Regulation of PP1 interaction with I-2, neurabin, and F-actin.

Reversible phosphorylation is a fundamental regulatory mechanism required for many biological processes and is coordinated by the opposing actions of protein kinases and phosphatases. Protein phosphatase 1 (PP1) is a major protein phosphatase that plays an important role in many fundamental physiological processes including synaptic transmission and memory formation. Here we investigate the regulation of PP1 by prominent signaling proteins and synaptic scaffolds including GSK3β, inhibitor-2 (I-2), neurabin (Nrb), and actin. While GSK3β is known to regulate PP1 via phosphorylation of the PP1-binding protein I-2, we found that GSK3β directly regulates PP1 via inhibitory phosphorylation in neurons. Additionally, using bioluminescence resonance energy transfer (BRET), we found that GSK3β alters PP1-I-2 interaction in living cells. The effect of GSK3β on PP1-I-2 interaction is independent of the PP1 C-terminal tail, contrary to predictions based on previous findings from purified proteins. I-2 has been shown to form a trimeric complex with PP1 and Nrb, a major synaptic scaffold for promoting PP1 localization to the actin cytoskeleton. Utilizing BRET, we found that Nrb promotes PP1-actin interaction, however no BRET was detected between I-2 and F-actin. Finally, we found that stabilizing F-actin promotes Nrb-PP1 binding and may also lead to conformational changes between Nrb-I-2 and Nrb-F-actin complexes. Overall, our findings elaborate the dynamic regulation of PP1 complexes by GSK3β, targeting proteins, and actin polymerization.