The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.     

The Arabidopsis kinase-associated protein phosphatase KAPP,interacting with protein kinases SnRK2.2/2.3/2.6, negatively regulates abscisic acid signaling

Plant Molecular Biology - The kinase-associated protein phosphatase, KAPP, is negatively involved in abscisic acid (ABA) signaling. KAPP interacts physically with SnRK2.2, SnRK2.3 and SnRK2.6, and...     

Phosphoprotein and phosphopeptide interactions with the FHA domain from Arabidopsis kinase-associated protein phosphatase

FHA domains are phosphoThr recognition modules found in diverse signaling proteins, including kinase-associated protein phosphatase (KAPP) from Arabidopsis thaliana. The kinase-interacting FHA domain (KI-FHA) of KAPP targets it to function as a negative regulator of some receptor-like kinase (RLK) signaling pathways important in plant development and environmental responses. To aid in the identification of potential binding sites for the KI-FHA domain, we predicted (i) the structure of a representative KAPP-binding RLK, CLAVATA1, and (ii) the functional surfaces of RLK kinase domains using evolutionary trace analysis. We selected phosphopeptides from KAPP-binding Arabidopsis RLKs for in vitro studies of association with KI-FHA from KAPP. Three phosphoThr peptide fragments from the kinase domain of CLV1 or BAK1 were found to bind KI-FHA with KD values of 8-20 microM, by NMR or titration calorimetry. Their affinity is driven by favorable enthalpy and solvation entropy gain. Mutagenesis of these three threonine sites suggests Thr546 in the C-lobe of the BAK1 kinase domain to be a principal but not sole site of KI-FHA binding in vitro. The brassinosteroid receptor BRI1 and KAPP are shown to associate in vivo and in vitro. Further genetic studies indicate that KAPP may be a negative regulator of the BRI1 signaling transduction pathway. 15N-Labeled KI-FHA was titrated with the GST-BRI1 kinase domain and monitored by NMR. BRI1 interacts with the same 3/4, 4/5, 6/7, 8/9, and 10/11 recognition loops of KI-FHA, with similar affinity as the phosphoThr peptides.     

The mitogen-activated protein kinase phosphatase PHS1 regulates flowering in Arabidopsis thaliana

Planta - Arabidopsis PHS1, initially known as an actor of cytoskeleton organization, is a positive regulator of flowering in the photoperiodic and autonomous pathways by modulating both CO and FLC...     

The CheC phosphatase regulates chemotactic adaptation through CheD

The bacterial chemotaxis system is one of the most extensively studied signal transduction systems in biology. The response regulator CheY controls flagellar rotation and is phosphorylated by the CheA histidine kinase to its active form. CheC is a CheY-P phosphatase, and this activity is enhanced in a CheC-CheD heterodimer. CheC is also critical for chemotactic adaptation, the return to the prestimulus system state despite persistent attractant concentrations. Here, CheC point mutants were examined in Bacillus subtilis for in vivo complementation and in vitro activity. The mutants were identified separating the three known abilities of CheC: CheD binding, CheY-P binding, and CheY-P phosphatase activity. Remarkably, the phosphatase ability was not as critical to the in vivo function of CheC as the ability to bind both CheY-P and CheD. Additionally, it was confirmed that CheY-P increases the affinity of CheC for CheD, the later of which is known to be necessary for receptor activation of CheA. These data suggest a model of CheC as a CheY-P-induced regulator of CheD. Here, CheY-P would cause CheC to sequester CheD from the chemoreceptors, inducing adaptation of the chemotaxis system. This model represents the first plausible means for feedback from the output of the system, CheY-P, to the receptors.     

PhosphoThr peptide binding globally rigidifies much of the FHA domain from Arabidopsis receptor kinase-associated protein phosphatase

