Okadaic acid and microcystin insensitive PPP-family phosphatases may represent novel biotechnology targets

Abstract

Reversible protein phosphorylation is of central importance to the proper cellular functioning of all living organisms. Catalyzed by the opposing reactions of protein kinases and phosphatases, dysfunction in reversible protein phosphorylation can result in a wide variety of cellular aberrations. In eukaryotic organisms there exists four classes of protein phosphatases, of which the PPP-family protein phosphatases have documented susceptibility to a range of protein and small molecule inhibitors. These inhibitors have been of great importance to the biochemical characterization of PPP-family protein phosphatases since their discovery, but also maintain in natura biological significance with their endogenous regulatory properties (protein inhibitors) and toxicity (small molecule inhibitors). Recently, two unique PPP-family protein phosphatases, named the Shewanella-like protein phosphatases (SLP phosphatases), from Arabidopsis thaliana were characterized and found to be phylogenetically similar to the PPP-family protein phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), while completely lacking sensitivity to the classic PPP-family phosphatase small molecule inhibitors okadaic acid and microcystin-LR. SLP phosphatases were also found to be absent in metazoans, but present in a wide range of bacteria, fungi and protozoa responsible for human disease. The unique biochemical properties and evolutionary heritage of SLP phosphatases suggests they could not only be potential biotechnology targets for agriculture, but may also prove to be of interest for future therapeutic drug development.

Introduction

The protein phosphatases constitute a large group of enzymes responsible for catalyzing the dephosphorylation of target phosphoproteins. Working in opposition to protein kinases, which catalyze the phosphorylation of target proteins, protein phosphatases and protein kinases form one of the most prolific post-translational regulatory systems (reversible phosphorylation) found in nature.^1,2 In Plantae and Metazoa alike the protein phosphatases are comprised of four main classes: the phospho-protein phosphatases (PPP), Mg²⁺-dependent phospho-protein phosphatases (PPM/PP2C), phospho-tyrosine phosphatases (PTP) and Asp-based phosphatases.^3-5 Previous efforts to directly compare the protein phosphatase complement of A. thaliana and H. sapiens found many similarities, emphasizing the central and conserved nature of protein phosphatases across diverse eukaryotes.³ However, this comparison also revealed a number of differences. One striking feature was the presence of PPP-family protein phosphatases in A. thaliana that were absent in H. sapiens. Two of these PPP-family protein phosphatases were the Shewanella-like (SLP) protein phosphatases, which were named after their relatedness to a protein phosphatase found in the marine bacterium, Shewanella.⁶ This addendum looks to further expand on a recent study which was the first to characterize the subcellular localization and biochemical characteristics of these unique PPP-family protein phosphatases, establishing that two SLP phosphatase isoforms exist in plants and that they maintain chloroplastic (SLP1) and cytosolic (SLP2) subcellular localizations, respectively.⁷

SLP phosphatases: Unique PPP-family protein phosphatases insensitive to inhibitors

Most eukaryotic genomes have a ‘basic’ complement of PPP-family phosphatases known as PP1, PP2A, PP2B (PP3), PP4, PP5, PP6 and PP7.^3,8 Each of these protein phosphatases are related at the primary sequence level and display a general sensitivity to an array of small molecule and protein inhibitors including microcystin,^9,10 okadaic acid¹¹ and inhibitor-2 protein.¹² Inhibitor-2 protein is a highly conserved, genomically encoded protein interactor specific to the type one (PP1) protein phosphatases.¹² As an innately flexible protein, inhibitor-2 exerts its inhibitory effect through a combination of induced conformational changes and direct occlusion of the PP1 active site.^13,14 Microcystin and okadaic acid however, are two representatives of a chemically diverse and naturally occurring group of small molecule toxins that target the PPP-family enzymes. Microcystins, produced by cyanobacteria (i.e., Microcystis aeruginosa) and okadaic acid, made by dinoflagellates (i.e., Dinophysis sp), each exert their inhibitory effects by directly binding to the active site of PPP-family phosphatases (Fig. 1A).^9,15,16 The general lack of inhibition exhibited by the PPP-family members PP2B and PP7 in the presence of these compounds, coupled with differential inhibition of the remaining PPP-family enzymes by select toxins, has made these small molecules invaluable tools in protein phosphatase research by helping implicate distinct protein phosphatases in various cellular processes. Recently, we reported two additional PPP-family protein phosphatases from A. thaliana (AtSLP1 and -2 phosphatases) that demonstrated complete insensitivity to inhibition by both microcystin and okadaic acid, with AtSLP1 exhibiting slight enzymatic activation.⁷ Bioinformatic analysis conclusively placed the AtSLPs as more closely related to the PPP-family phosphatases than any other microcystin or okadaic acid insensitive protein phosphatase class (i.e., PPM, PTP and Asp-based family phosphatases). As well, within the PPP-family as a whole, the inhibitor insensitive SLP phosphatases were unexpectedly found to be most related to the microcystin and okadaic acid sensitive PP1 and PP2A enzymes (Fig. 1B).⁷

