Structural basis for phosphatase regulation by the anchoring protein AKAP79
Nygren, Patrick J.
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The calcium/calmodulin-activated protein phosphatase 2B (PP2B, or calcineurin) regulates diverse biological processes including glucose homeostasis and synaptic plasticity. PP2B is targeted to specific substrates, such as the GluRI subunit of the AMPA receptor, by the scaffolding molecule A-kinase anchoring protein 79 (AKAP79), which also interacts with protein kinase A (PKA). AKAP79 contains a short linear motif that is the primary site of interaction with PP2B. We have identified an auxiliary interaction site for PP2B at the N-terminal region of AKAP79. Using hybrid approaches including single particle EM, chemical crosslinking/mass spectrometry, biophysical techniques, and in vitro protein-protein interaction assays, I show that this additional interaction exclusively binds activated PP2B through a conserved LxVP mechanism. I mapped a 16 amino acid region in AKAP79 that is necessary for the LxVP interaction and show that the peptide can effectively compete for binding. I use FRET reporters for PP2B activity to show that this auxiliary interaction is important for tuning the activity of PP2B towards specific substrates. Preliminary negative stain EM studies reveal that AKAP79 forms multiple simultaneous contacts with PP2B in the presence of calcium and calmodulin. This structural model shows that AKAP79 has a greater role in regulating PP2B activity and targeting than previously appreciated. This work describes a dynamic model of PP2B anchoring in which the active phosphatase is anchored differently than the inactive form. A bipartite interaction allows control of localization through the PxIxIT motif, while simultaneously allowing fine control of PP2B sensitivity to calcium through the dynamic LxVP motif.
- Pharmacology