Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.     

Nucleoside diphosphate kinase from Escherichia coli.

Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [gamma-32P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists. These results are discussed in light of the recent report of N. J. MacDonald et al. on the autophosphorylation of human NDP kinase (J. Biol. Chem. 268:25780-25789, 1993).     

Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth

The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.     

Phosphorylation of CTP synthetase on Ser36, Ser330, Ser354, and Ser454 regulates the levels of CTP and phosphatidylcholine synthesis in Saccharomyces cerevisiae

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C. We examined the hypothesis that Ser36, Ser330, Ser354, and Ser454, contained in a protein kinase C sequence motif in CTP synthetase, were target sites for the kinase. Synthetic peptides containing a phosphorylation motif at these serine residues served as substrates for protein kinase C in vitro. Ser --> Ala (S36A, S330A, S354A, and S454A) mutations in CTP synthetase were constructed by site-directed mutagenesis and expressed normally in a ura7 ura8 double mutant that lacks CTP synthetase activity. The CTP synthetase activity in extracts from cells bearing the S36A, S354A, and S454A mutant enzymes was reduced when compared with cells bearing the wild type enzyme. Kinetic analysis of purified mutant enzymes showed that the S36A and S354A mutations caused a decrease in the Vmax of the reaction. This regulation could be attributed in part by the effects phosphorylation has on the nucleotide-dependent oligomerization of CTP synthetase. In contrast, CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of purified enzyme showed that the S330A mutation caused an elevation in the Vmax of the reaction. In vitro data indicated that phosphorylation of CTP synthetase at Ser330 affected the phosphorylation of the enzyme at another site. The phosphorylation of CTP synthetase at Ser36, Ser330, Ser354, and Ser454 residues was physiologically relevant. Cells bearing the S36A, S354A, and S454A mutations had reduced CTP levels, whereas cells with the S330A mutation had elevated CTP levels. The alterations in CTP levels correlated with the regulatory effects CTP has on the pathways responsible for the synthesis of the membrane phospholipid phosphatidylcholine.     

Specific phosphorylation of exogenous protein and peptide substrates by the human cytomegalovirus UL97 protein kinase. Importance of the P+5 position

Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathione S-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, maribavir. Sequencing and mass spectrometric analyses of purified (32)P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P+5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P+5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.     

Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings

The mitogen-activated protein kinase (MAPK) cascade, consisting of MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK), is the signaling system that relays various external signals, including mitogens and stresses in eukaryotes. MAPKK is activated by phosphorylation in the consensus motif, SXXXS/T, in animals, but the regulation mechanism for the plant MAPKK by phosphorylation, having the putative phosphorylation motif of S/TXXXXXS/T, is not yet fully clarified. Here we constructed a series of mutants of AtMEK1, an Arabidopsis MAPKK, having the sequence T218-X-S220-X-X-X-S224 that fits both of the plant- and animal-type motifs. We show that the two double-mutant proteins replacing Thr-218/Ser-224 and Ser-220/Ser-224 by Glu expressed in Escherichia coli show a constitutive activity to phosphorylate the Thr and Tyr residues of the kinase-negative mutant of an Arabidopsis MAPK, named ATMPK4, in vitro. The mutation analysis of AtMEK1 replacing Thr-218 and Ser-220 to Ala suggested that Thr-218 is autophosphorylated by the enzyme. The wild-type ATMPK4 was also phosphorylated by the active mutants of AtMEK1 and showed a high protein kinase activity toward myelin basic proteins. In contrast, ATMPK3, another Arabidopsis MAPK, was a poor substrate of this plant MAPKK, indicating that AtMEK1 has a substrate specificity preferring ATMPK4 to ATMPK3, at least in vitro. Furthermore, AtMEK1 immunoprecipitated from Arabidopsis seedlings stimulated with wounding, cold, drought, and high salt showed an elevated protein kinase activity toward the kinase-negative ATMPK4, while the amounts of the AtMEK1 protein did not change significantly. These data indicate that the AtMEK1 becomes an active form through phosphorylation and activates its downstream target ATMPK4 in stress response in Arabidopsis.     

Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation.

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.     

Regulation of neuronal nitric-oxide synthase by calmodulin kinases.

Phosphorylation of neuronal nitric-oxide synthase (nNOS) by Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) including CaM kinase Ialpha (CaM-K Ialpha), CaM kinase IIalpha (CaM-K IIalpha), and CaM kinase IV (CaM-K IV), was studied. It was found that purified recombinant nNOS was phosphorylated by CaM-K Ialpha, CaM-K IIalpha, and CaM-K IV at Ser847 in vitro. Replacement of Ser847 with Ala (S847A) prevented phosphorylation by CaM kinases. Phosphorylated recombinant wild-type nNOS at Ser847 (approximately 0.5 mol of phosphate incorporation into nNOS) exhibited a 30% decrease of Vmax with little change of both the Km for L-arginine and Kact for CaM relative to unphosphorylated enzyme. The activity of mutant S847D was decreased to a level 50-60% as much as the wild-type enzyme. The decreased NOS enzyme activity of phosphorylated nNOS at Ser847 and mutant S847D was partially due to suppression of CaM binding, but not to impairment of dimer formation which is thought to be essential for enzyme activation. Inactive nNOS lacking CaM-binding ability was generated by mutation of Lys732-Lys-Leu to Asp732-Asp-Glu (Watanabe, Y., Hu, Y., and Hidaka, H. (1997) FEBS Lett. 403, 75-78). It was phosphorylated by CaM kinases, as was the wild-type enzyme, indicating that CaM-nNOS binding was not required for the phosphorylation reaction. We developed antibody NP847, which specifically recognize nNOS in its phosphorylated state at Ser847. Using the antibody NP847, we obtained evidence that nNOS is phosphorylated at Ser847 in rat brain. Thus, our results suggest that CaM kinase-induced phosphorylation of nNOS at Ser847 alters the activity control of this enzyme.     