A net increase in the backbone rigidity of the kinase-interacting FHA domain (KI-FHA) from the Arabidopsis receptor kinase-associated protein phosphatase (KAPP) accompanies the binding of a phosphoThr peptide from its CLV1 receptor-like kinase partner, according to (15)N NMR relaxation at 11.7 and 14.1 T. All of the loops of free KI-FHA display evidence of nanosecond-scale motions. Many of these same residues have residual dipolar couplings that deviate from structural predictions. Binding of the CLV1 pT868 peptide seems to reduce nanosecond-scale fluctuations of all loops, including half of the residues of recognition loops. Residues important for affinity are found to be rigid, i.e., conserved residues and residues of the subsite for the key pT+3 peptide position. This behavior parallels SH2 and PTB domain recognition of pTyr peptides. PhosphoThr peptide binding increases KI-FHA backbone rigidity (S(2)) of three recognition loops, a loop nearby, seven strands from the beta-sandwich, and a distal loop. Compensating the trend of increased rigidity, binding enhances fast mobility at a few sites in four loops on the periphery of the recognition surface and in two loops on the far side of the beta-sandwich. Line broadening evidence of microsecond- to millisecond-scale fluctuations occurs across the six-stranded beta-sheet and nearby edges of the beta-sandwich; this forms a network connected by packing of interior side chains and H-bonding. A patch of the slowly fluctuating residues coincides with the site of segment-swapped dimerization in crystals of the FHA domain of human Chfr. Phosphopeptide binding introduces microsecond- to millisecond-scale fluctuations to more residues of the long 8/9 recognition loop of KI-FHA. The rigidity of this FHA domain appears to couple as a whole to pThr peptide binding.     

The Schizosaccharomyces pombe Pzh1 protein phosphatase regulates Na+ ion influx in a Trk1-independent fashion.

We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzhl, which is an important determinant of cation homeostasis. pzh1 delta mutants display increased tolerance to Na+ ions, but they are hypersensitive to KC1 [Balcells, L., Gómez, N., Casamayor, A., Clotet, J. & Ari?o, J. (1997) Eur. J. Biochem. 250, 476-483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild-type cells. K+ ion content of pzh1 delta cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K+. Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast. To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high-affinity transporter of K+ ions. trkl delta mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions. However, they showed a Na(+)-sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions.     

Partially unfolded forms and non-two-state folding of a beta-sandwich: FHA domain from Arabidopsis receptor kinase-associated protein phosphatase

FHA domains adopt a beta-sandwich fold with 11 strands. The first evidence of partially unfolded forms of a beta-sandwich is derived from native-state hydrogen exchange (NHX) of the forkhead-associated (FHA) domain from kinase-associated protein phosphatase from Arabidopsis. The folding kinetics of this FHA domain indicate that EX2 behavior prevails at pH 6.3. In the chevron plot, rollover in the folding arm and bends in the unfolding arm suggest folding intermediates. NHX of this FHA domain suggests a core of six most stable beta-strands and two loops, characterized by rare global unfolding events. Flanking this stable core are beta-strands and recognition loops with less stability, termed subglobal motifs. These suggest partially unfolded forms (near-native intermediates) with two levels of stability. The spatial separation of the subglobal motifs on the flanks suggests possible parallelism in their folding as additional beta-strands align with the stable core of six strands. Intermediates may contribute to differences in stabilities and m-values suggested by NHX or kinetics relative to chemical denaturation. Residual structure in the unfolded regime is suggested by superprotection of beta-strand 6 and by GdmCl-dependence of adjustments in amide NMR spectra and residual optical signal. The global folding stability depends strongly on pH, with at least 3 kcal/mol more stability at pH 7.3 than at pH 6.3. This FHA domain is hypothesized to fold progressively with initial hydrophobic collapse of its stable six-stranded core followed by addition of less stable flanking beta-strands and ordering of recognition loops.     

Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in β cells

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The F box protein S phase kinase-associated protein 2 regulates adipose mass and adipocyte number in vivo

Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin‐dependent kinase inhibitor p27 regulates activity of cyclin/cyclin‐dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1‐Cullin‐F‐box protein) complex targets proteins such as p27 for ubiquitin‐proteosome degradation; the F box protein S phase kinase‐associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2^?/?) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild‐type (WT), Skp2^?/?, and p27^?/?Skp2^?/? mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2^?/? fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2^?/? mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27^?/?Skp2^?/? double knockout mice. The Skp2^?/? decrements in adipocyte number and fat pad mass were totally reversed in p27^?/?Skp2^?/? mice. Adipogenesis was inhibited in MEFs from Skp2^?/? vs. WT mice, and this inhibition was absent in MEFs from p27^?/?Skp2^?/? mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia.     

