Kinase-phosphatase Pair Research Articles

A disease-associated PPP2R3C-MAP3K1 phospho-regulatory module controls centrosome function

Centrosomes have critical roles in microtubule organization, ciliogenesis, and cell signaling.1,2,3,4,5,6,7,8 Centrosomal alterations also contribute to diseases, including microcephaly, cancer, and ciliopathies.9,10,11,12,13 To date, over 150 centrosomal proteins have been identified, including several kinases and phosphatases that control centrosome biogenesis, function, and maintenance.2,3,4,5,14,15,16,17,18,19,20,21 However, the regulatory mechanisms that govern centrosome function are not fully defined, and thus how defects in centrosomal regulation contribute to disease is incompletely understood. Using a systems genetics approach, we find here that PPP2R3C, a poorly characterized PP2A phosphatase subunit, is a distal centriole protein and functional partner of centriolar proteins CEP350 and FOP. We further show that a key function of PPP2R3C is to counteract the kinase activity of MAP3K1. In support of this model, MAP3K1 knockout suppresses growth defects caused by PPP2R3C inactivation, and MAP3K1 and PPP2R3C have opposing effects on basal and microtubule stress-induced JNK signaling. Illustrating the importance of balanced MAP3K1 and PPP2R3C activities, acute overexpression of MAP3K1 severely inhibits centrosome function and triggers rapid centriole disintegration. Additionally, inactivating PPP2R3C mutations and activating MAP3K1 mutations both cause congenital syndromes characterized by gonadal dysgenesis.22,23,24,25,26,27,28 As a syndromic PPP2R3C variant is defective in centriolar localization and binding to centriolar protein FOP, we propose that imbalanced activity of this centrosomal kinase-phosphatase pair is the shared cause of these disorders. Thus, our findings reveal a new centrosomal phospho-regulatory module, shed light on disorders of gonadal development, and illustrate the power of systems genetics to identify previously unrecognized gene functions.

A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.

Sclerotinia sclerotiorum Response to Long Exposure to Glucosinolate Hydrolysis Products by Transcriptomic Approach.

ABSTRACTWhite mold disease, caused by the necrotrophic fungus Sclerotinia sclerotiorum, affects Brassica crops. Brassica crops produce a broad array of compounds, such as glucosinolates, which contribute to the defense against pathogens. From their hydrolysis, several products arise that have antimicrobial activity (GHPs) whose toxicity is structure dependent. S. sclerotiorum may overcome the toxic effect of moderate GHP concentrations after prolonged exposure to their action. Our objective was to identify the molecular mechanism underlying S. sclerotiorum response to long exposure to two chemically diverse GHPs: aliphatic GHP allyl-isothiocyanate (AITC) and indole GHP indol-3-carbinol (I3C). We found that the transcriptomic response is dependent on the type of GHP and on their initial target, involving cell membranes in the case of AITC or DNA in the case of I3C. Response mechanisms include the reorganization of chromatin, mediated by histone chaperones hip4 and cia1, ribosome synthesis controlled by the kinase-phosphatase pair aps1-ppn1, catabolism of proteins, ergosterol synthesis, and induction of detoxification systems. These mechanisms probably help S. sclerotiorum to grow and survive in an environment where GHPs are constantly produced by Brassica plants upon glucosinolate breakdown.IMPORTANCE Brassica species, including important vegetable crops, such as cabbage, cauliflower, or broccoli, or oil crops, such as rapeseed, produce specific chemical compounds useful to protect them against pests and pathogens. One of the most destructive Brassica diseases in temperate areas around the world is Sclerotinia stem rot, caused by the fungus Sclerotinia sclerotiorum. This is a generalist pathogen that causes disease over more than 400 plant species, being a serious threat to economically important crops worldwide, including potato, bean, soybean, and sunflower, among many others. Understanding the mechanisms utilized by pathogens to overcome specific plant defensive compounds can be useful to increase plant resistance. Our study demonstrated that Sclerotinia shows different adaptation mechanisms, including detoxification systems, to grow and survive when plant protective compounds are present.

