Neurabin is a brain-specific actin and protein phosphatase-1 (PP-1) binding protein that inhibits the purified catalytic subunit of protein phosphatase-1 (PP-1(C)). However, endogenous PP-1 exists primarily as multimeric complexes of PP-1(C) bound to various regulatory proteins that determine its activity, substrate specificity, subcellular localization and function. The major form of endogenous PP-1 in brain is protein phosphatase-1(I) (PP-1(I)), a Mg2+/ATP-dependent form of PP-1 that consists of PP-1(C), the inhibitor-2 regulatory subunit, an activating protein kinase and other unidentified proteins. We have identified four PP-11 holoenzyme fractions (PP-1(A), PP-1(B), PP-1(IC), and PP-1(ID)) in freshly harvested pig brain separable by poly-L-lysine chromatography. Purified recombinant neurabin (amino acid residues 1-485) inhibited PP-1(IB) (IC50= 1-1 mu M), PP-1(IC) (IC50 = 0.1 mu M), and PP-1(ID) (IC50 = 0.2 mu M), but activated PP-1(IA) by up to threefold (EC50 = 40 nM). The PP-1(IA) activation domain was localized to neurabin(1-210). Our results indicate a novel mechanism of PP-1 regulation by neurabin as both an inhibitor and an activator of distinct forms of PP-1(I) in brain. (c) 2007 Elsevier Inc. All rights reserved.