The protein tyrosine phosphatase Shp-2 regulates RhoA activity

Remodeling of filamentous actin into distinct arrangements is precisely controlled by members of the Rho family of small GTPases [1]. A well characterized member of this family is RhoA, whose activation results in reorganization of the cytoskeleton into thick actin stress fibers terminating in integrin-rich focal adhesions [2]. Regulation of RhoA is required to maintain adhesion in stationary cells, but is also critical for cell spreading and migration [3]. Despite its biological importance, the signaling events leading to RhoA activation are not fully understood. Several independent studies have implicated tyrosine phosphorylation as a critical event upstream of RhoA [4]. Consistent with this, our recent studies have demonstrated the existence of a protein tyrosine phosphatase (PTPase), sensitive to the dipeptide aldehyde calpeptin, acting upstream of RhoA [5]. Here we identify the SH2 (Src homology region 2)-containing PTPase Shp-2 as a calpeptin-sensitive PTPase, and show that calpeptin interferes with the catalytic activity of Shp-2 in vitro and with Shp-2 signaling in vivo. Finally, we show that perturbation of Shp-2 activity by a variety of genetic manipulations results in raised levels of active RhoA. Together, these studies identify Shp-2 as a PTPase acting upstream of RhoA to regulate its activity and contribute to the coordinated control of cell movement.     

A new protein kinase gene SSG1 is essential for adaptation of Arabidopsis to salt stress

Soil salinity significantly limits plant productivity on agricultural lands, and salt tolerance of plant requires a complex mechanism in which many genes are involved. From an activation-tagging mutant collection, we identified a loss-of-function Arabidopsis mutant which exhibits hypersensitivity to NaCl during seed germination and is designated ssg1 (salt sensitive during seed germination 1). Knocking down the expression of SSG1 by RNAi recapitulated the phenotype of ssg1, suggesting that functional SSG1 is necessary for normal seed germination in the presence of NaCl. The seed germination of ssg1 was not sensitive to K⁺, but was hypersensitive to osmotic stress. SSG1 encodes a protein kinase, possibly with alternatively spliced forms. In vitro kinase assay indicated that SSG1 possessed protein kinase activity which can both auto-phosphorylate and phosphorylate substrate. Quantitative real-time RT-PCR analysis showed that, in the mutants ssg1-1 and ssg1-2, the expression level of some salt-responsive marker genes, i.e., SOS1, SOS2, SOS3, SOS4, AtNHX and AtNKT, was down-regulated, whereas SSG1 expression level was not changed in sos mutants. These results suggest that SSG1 is possibly a component in the signaling pathway in response of plants to salt stress.     

Angiotensin regulates the selectivity of the Na+-K+ pump for intracellular Na+

Treatment of rabbits with angiotensin-converting enzyme (ACE)inhibitors increases the apparent affinity of theNa⁺-K⁺pump for Na⁺. To explore themechanism, we voltage clamped myocytes from control rabbits and rabbitstreated with captopril with patch pipettes containing 10 mMNa⁺. When pipette solutions wereK⁺ free, pump current(I_p) formyocytes from captopril-treated rabbits was nearly identical to thatfor myocytes from controls. However, treatment caused a significantincrease in I_pmeasured with pipettes containingK⁺. A similar difference wasobserved when myocytes from rabbits treated with the ANG II receptorantagonist losartan and myocytes from controls were compared.Treatment-induced differences in I_p wereeliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed ofamino acids 530-558 in pipette solutions. Treatmentwith captopril had no effect on the voltage dependence ofI_p. We concludethat ANG II regulates the pump's selectivity for intracellularNa⁺ at sites near the cytoplasmicsurface. Protein kinase C is implicated in the messenger cascade.

     

The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis

HKT-type transporters appear to play key roles in Na(+) accumulation and salt sensitivity in plants. In Arabidopsis HKT1;1 has been proposed to influx Na(+) into roots, recirculate Na(+) in the phloem and control root : shoot allocation of Na(+). We tested these hypotheses using (22)Na(+) flux measurements and ion accumulation assays in an hkt1;1 mutant and demonstrated that AtHKT1;1 contributes to the control of both root accumulation of Na(+) and retrieval of Na(+) from the xylem, but is not involved in root influx or recirculation in the phloem. Mathematical modelling indicated that the effects of the hkt1;1 mutation on root accumulation and xylem retrieval were independent. Although AtHKT1;1 has been implicated in regulation of K(+) transport and the hkt1;1 mutant showed altered net K(+) accumulation, (86)Rb(+) uptake was unaffected by the hkt1;1 mutation. The hkt1;1 mutation has been shown previously to rescue growth of the sos1 mutant on low K(+); however, HKT1;1 knockout did not alter K(+) or (86)Rb(+) accumulation in sos1.