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the k_cat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme.     

Phosphorylation and activation of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) by Ca2+/calmodulin-dependent protein kinase I (CaMKI)

Nuclear Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol.     

Phosphorylation of Saccharomyces cerevisiae choline kinase on Ser30 and Ser85 by protein kinase A regulates phosphatidylcholine synthesis by the CDP-choline pathway

The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser(30) and Ser(85) contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (V(max)/K(m) = 10.8 microm(-1) nmol min(-1) mg(-1)) and GPRRASATDV (V(max)/K(m) = 0.15 microm(-1) nmol min(-1) mg(-1)) containing the protein kinase A motif for Ser(30) and Ser(85), respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser(30) to Ala (S30A) and Ser(85) to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Delta eki1Delta double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of (3)H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser(30) and Ser(85) by protein kinase A regulates PC synthesis by the CDP-choline pathway.     

Soybean nodule sucrose synthase (nodulin-100): further analysis of its phosphorylation using recombinant and authentic root-nodule enzymes.

Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant "sink" tissues. In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli. Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized. We document that a conserved serine near the N-terminus (Ser(11)) is the primary phosphorylation site for a nodule Ca(2+)-dependent protein kinase (CDPK) in vitro. Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100. In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala(2) and Arg(13)) were phosphorylated, albeit weakly, by the CDPK. This secondary site(s) likely resides between Glu(14) and Met(193) as evidenced by CNBr cleavage and phosphopeptide mapping. Phosphorylation of the recombinant and authentic nodule Ser(11) enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties. However, phosphorylation decreased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin-100 between the nodule symbiosome/plasma membranes and cytosol in planta.     

Functional analyses of the activation loop of phototropin2 in Arabidopsis

Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2-mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses.     

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.     

Phosphorylation of the yeast choline kinase by protein kinase C. Identification of Ser25 and Ser30 as major sites of phosphorylation

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C.     

Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)-K(+)-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.     

Host-mediated phosphorylation of type III effector AvrPto promotes Pseudomonas virulence and avirulence in tomato

The AvrPto protein from Pseudomonas syringae pv tomato is delivered into plant cells by the bacterial type III secretion system, where it either promotes host susceptibility or, in tomato plants expressing the Pto kinase, elicits disease resistance. Using two-dimensional gel electrophoresis, we obtained evidence that AvrPto is phosphorylated when expressed in plant leaves. In vitro phosphorylation of AvrPto by plant extracts occurs independently of Pto and is due to a kinase activity that is conserved in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), and Arabidopsis thaliana. Three Ser residues clustered in the C-terminal 18 amino acids of AvrPto were identified in vitro as putative phosphorylation sites, and one site at S149 was directly confirmed as an in vivo phosphorylation site by mass spectrometry. Substitution of Ala for S149 significantly decreased the ability of AvrPto to enhance disease symptoms and promote growth of P. s. tomato in susceptible tomato leaves. In addition, S149A significantly decreased the avirulence activity of AvrPto in resistant tomato plants. Our observations support a model in which AvrPto has evolved to mimic a substrate of a highly conserved plant kinase to enhance its virulence activity. Furthermore, residues of AvrPto that promote virulence are also monitored by plant defenses.     

Mutational analysis of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism

Nucleotide pyrophosphatases/phosphodiesterases (NPPs, PF01663) release nucleoside 5′-monophosphates from a wide range of nucleotide substrates. Only very recently, the first plant members of the NPP family were characterised (Joye et al. J Cereal Sci 51: 326–336, 2010), and little is known about their substrate-specifying residues. We elucidated the role of six amino acid residues of the recently identified and characterised Triticum aestivum L. NPP (Joye et al. J Cereal Sci 51: 326–336, 2010). Substitution of the highly conserved catalytic Thr132 into Ser or Ala completely abolished enzyme activity. Mutation of a highly conserved His255 residue into an apolar Ala suprisingly increased enzyme activity against most phosphodiester substrates. Four other residues moderately to highly conserved over NPPs of different organisms were studied as well. Mutation of the Asn153, Asn165 and Glu199 into an Arg, Ser and Asp residue, respectively, increased the relative enzyme activity against p-nitrophenyl phosphate. Furthermore, mutation of Phe194 into Ser increased the relative enzyme activity against adenosine 5′-monophosphate-containing substrates, although the overall enzyme activity of this mutant enzyme decreased. We conclude that the structural requirements and the conservation of the amino acids of the catalytic site of TaNPPr and, by extension, probably of all NPPs, are very stringent.