The split protein phosphatase system.

Biochemical Journal (2018) 475 3707–3723 https://doi.org/10.1042/BCJ20170726 Review Article The split protein phosphatase system Anne Bertolotti MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, U.K. Correspondence: Anne Bertolotti () Reversible phosphorylation of proteins is a post-translational modification that regulates all aspect of life through the antagonistic action of kinases and phosphatases. Protein kinases are well characterized, but protein phosphatases have been relatively neglected. Protein phosphatase 1 (PP1) catalyzes the dephosphorylation of a major fraction of phospho-serines and phospho-threonines in cells and thereby controls a broad range of cellular processes. In this review, I will discuss how phosphatases were discovered, how the view that they were unselective emerged and how recent findings have revealed their exquisite selectivity. Unlike kinases, PP1 phosphatases are obligatory heteromers composed of a catalytic subunit bound to one (or two) non-catalytic subunit(s). Based on an in-depth study of two holophosphatases, I propose the following: selective dephosphorylation depends on the assembly of two components, the catalytic subunit and the non-catalytic subunit, which serves as a high-affinity substrate receptor. Because functional complementation of the two modules is required to produce a selective holophosphatase, one can consider that they are split enzymes. The non-catalytic subunit was often referred to as a regulatory subunit, but it is, in fact, an essential component of the holoenzyme. In this model, a phosphatase and its array of mostly orphan substrate receptors constitute the split protein phosphatase system. The set of potentially generalizable principles outlined in this review may facilitate the study of these poorly understood enzymes and the identification of their physiological substrates. Introduction Received: 29 August 2018 Revised: 29 October 2018 Accepted: 1 November 2018 Version of Record published: 6 December 2018 Life depends on the controlled regulation of the activity of thousands of proteins. Protein phosphorylation is a post-translational modification that controls the fate, location and activity of the majority of cellular proteins. Protein phosphorylation occurs predominantly on serines and threonines, with kinases catalyzing the addition of a phosphate group and phosphatases reversing this. One can think of protein phosphorylation as a switch to turn signaling on or off through the antagonistic action of kinases and phosphatases. However, the reality may be more nuanced. It is likely that protein phosphorylation provides a versatile way to control protein function and fate through the constant antagonistic actions of kinases and phosphatases, both being most probably highly regulated. In that sense, the state of a given protein would oscillate between a phosphorylated and a non-phosphorylated state to adjust cellular functions to various signals resulting from changes in conditions. There are ∼500 kinases in humans [1] and ∼189 phosphatases [2]. Unlike kinases which share a common catalytic fold and mechanism [1], phosphatases exhibit greater diversity of structures and catalytic mechanisms [2]. In contrast to kinases, which consist of a single polypeptide chain, phosphatases are found in complex with one or two non-catalytic subunits. Protein phosphatase 1 (PP1) is an abundant protein that catalyzes most serine–threonine dephosphorylation in cells. Historically, PP1 has been purified following a procedure that dissociated it from its interactors [3]. The resulting enzyme is active against a variety of substrates leading to the erroneous notion that phosphatases are not selective. Because PP1 controls a large number of signaling events, it is hard to imagine why evolution would have designed a nonselective enzyme that controls so many aspects of life. I will argue that while PP1 is ubiquitous, it is not promiscuous. In contrast to © 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). 3707 Biochemical Journal (2018) 475 3707–3723 https://doi.org/10.1042/BCJ20170726 the in vitro situation, it is believed that there is no free PP1 in cells, which is instead associated with one or two among an array of diverse non-catalytic subunits [4]. These complexes are the physiological holophosphatases and I will discuss how this heteromeric design led to exquisite selectivity. I will begin this review with a historical overview of the discovery of protein phosphatases and integrate historical findings with recent observations. Discovery of protein phosphatase Gerty Cori and Arda Green, working on glycogen phosphorylase, an enzyme that catalyzes the rate-limiting step of glycogenolysis, discovered protein phosphorylation by discovering the first phosphatase activity [5]. Phosphorylase can be isolated from muscle in two forms: an active form, phosphorylase a and an inactive form, phosphorylase b. Phosphorylase b becomes active upon the addition of AMP, whereas a is active without it. Remarkably, Cori and Green figured out that a contains a non-protein prosthetic group, which they reported correctly was covalently attached, because it could not be dissociated easily. Its removal needed an enzyme, contained in muscles and other tissues, which they named PR: the prosthetic group-removing enzyme [5]. The prosthetic group was, in fact, a phosphate and PR was the phosphate-removing enzyme (the phosphatase PP1). This discovery was also the first report of an allosteric regulation of an enzyme, although the concept of allostery was only formally articulated later [6]. Now, 75 years later, phosphorylase a is still widely used as a model substrate to study protein phosphatases. Yet, as we know, protein phosphorylation reversibly regulates most cellular proteins and there are thousands of phosphorylated sites [7]. Thus, many phosphatase substrates remained to be characterized. The discovery of the first protein kinase followed, as often in science, a rather tortuous path. Ed Krebs trained as a post-doc with Cori and Green, but he struggled to produce active phosphorylase in his own laboratory. He was not the only one to fail replicating the results of Cori and Green (see references within [8]). Ed Krebs and Eddy Fischer converted their frustrations and misfortune into a puzzle: what makes phosphorylase a active in Cori and Green’s preparation? Therein lies a frequent pattern underlying important scientific discoveries: Krebs and Fischer did not give up in the face of failure, they worked to understand what causes their problem. Scrutinizing the differences between procedures, with rigor and diligence, Krebs and Fischer realized that the a form of phosphorylase appeared following filtration through filter paper. If this step was omitted, phosphorylase b was reco (...truncated)