Conformational Change of Tetratricopeptide Repeats Region Triggers Activation of Phytochrome-Associated Protein Phosphatase 5

Phytochrome activity is not only controlled by light but also by post-translational modifications, e. g. phosphorylation. One of the phosphatases responsible for plant phytochrome dephosphorylation and thereby increased activity is the phytochrome-associated protein phosphatase 5 (PAPP5). We show that PAPP5 recognizes phospho-site mimicking mutants of phytochrome B, when being activated by arachidonic acid (AA). Addition of AA to PAPP5 decreases the α-helical content as tracked by CD-spectroscopy. These changes correspond to conformational changes of the regulatory tetratricopeptide repeats (TPR) region as shown by mapping data from hydrogen deuterium exchange mass spectrometry onto a 3.0 Å crystal structure of PAPP5. Surprisingly, parts of the linker between the TPR and PP2A domains and of the so-called C-terminal inhibitory motif exhibit reduced deuterium uptake upon AA-binding. Molecular dynamics analyses of PAPP5 complexed to a phyB phosphopeptide show that this C-terminal motif remains associated with the TPR region in the substrate bound state, suggesting that this motif merely serves for restricting the orientations of the TPR region relative to the catalytic PP2A domain. Given the high similarity to mammalian PP5 these data from a plant ortholog show that the activation mode of these PPP-type protein phosphatases is highly conserved.

Unraveling the MAX2 Protein Network in Arabidopsis thaliana: Identification of the Protein Phosphatase PAPP5 as a Novel MAX2 Interactor

The F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/β-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/ HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. Additionally, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1) – LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14 and KAI2 protein network by tandem affinity purification using Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were co-purified among which general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination in suboptimal conditions and seedling development. Additionally, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic strigolactone analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14 and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL- related phenotypes and, hence, provide another link with the light pathway.

Quantum-Based Modeling of Dephosphorylation in the Catalytic Site of Serine/Threonine Protein Phosphatase-5 (PPP5C)

Serine/threonine protein phosphatase-5 (PP5; PPP5C) is a member of the phosphoprotein phosphatase (PPP) gene family. The PPP catalytic domains feature a bimetal system (M1/M2), an associated bridge hydroxide (W1(OH−)), an M1-bound water/hydroxide (W2), and a highly conserved core sequence. The PPPs are presumed to share a common mechanism: The seryl/threonyl phosphoryl group of the phosphoprotein coordinates the metal ions, W1(OH−) attacks the central phosphorous atom, rupturing the antipodal phosphoester bond and releasing the phosphate-free protein. Also, a histidine/aspartate tandem is responsible for protonating the exiting seryl/threonyl alkoxide. Here, we employed quantum-based computations on a large section of the PP5 catalytic site. A 33-residue, ONIOM(UB3LYP/6-31G(d):UPM7) model was built to perform computations using methylphosphate dianion as a stand-in substrate for phosphoserine/phosphothreonine. We present a concerted transition state (TS) in which W1(OH−) attacks the phosphate center at the same time that the exiting seryl/threonyl alkoxide is protonated directly by the His304/Asp274 tandem, with W2 assigned as a water molecule: W2(H2O). Arg275, proximal to M1, stabilizes the substrate and TS by binding both the ester oxygen (Oγ) and a phosphoryl oxygen (O1) in a bidentate fashion; in the product state, Tyr451 aids in decoupling Arg275 from O1 of the product phosphate ion. The reaction is exothermic (ΔH = −2.0 kcal/mol), occurs in a single step, and has a low activation barrier (ΔH‡ = +10.0 kcal/mol). Our work is an improvement over an earlier computational study that also found bond rupture and alkoxide protonation to be concerted, but concluded that Arg275 is deprotonated during the reactant and TS stages of the pathway. In that earlier study, the critical electron-withdrawal role that Arg275 plays during the hydroxide attack was not correctly accounted for.

Protein Phosphatase 5 Is a Negative Regulator of Estrogen Receptor-Mediated Transcription

Estrogen receptors (ERs) are transcription factorsthat can be modulated by both estrogen-dependentand growth factor-dependent phosphorylation.A yeast two-hybrid screening identified aserine/threonine protein phosphatase (PP5) as aninteractant of ER? (1–481), a dominant negativeER? mutant. Glutathione S-transferase pull-downassays, mammalian two-hybrid assays, and immunoprecipitationstudies showed that PP5 directlybinds to both ER? and ER? via its tetratricopeptiderepeat domain. E domains of ER? and ER?, withoutcontaining activation domain core regions in transcriptionactivation function 2, were required forthe binding to PP5. In ER?-positive breast cancerMCF7 cells, estrogen- and epidermal growth factordependentphosphorylation of ER? on serine residue118, a major phosphorylation site of the receptor,was reduced by expressing PP5 but enhanced byPP5 antisense oligonucleotide. Estrogen-inducedtranscriptional activities of both ER? and ER? andmRNA expression of estrogen-responsive genes,including pS2, c-myc, and cyclin D1, were suppressedby PP5 but enhanced by PP5 antisenseoligonucleotide. A truncated PP5 mutant consistingonly of its tetratricopeptide repeat domainacted as a dominant negative PP5 that enhancedserine residue 118 phosphorylation of ER? andtransactivations by ER? and ER?. We present thefirst evidence that PP5 functions as an inhibitoryregulator of ER phosphorylation and transcriptionalactivation in vivo. (Molecular