Importance of a Surface Hydrophobic Pocket on Protein Phosphatase-1 Catalytic Subunit in Recognizing Cellular Regulators

Abstract

Cellular functions of protein phosphatase-1 (PP1), a major eukaryotic serine/threonine phosphatase, are defined by the association of PP1 catalytic subunits with endogenous protein inhibitors and regulatory subunits. Many PP1 regulators share a consensus RVXF motif, which docks within a hydrophobic pocket on the surface of the PP1 catalytic subunit. Although these regulatory proteins also possess additional PP1-binding sites, mutations of the RVXF sequence established a key role of this PP1-binding sequence in the function of PP1 regulators. WT PP1␣, the C-terminal truncated PP1␣-(1-306), a chimeric PP1␣ containing C-terminal sequences from PP2A, another phosphatase, PP1␣-(1-306) with the RVXF-binding pocket substitutions L289R, M290K, and C291R, and PP2A were analyzed for their regulation by several mammalian proteins. These studies established that modifications of the RVXF-binding pocket had modest effects on the catalytic activity of PP1, as judged by recognition of substrates and sensitivity to toxins. However, the selected modifications impaired the sensitivity of PP1 to the inhibitor proteins, inhibitor-1 and inhibitor-2. In addition, they impaired the ability of PP1 to bind neurabin-I, the neuronal regulatory subunit, and G M , the skeletal muscle glycogen-targeting subunit. These data suggested that differences in RVXF interactions with the hydrophobic pocket dictate the affinity of PP1 for cellular regulators. Substitution of a distinct RVXF sequence in inhibitor-1 that enhanced its binding and potency as a PP1 inhibitor emphasized the importance of the RVXF sequence in defining the function of this and other PP1 regulators. Our studies suggest that the diversity of RVXF sequences provides for dynamic physiological regulation of PP1 functions in eukaryotic cells.Early studies by Ingebritsen and Cohen (1, 2) classified mammalian protein serine/threonine phosphatases into two major groups, type-1 and type-2, based in large part on their sensitivity to two endogenous protein inhibitors, inhibitor-1 (I-1) 1 and inhibitor-2 (I-2). Type-I protein serine/threonine phosphatase or protein phosphatase-1 (PP1), displays unique and potent inhibition by nanomolar concentrations of I-1 and I-2. In contrast, PP2A and other type-2 protein phosphatases are essentially resistant to these inhibitor proteins. To date, more than 50 PP1-interacting proteins have been identified (3). Some, like I-1 and I-2, function as PP1 inhibitors, whereas others represent targeting subunits that direct subcellular localization and substrate recognition by multiprotein complexes containing PP1 catalytic subunits. Remarkably, PP1 and PP2A catalytic subunits share nearly 50% primary sequence identity, which may account for their overlapping substrate specificity in vitro (1). However, all PP1 regulators thus far analyzed demonstrate the unique ability displayed by I-1 and I-2 to selectively associate with PP1 and modify its catalytic function.Detailed structure-function analyses of I-1 (4) and its neuronal homologue, DARPP-32 (5), first hig...

Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2

“…Despite the apparent dissimilarity between Gln and Val, the aliphatic C ␤ and C ␥ atoms of I-2 Gln 46 interact with PP1c similarly to Val, and most importantly, the side chain amide nitrogen of I-2 Gln 46 hydrogen bonds with the sulfhydryl of PP1c Cys 291 (Fig. 4d), which is highly conserved in all PP1c isoforms (45) and may contribute to the ability of I-2 to potently inhibit all PP1 catalytic subunits. In contrast, the equivalent position in PP2A is Tyr 284 , and this substitution as well as others in and around the corresponding binding site for residues 12-17 of I-2 accounts for the inability of I-2 to inhibit PP2A activity, despite the highly conserved active site structures in the PP1 and PP2A phosphatases (31,32).…”

Expression of Human Protein Phosphatase-1 in Saccharomyces cerevisiae Highlights the Role of Phosphatase Isoforms in Regulating Eukaryotic Functions

“…PP1 Catalytic Subunits-Recombinant PP1 catalytic subunits were expressed in E. coli as described (26). Native PP1 catalytic subunits were purified from rabbit skeletal muscle (27).…”

Direct Interaction between Myocyte Enhancer Factor 2 (MEF2) and Protein Phosphatase 1α Represses MEF2-Dependent Gene Expression

“…5B). While tPP1␣ has increased phosphatase activity, essentially normal RVXF binding (19), and normal subcellular localization (not shown), the inability to negatively regulate MEF2 suggests the far C terminus of PP1␣ is required for regulation of MEF2-dependent transcriptional activity. Since the C terminus of PP1 contains an important regulatory threonine residue (T320), we examined PP1␣ mutants that are constitutively active (threonine 320 mutated to alanine [T320A]) (2) or a phospho-mimetic, inactive phosphatase (T320 mutated to glutamic acid [T320E]) (Fig.…”

“…The three PP1 forms (␣, ␤, and ␥) show tissue-specific expression patterns and R-subunit binding specificity (11), and the necessary regions of PP1␣ involved in R-subunit interaction have been well characterized (19). Coexpression of PP1 [tPP1␣]) demonstrated that all three PP1 isoforms repress MEF2 transactivation activity, whereas tPP1␣ exerted no effect (Fig.…”