Identification of conserved tyrosine residues important for gibberellin sensitivity of <Emphasis Type="Italic">Arabidopsis</Emphasis> RGL2 protein

DELLA proteins are regulators in the signaling pathway of gibberellin (GA), a plant growth regulator of diverse functions. GA typically induces the degradation of DELLA proteins to overcome their repressive roles in growth and development. We have previously evaluated the likely roles of Ser–Thr phosphorylation of DELLA proteins in GA signaling (Hussain et al., Plant J 44:88–99, 2005). Here we report that four DELLA proteins of Arabidopsis, namely GAI, RGL1, RGL2 and RGL3, expressed in tobacco BY2 cells, are degradable by GA. Both, proteasome inhibitor and protein tyrosine (Tyr) kinase inhibitors, strongly inhibit GA-induced DELLA degradation whereas phospho-Tyr phosphatase inhibitors have no effect, suggesting that Tyr phosphorylation is critical in GA-induced DELLA degradation. Mutation of eight conserved Tyr residues of RGL2 into alanine shows four mutant proteins (Y52A, Y89A, Y223A and Y435A) are resistant to GA-induced degradation. Substitution of these four critical Tyr residues into negatively charged glutamate (Y → E) also resulted in stabilization of these mutants against GA treatment. However, further mutation of these four Tyrs into conservative phenylalanine (Y → F) rendered the mutant proteins sensitive to GA like the wild-type RGL2. Since Y → E mutations sometimes mimic phosphor-Tyr whereas Y → F mutations render the protein unphosphorylatable at these Tyr sites, we conclude that these four conserved Tyrs, despite being critical for GA-sensitivity, are unlikely to be sites of Tyr phosphorylation but instead play important roles in maintaining the structure integrity of RGL2 for GA-sensitivity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression in Escherichia coli and in vitro refolding of the plant transcription factor Arabidopsis thaliana RGL3

Recombinant Arabidopsis thaliana (At) RGL-3, using two vectors pMAL-c2 and pET 21, was expressed as inclusion bodies in Escherichia coli under a range of temperature conditions. Only low levels (8-12% of total protein) of soluble protein were produced. The "soluble" fraction was shown by native PAGE to exist as soluble aggregates of RGL-3. A method was developed, consisting of induction of expression at various temperatures that yielded high levels of refoldable inclusion bodies using the pET vector. (At) RGL-3, as inclusion bodies, was solubilized in 8M urea and refolding was initiated by 20-fold direct dilution of denaturant. Under optimal conditions, 87% of the denatured protein of inclusion bodies was successfully re-natured. Refolding was monitored by "native" PAGE. Refolded RGL-3 was shown to be present as monomers and dimers. Attempts to further purify His-tagged RGL-3 using Ni/NTA chromatography resulted in the formation of higher polymers.     

Identification and characterization of Arabidopsis gibberellin receptors

Three gibberellin (GA) receptor genes (AtGID1a, AtGID1b and AtGID1c), each an ortholog of the rice GA receptor gene (OsGID1), were cloned from Arabidopsis, and the characteristics of their recombinant proteins were examined. The GA-binding activities of the three recombinant proteins were confirmed by an in vitro assay. Biochemical analyses revealed similar ligand selectivity among the recombinants, and all recombinants showed higher affinity to GA(4) than to other GAs. AtGID1b was unique in its binding affinity to GA(4) and in its pH dependence when compared with the other two, by only showing binding in a narrow pH range (pH 6.4-7.5) with 10-fold higher affinity (apparent K(d) for GA(4) = 3 x 10(-8) m) than AtGID1a and AtGID1c. A two-hybrid yeast system only showed in vivo interaction in the presence of GA(4) between each AtGID1 and the Arabidopsis DELLA proteins (AtDELLAs), negative regulators of GA signaling. For this interaction with AtDELLAs, AtGID1b required only one-tenth of the amount of GA(4) that was necessary for interaction between the other AtGID1s and AtDELLAs, reflecting its lower K(d) value. AtDELLA boosted the GA-binding activity of AtGID1 in vitro, which suggests the formation of a complex between AtDELLA and AtGID1-GA that binds AtGID1 to GA more tightly. The expression of each AtGID1 clone in the rice gid1-1 mutant rescued the GA-insensitive dwarf phenotype. These results demonstrate that all three AtGID1s functioned as GA receptors in Arabidopsis.     

