ABSTRACT
Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.
Subject(s)
Fungi/enzymology , Protein Tyrosine Phosphatases/classification , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Cysteine/metabolism , Disulfides/metabolism , Dual-Specificity Phosphatases , Histidine/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction/drug effects , Oxidative Stress , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/classification , Phosphoprotein Phosphatases/metabolism , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/classification , Saccharomyces cerevisiae Proteins/metabolism , Substrate SpecificityABSTRACT
AIM: To identify novel microbial inhibitors of protein phosphatase 1 (PP1). METHODS AND RESULTS: 750 actinomycetes and 408 microfungi were isolated from Sabah forest soils and screened for production of potential PP1 inhibitors using an in vivo screening system, in which candidate inhibitors were identified through mimicking the properties of PP1-deficient yeast cells. Acetone extracts of two fungi, H9318 (Penicillium) and H9978 (non-Penicillium) identified in this way showed inhibitory activity towards both mammalian PP1 and PP2A in an in vitro phosphatase assay, while extract from H7520 (Streptomyces) inhibited PP2A but not PP1. Consistently, using a drug-induced haploinsufficiency test, strains with either reduced PP1 or PP2A function were hypersensitive to H9318 and H9978 extracts whereas only the latter strain showed hypersensitivity to H7250 extract. H9318 extract was fractionated using RP-HPLC into two active peaks (S1 and S2). A yeast strain with reduced PP1 function showed hypersensitivity to fraction S2 whereas a strain with reduced PP2A function was hypersensitive to fraction S1. However, S1 and S2 inhibited both PP1 and PP2A activities to a similar extent. CONCLUSION: Three candidate PP inhibitors have been identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Further development may generate useful research tools and ultimately therapeutic agents.