Evolution of opposing regulatory interactions underlies the emergence of eukaryotic cell cycle checkpoints

In eukaryotes the entry into mitosis is initiated by activation of cyclin-dependent kinases (CDKs), which in turn activate a large number of protein kinases to induce all mitotic processes. The general view is that kinases are active in mitosis and phosphatases turn them off in interphase. Kinases activate each other by cross- and self-phosphorylation, while phosphatases remove these phosphate groups to inactivate kinases. Crucial exceptions to this general rule are the interphase kinase Wee1 and the mitotic phosphatase Cdc25. Together they directly control CDK in an opposite way of the general rule of mitotic phosphorylation and interphase dephosphorylation. Here we investigate why this opposite system emerged and got fixed in almost all eukaryotes. Our results show that this reversed action of a kinase-phosphatase pair, Wee1 and Cdc25, on CDK is particularly suited to establish a stable G2 phase and to add checkpoints to the cell cycle. We show that all these regulators appeared together in LECA (Last Eukaryote Common Ancestor) and co-evolved in eukaryotes, suggesting that this twist in kinase-phosphatase regulation was a crucial step happening at the emergence of eukaryotes.

Multisite phosphorylation drives phenotypic variation in (p)ppGpp synthetase-dependent antibiotic tolerance

Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. Individual bacteria within a population can differ in antibiotic tolerance, but whether this variability can be regulated or is generally an unavoidable consequence of stochastic fluctuations is unclear. Here we report that a gene encoding a bacterial (p)ppGpp synthetase in Bacillus subtilis, sasA, exhibits high levels of extrinsic noise in expression. We find that sasA is regulated by multisite phosphorylation of the transcription factor WalR, mediated by a Ser/Thr kinase-phosphatase pair PrkC/PrpC, and a Histidine kinase WalK of a two-component system. This regulatory intersection is crucial for controlling the appearance of outliers; rare cells with unusually high levels of sasA expression, having increased antibiotic tolerance. We create a predictive model demonstrating that the probability of a given cell surviving antibiotic treatment increases with sasA expression. Therefore, multisite phosphorylation can be used to strongly regulate variability in antibiotic tolerance.

GroEL Mediates Folding of Bacillus anthracis Serine/Threonine Protein Kinase, PrkC

Bacillus anthracis causes anthrax in human and animals. Both, signaling system such as two component system and endogenous chaperone system such as GroEL-GroES help bacteria to cope with the environmental challenges. Such molecular chaperones are the stress induced proteins that help bacteria to override unfavorable conditions by their moonlighting functions. Previous reports showed that PrkC and PrpC, the Ser/Thr kinase-phosphatase pair in B. anthracis, control phosphorylation of GroEL and regulate biofilm formation. In this study, we show that GroEL is involved in the folding of PrkC to active form. The proteins (GroEL, PrkC and PrpC) were expressed and purified by affinity chromatography. Purified GroEL was used for refolding of denatured PrkC and PrpC and observed that GroEL refolds PrkC but not PrpC as measured by their enzymatic activity. We also observed that purification of GroEL with six histidine tag using Cobalt-Agarose resin yielded superior quality GroEL protein with negligible contamination of non-specific proteins. Thus, cobalt resin can be a better choice for purification of many histidine tagged proteins, where Ni-NTA does not work very well.

Lysosomal mTORC2/PHLPP1/Akt Regulate Chaperone-Mediated Autophagy

Chaperone-mediated autophagy (CMA), a selective form of degradation of cytosolic proteins in lysosomes, contributes to maintenance of proteostasis and to the cellular adaptation to stress. CMA substrates are delivered by a cytosolic chaperone to the lysosomal surface, where, upon unfolding, they are internalized through a membrane translocation complex. The molecular components that participate in CMA substrate targeting and translocation are well characterized, but those involved in CMA regulation remain mostly unknown. In this study, we have identified that CMA is under the positive control of the phosphatase PHLPP1 that associates with the lysosomal membrane and counteracts the inhibitory effect of mTORC2 on CMA. Lysosomal Akt, a target of the mTORC2/PHLPP1 kinase-phosphatase pair, modulates CMA activity by controlling the dynamics of assembly and disassembly of the CMA translocation complex at the lysosomal membrane. The lysosomal mTORC2/PHLPP1/Akt axis could become a target to restore CMA dysfunction in aging and disease.