Evidence that the Arabidopsis nuclear gibberellin signalling protein GAI is not destabilised by gibberellin

Plant growth is regulated by bioactive gibberellin (GA), although there is an unexplained diversity in the magnitude of the GA responses exhibited by different plant species. GA acts via a group of orthologous proteins known as the DELLA proteins. The Arabidopsis genome contains genes encoding five different DELLA proteins, the best known of which are GAI and RGA. The DELLA proteins are thought to act as repressors of GA-regulated processes, whilst GA is thought to act as a negative regulator of DELLA protein function. Recent experiments have shown that GA induces rapid disappearance of nuclear RGA, SLR1 and SLN1 (DELLA proteins from rice and barley), suggesting that GA signalling and degradation of DELLA proteins are coupled. However, RGL1, another Arabidopsis DELLA protein, does not disappear from the nucleus in response to GA treatment. Here, we present evidence suggesting that GAI, like RGL1, is stable in response to GA treatment, and show that transgenic Arabidopsis plants containing constructs that enable high-level expression of GAI exhibit a dwarf, GA non-responsive phenotype. Thus, GAI appears to be less affected by GA than RGA, SLR1 or SLN1. We also show that neither of the two putative nuclear localisation signals contained in DELLA proteins are individually necessary for nuclear localisation of GAI. The various DELLA proteins have different properties, and we suggest that this functional diversity may explain, at least in part, why plant species differ widely in their GA response magnitudes.     

Role of threonine residues in the regulation of manganese-dependent arabidopsis serine/threonine/tyrosine protein kinase activity

Tyrosine phosphorylation in plants could be performed only by dual-specificity kinases. Arabidopsis thaliana dual-specificity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors manganese dependent serine/threonine kinase domain, COG3642. His248 and Ser265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr208 in the TEY motif and Thr293 of the activation loop were converted to alanine. The conversion of Thr284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the first identification of a manganese dependent dual-specificity kinase and the importance of Thr208, Thr284, and Thr293 residues in the regulation of kinase activity.     

Probing the function of conserved residues in the serine/threonine phosphatase PP2Calpha

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Br?nsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.     

Molecular cloning and chromosomal mapping of type one serine/threonine protein phosphatases in Arabidopsis thaliana

Type one serine/threonine protein phosphatases (PP1s) have been implicated in various processes of plant growth and development. In all plant species studied, PP1s are encoded by multigene families. Previous studies in our laboratory identified five Arabidopsis thaliana PP1 genes (TOPP1, TOPP2, TOPP3, TOPP4 and TOPP5). In the present study, we report the isolation of three additional PP1 genes (TOPP6, TOPP7 and TOPP8). Southern blot analyses indicate that these three newly isolated genes are single-copy genes in A. thaliana genome. All the three genes are expressed in roots, rosettes and flowers, although their expression levels appear to be lower than those of the five previously identified TOPP genes. Six of the eight TOPP genes were mapped to different positions on four of five A. thaliana chromosomes. Sequence comparison revealed that TOPP genes belong to different subgroups of plant PP1 genes, suggesting that they may encode proteins with distinct functions.     

Multiple phosphorylation of membrane-associated calcium-dependent protein serine/threonine kinase in Streptomyces fradiae

In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate aerial mycelium formation and sporulation. Using in vitro labeling, we demonstrate that in S. fradiae in the late exponential growth phosphorylation of 65-kDa membrane-associated protein is also influenced by Ca(2+) added exogenously. Calcium ions at physiological concentration stimulate intensive Ca(2+)-dependent phosphorylation of 65-kDa protein at multiple sites on serine, threonine, and tyrosine residues. Assay of protein kinases in situ demonstrated in the fraction of membrane-associated proteins the presence of two autophosphorylating protein serine/threonine kinases with molecular masses of 127 kDa and 65 kDa. Autophosphorylation of both proteins is also Ca(2+)-dependent.     

Dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain and its chloramphenicol-resistant mutant

The dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain 964 and its chloramphenicol-resistant (Cml^r) pleiotropic mutant with an enhanced production of avermectins was studied by measuring the transfer of radiolabeled phosphate from [y-³²P]ATP to the serine and threonine residues of proteins in cell-free extracts. In both of the strains studied, radiolabeled phosphate was found to incorporate into polypeptides with molecular masses of 32, 35, 41, 68, 75, 79, 83, and 137 kDa; however, the degree and the dynamics of phosphorylation of particular peptides were different in these strains. The differences revealed could not be accounted for by the interference of ATPases or phosphoprotein phosphatases. The data obtained may be interpreted as evidence that Cml^r mutation activates the protein kinase signalling system ofS.avermitilis cells in the early stationary growth phase and thus enhances the production of avermectins and leads to some other physiological changes in the mutant strain.     

Potential involvement of serine/threonine protein phosphatases in apoptosis of HepG2 cells during selenite treatment

Selenium, an essential biological trace element present in both prokaryotic and eukaryotic cells, exerts its regulatory effect in a variety of cellular events, including cell growth, survival, and death. Selenium compunds have been shown in different cell lines to inhibit apoptosis by several mechanisms. Serine/threonine phosphatases (STPs) are potentially important in selenite-induced apoptosis because of their role in regulation of diverse set of cellular processes. In this study, the regulatory role of STPs in selenite-induced apoptosis has been implied by the use of two specific inhibitors: ocadaic acid and calyculin A. Our results show a decrease in cell density in HepG2 cells under selenite treatment. Resulting specific enzyme activities showed a concentration-dependent increase in all three phosphatase activities after 24 h in cells treated with 5 μM selenite and these activities decreased at 48 and 72 h. However, in cells treated with 10μM selenite, PP2A and PP2B decreased at 48 h, whereas PP2C activity did not change at this dose. In cells treated with 25μM, there was not a significant change in PP2C activity. These data suggest that the most specific response to selenite treatment was in PP2A and PP2B activities in a dose-dependent manner. Our results with OA and Cal-A further support the view that PP1 and PP2A might act as negative regulators of growth. With these data, we have first demonstrated the role of serine/threonine protein phosphatases in the signaling pathway of selenite-induced apoptosis and resulting cytotoxicity     

Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases

Fluorescent chemosensors of protein kinase activity provide a continuous, high-throughput sensing format for the study of the roles of these enzymes, which are crucial for regulating cellular function. Specifically, chemosensors using the nonnatural amino acid, Sox, and physiological Mg(2+) levels report phosphorylation with dramatic fluorescence changes that are amenable to real-time and high-throughput analysis. In this article, we report 15 probes for a total of six distinct serine/threonine kinases with large fluorescence increases and good reactivity toward the target kinase. The sensing mechanism is detailed, and the optimal sensing motif is determined. These versatile and powerful sensors provide tools for researchers studying the roles of the targeted kinases in signal transduction, and the design principles provide guidelines for the generation of future fluorescent chemosensors for any serine/threonine kinase.     

Phosphorylation of the LCB1 subunit of Arabidopsis serine palmitoyltransferase stimulates its activity and modulates sphingolipid biosynthesis

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes.Serine palmitoyltransferase(SPT) is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway.The core SPT enzyme is a heterodimer consisting of LONG-CHAIN BASE1(LCB1) and LCB2 subunits.SPT activity is inhibited by orosomucoid proteins and stimulated by small subunits of SPT(ssSPTs).However,whether LCB1 is modified and how such modi...     

Identification and characterization of the serine/threonine protein kinases in Bifidobacterium

Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.     

Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine,serine and threonine

An intact cDNA fromArabidopsis thaliana for adenine phosphoribosyltransferase (APRT) was isolated and sequenced. The cDNA is 729 nucleotides in length and predicts a protein ofM _r 27140. The deduced amino acid sequence has been compared with those of other APRTs and shown to be most similar to theEscherichia coli protein. Construction of a molecular tree of the known APRT amino acid sequences indicates theA. thaliana andE. coli APRT sequences form one cluster and the currently available vertebrate and invertebrate sequences form a separate grouping. Since it is possible to select either for or against the expression of APRT, the isolation of this APRT cDNA clone will allow these selection schemes to be used in plant genetic experiments.     

Modulation of hippocampal calcium signalling and plasticity by serine/threonine protein phosphatases

Kinases and phosphatases act antagonistically to maintain physiological phosphorylation/dephosphorylation at numerous intracellular sites critical for neuronal signalling. In this study, it was found that inhibition of serine/threonine phosphatases by exposure of hippocampal slices to okadaic acid (OA) or cantharidin (CA; 100 nmol/L) for 2 h resulted in reduced basal synaptic transmission and blocked the induction of synaptic plasticity in the form of long-term potentiation as determined by electrophysiological analysis. Fura-2 Ca(2+) imaging revealed a bidirectional modulation of N-methyl-D-aspartate (NMDA) -mediated Ca(2+) responses and reduced KCl-mediated Ca(2+) responses in neonatal cultured hippocampal neurons after phosphatase inhibition. While OA inhibited NMDA-induced Ca(2+) influx both acutely and after incubation, CA-enhanced receptor-mediated Ca(2+) signalling at low concentrations (1 nmol/L) but reduced NMDA and KCl-mediated Ca(2+) responses at higher concentrations (100 nmol/L). Changes in Ca(2+) signalling were accompanied by increased phosphorylation of cytoskeletal proteins tau and neurofilament and the NMDA receptor subunit NR1 in selective treatments. Incubation with OA (100 nmol/L) also led to the disruption of the microtubule network. This study highlights novel signalling effects of prolonged inhibition of protein phosphatases and suggests reduced post-synaptic signalling as a major mechanism for basal synaptic transmission and long-term potentiation impairments.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

Determination of serine/threonine protein phosphatase type 2B PP2B in lymphocytes by HPLC

Current cyclosporin (CsA) and tacrolimus (FK506) monitoring methods are based on blood concentrations determination, but the optimal sampling times are not clearly defined. An alternative pharmacodynamic approach has recently been introduced, based on assaying lymphocytes activity of calcineurin (PP2B), a phosphatase that is partially inhibited by CsA and FK506. However, the princeps method uses large amounts of radioactive [32P]substrate, raising a number of safety issues. Here we describe an alternative method for PP2B activity determination, based on HPLC coupled with spectrophotometric detection. A 19-amino-acid peptide is phosphorylated by a protein kinase, and further dephosphorylated by lymphocyte PP2B in the presence of okadaic acid. The two peptides are separated by using reverse-phase chromatography with a detection wavelength of 205 nm. After lymphocyte isolation by density-gradient centrifugation, PP2B activity is derived from the dephosphorylated peptide concentration measured during the first 6 min of the enzymatic reaction. This technique gives reproducible results and good analytical sensitivity with 10(6) lymphocytes. With an average isolation rate of 59.6%, only 7 ml of blood is required, making the method suitable for lymphopenic patients. Moreover, PP2B activity is stable in blood samples kept for 24h at room temperature and in isolated lymphocytes stored for 48 h at -20 degrees C. We validated the method by comparing median PP2B activity in 10 healthy volunteers (285.0+/-46.5 pmol/min/10(6)lymphocytes) and in 10 liver transplant patients (147.8+/-71.0 pmol/min/10(6)lymphocytes) (p<0.001). The relation between calcineurin activity and tacrolimus blood concentrations was also studied.