite extensive study, details of the allosteric mechanisms underlying the cooperativity of holoenzyme activation remain unclear. Several Markov state models of PKA-RIα were developed to test competing theories of activation for the R(2)C(2) complex. We found that CBD-B plays an essential role in R-C interaction and promotes the release of the first C-subunit prior to the binding to CBD-A. This favors a conformational selection mechanism for release of the first C-subunit of PKA. However, the release of the second C-subunit requires all four cAMP sites to be occupied. These analyses elucidate R-C heterodimer interactions in the cooperative activation of PKA and cAMP binding and represent a new mechanistic model of R(2)C(2) PKA-RIα activation.

Highlights

Binding four cAMP molecules activates protein kinase A (PKA)
It was able to reproduce the free R-subunit cAMP binding with no predicted value further than two S.E. from the experimental data, suggesting that the R-C heterodimer interactions necessary for data sets I, II, III, and V are less significant for binding to free R-subunit
cyclic nucleotide binding domains (CBDs)-BЈ did not affect the C-subunit (F1 ϭ 1) even when it was not constrained. These results reduce the unconstrained model to the form of the dually regulated model, suggesting that these additional degrees of freedom are not needed to explain the current data (Tables 2 and 3)

Summary

Introduction

Results: Mechanistic Markov models (MM) show that one cAMP activates one catalytic subunit four are necessary for full activation. We found that CBD-B plays an essential role in R-C interaction and promotes the release of the first C-subunit prior to the binding to CBD-A. This favors a conformational selection mechanism for release of the first C-subunit of PKA. The release of the second C-subunit requires all four cAMP sites to be occupied These analyses elucidate R-C heterodimer interactions in the cooperative activation of PKA and cAMP binding and represent a new mechanistic model of R2C2 PKA-RI␣ activation

Results

Discussion

Conclusion