Phosphorylation of theStreptococcus pneumoniaecell wall biosynthesis enzyme MurC by a eukaryotic-like Ser/Thr kinase

Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S.pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway. Combinatorial phage display using StkP as target selected the peptide sequence YEVCGSDTVGC as an interacting partner and subsequently confirmed by ELISA. The phage peptide sequence YEVCGSDTVGC aligns closely with the MurC motif spanning S.pneumoniae amino acid coordinates 31-37. We show that MurC is phosphorylated by StkP and that phosphoMurC is dephosphorylated by PhpP. These data suggest a link between StkP-PhpP with the coordinated regulation of cell wall biosynthesis via MurC.

Protein cysteine phosphorylation of SarA/MgrA family transcriptional regulators mediates bacterial virulence and antibiotic resistance

Protein posttranslational modifications (PTMs), particularly phosphorylation, dramatically expand the complexity of cellular regulatory networks. Although cysteine (Cys) in various proteins can be subject to multiple PTMs, its phosphorylation was previously considered a rare PTM with almost no regulatory role assigned. We report here that phosphorylation occurs to a reactive cysteine residue conserved in the staphylococcal accessary regulator A (SarA)/MarR family global transcriptional regulator A (MgrA) family of proteins, and is mediated by the eukaryotic-like kinase-phosphatase pair Stk1-Stp1 in Staphylococcus aureus. Cys-phosphorylation is crucial in regulating virulence determinant production and bacterial resistance to vancomycin. Cell wall-targeting antibiotics, such as vancomycin and ceftriaxone, inhibit the kinase activity of Stk1 and lead to decreased Cys-phosphorylation of SarA and MgrA. An in vivo mouse model of infection established that the absence of stp1, which results in elevated protein Cys-phosphorylation, significantly reduces staphylococcal virulence. Our data indicate that Cys-phosphorylation is a unique PTM that can play crucial roles in bacterial signaling and regulation.

Biochemical Characterization of In Vitro Phosphorylation and Dephosphorylation of the Plasma Membrane H+-ATPase

Stomatal opening, which is mediated by blue light receptor phototropins, is driven by activation of the plasma membrane H(+)-ATPase via phosphorylation of the penultimate threonine in the C-terminus and subsequent binding of a 14-3-3 protein. However, the biochemical properties of the protein kinase and protein phosphatase for H(+)-ATPase are largely unknown. We therefore investigated in vitro phosphorylation and dephosphorylation of H(+)-ATPase. H(+)-ATPase was phosphorylated in vitro on the penultimate threonine in the C-terminus in isolated microsomes from guard cell protoplasts of Vicia faba. Phosphorylated H(+)-ATPase was dephosphorylated in vitro, and the dephosphorylation was inhibited by EDTA, a divalent cation chelator, but not by calyculin A, an inhibitor of type 1 and 2A protein phosphatases. Essentially the same results were obtained in purified plasma membranes from etiolated Arabidopsis seedlings, indicating that a similar protein kinase and phosphatase are involved in plant cells. Further analyses revealed that phosphorylation of the H(+)-ATPase is insensitive to K-252a, a potent inhibitor of protein kinase, and is hypersensitive to Triton X-100, a non-ionic detergent. Moreover, dephosphorylation required Mg(2+) but not Ca(2+), and protein phosphatase was localized in the 1% Triton X-100-insoluble fraction. These results demonstrate that a protein kinase-phosphatase pair, K-252a-insensitive protein kinase and Mg(2+)-dependent type 2C protein phosphatase, co-localizes at least in part with the H(+)-ATPase in the plasma membrane and regulates the phosphorylation status of the penultimate threonine of the H(+)-ATPase.

CD45-Csk Phosphatase-Kinase Titration Uncouples Basal and Inducible T Cell Receptor Signaling during Thymic Development

The kinase-phosphatase pair Csk and CD45 reciprocally regulate phosphorylation of the inhibitory tyrosine of the Src family kinases Lck and Fyn. T cell receptor (TCR) signaling and thymic development require CD45 expression but proceed constitutively in the absence of Csk. Here, we show that relative titration of CD45 and Csk expression reveals distinct regulation of basal and inducible TCR signaling during thymic development. Low CD45 expression is sufficient to rescue inducible TCR signaling and positive selection, whereas high expression is required to reconstitute basal TCR signaling and beta selection. CD45 has a dual positive and negative regulatory role during inducible but not basal TCR signaling. By contrast, Csk titration regulates basal but not inducible signaling. High physiologic expression of CD45 is thus required for two reasons-to downmodulate inducible TCR signaling during positive selection and to counteract Csk during basal TCR signaling.

Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair.

In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.

A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells

The plant hormone abscisic acid (ABA) serves as a physiological monitor to assess the water status of plants and, under drought conditions, induces stomatal pore closure by activating specific ion channels, such as a slow-anion channel (SLAC1) that, in turn, mediate ion efflux from the guard cells. Earlier genetic analyses uncovered a protein kinase (OST1) and several 2C-type phosphatases, as respective positive and negative regulators of ABA-induced stomatal closure. Here we show that the OST1 kinase interacts with the SLAC1 anion channel, leading to its activation via phosphorylation. PP2CA, one of the PP2C phosphatase family members acts in an opposing manner and inhibits the activity of SLAC1 by two mechanisms: (1) direct interaction with SLAC1 itself, and (2) physical interaction with OSTI leading to inhibition of the kinase independently of phosphatase activity. The results suggest that ABA signaling is mediated by a physical interaction chain consisting of several components, including a PP2C member, SnRK2-type kinase (OST1), and an ion channel, SLAC1, to regulate stomatal movements. The findings are in keeping with a paradigm in which a protein kinase-phosphatase pair interacts physically with a target protein to couple a signal with a specific response.

Coordinated control of volume regulatory Na+/H+ and K+/H+ exchange pathways in Amphiuma red blood cells

The Na(+)/H(+) and K(+)/H(+) exchange pathways of Amphiuma tridactylum red blood cells (RBCs) are quiescent at normal resting cell volume yet are selectively activated in response to cell shrinkage and swelling, respectively. These alkali metal/H(+) exchangers are activated by net kinase activity and deactivated by net phosphatase activity. We employed relaxation kinetic analyses to gain insight into the basis for coordinated control of these volume regulatory ion flux pathways. This approach enabled us to develop a model explaining how phosphorylation/dephosphorylation-dependent events control and coordinate the activity of the Na(+)/H(+) and K(+)/H(+) exchangers around the cell volume set point. We found that the transition between initial and final steady state for both activation and deactivation of the volume-induced Na(+)/H(+) and K(+)/H(+) exchange pathways in Amphiuma RBCs proceed as a single exponential function of time. The rate of Na(+)/H(+) exchange activation increases with cell shrinkage, whereas the rate of Na(+)/H(+) exchange deactivation increases as preshrunken cells are progressively swollen. Similarly, the rate of K(+)/H(+) exchange activation increases with cell swelling, whereas the rate of K(+)/H(+) exchange deactivation increases as preswollen cells are progressively shrunken. We propose a model in which the activities of the controlling kinases and phosphatases are volume sensitive and reciprocally regulated. Briefly, the activity of each kinase-phosphatase pair is reciprocally related, as a function of volume, and the volume sensitivities of kinases and phosphatases controlling K(+)/H(+) exchange are reciprocally related to those controlling Na(+)/H(+) exchange.

Mathematical model of the fission yeast cell cycle with checkpoint controls at the G1/S, G2/M and metaphase/anaphase transitions

All events of the fission yeast cell cycle can be orchestrated by fluctuations of a single cyclin-dependent protein kinase, the Cdc13/Cdc2 heterodimer. The G1/S transition is controlled by interactions of Cdc13/Cdc2 and its stoichiometric inhibitor, Rum1. The G2/M transition is regulated by a kinase-phosphatase pair, Wee1 and Cdc25, which determine the phosphorylation state of the Tyr-15 residue of Cdc2. The meta/anaphase transition is controlled by interactions between Cdc13/Cdc2 and the anaphase promoting complex, which labels Cdc13 subunits for proteolysis. We construct a mathematical model of fission yeast growth and division that encompasses all three crucial checkpoint controls. By numerical simulations we show that the model is consistent with a broad selection of cell cycle mutants, and we predict the phenotypes of several multiple-mutant strains that have not yet been constructed.