Figure 1.

Comparison of PP1 / PP2A protein phosphatase complexes to SLP1 and 2. (A) Chemical structures of PPP-family protein phosphatase inhibitors microcystin (left) and okadaic acid (right). (B) Protein phosphatase one catalytic subunits (PP1) interact with hundreds of regulatory subunits through their RVxF motif (labeled in red) to form numerous protein phosphatase complexes. Although PP1 complexes have been demonstrated to control a plethora of events in other eukaryotes, in plants to date the only defined PP1 functions are linked to cell cycle control. PP2A catalytic subunits (PP2Ac) however, interact with a select number of both regulatory ‘B’ subunits (B’, B’’ and B’’’) and scaffolding ‘A’ subunits (A1, A2 and A3) to form a variety of trimeric protein phosphatases complexes. These trimeric protein complexes have been shown to regulate aspects of plant growth and metabolism. Unlike PP1 and PP2A, regulatory or scaffolding (Reg) subunits have not yet been identified for SLP protein phosphatases. The dashed line represents a currently possible, unidentified SLP interaction motif. As well, the biological role of SLP protein phosphatases has not yet been uncovered. Question marks represent events not yet resolved.

SLP phosphatases identified in human pathogens

In addition to an unique insensitivity to classic PPP-family protein phosphatases inhibitors, examination of SLP phosphatase phylogenetic history uncovered a complete absence of SLP phosphatases in metazoans, but a presence in a select number of bacteria, fungi and parasitic protozoa responsible for human disease.^6,7,17 Two such SLP phosphatase containing protozoa are Trypanosoma and Plasmodium, each responsible for African sleeping sickness and malaria respectively. Interestingly, these protozoa are also known to possess a vestigial plastid (chloroplast remnant) called an apicoplast as a result of their evolution from early photosynthetic, chloroplast containing eukaryotes.^18,19 Moreover, both protozoa have multiple copies of SLP phosphatases, which may be reflective of different biological roles needed to accommodate the complex life cycles of these organisms. Conversely, SLP phosphatase containing bacteria and fungi were found to possess only one SLP phosphatase.^6,7 Having a single SLP enzyme appears to parallel a lack of a plastid or vestigial plastid and may account for an evolutionary history that determines whether an organism has one vs. two SLPs.

Drugs and Crops: SLP phosphatases may represent future biotechnology targets

SLP insensitivity to microcystin and okadaic acid combined with their complete exclusion from metazoans, but presence in select bacteria, fungi, and protozoa, renders these protein phosphatases potential therapeutic drug targets for human disease caused by SLP phosphatase containing organisms.^6,7,17,20 Furthermore, the complete conservation and lack of genetic redundancy of SLP phosphatases in plants may also render them targets for rational agricultural crop engineering efforts.⁷ However, despite these unique traits and recent speculation as to the possible biological function(s) of the SLP phosphatases,²⁰ significantly more work, in a number of organismal models, will be required to completely understand the role(s) of the SLP phosphatases before any engineering efforts can be undertaken. In particular, complete understanding of the SLP protein phosphatases will require resolving their target substrates as well as the presence or absence of regulatory proteins that may be directing intracellular functionality (Fig. 1B).²⁰ As is the case with PP1 and PP2A, regulatory proteins may be central to determining SLP protein phosphatase substrate specificity.^21-24

Uhrig R G, Moorhead G B. Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties. Plant Physiol. 2011;157 doi: 10.1104/pp.111.182493.