A mitogen-activated protein kinase NtMPK4 activated by SIPKK is required for jasmonic acid signaling and involved in ozone tolerance via stomatal movement in tobacco

The mitogen-activated protein kinase (MAPK) cascade is involved in responses to biotic and abiotic stress in plants. In this study, we isolated a new MAPK, NtMPK4, which is a tobacco homolog of Arabidopsis MPK4 (AtMPK4). NtMPK4 was activated by wounding along with two other wound-responsive tobacco MAPKs, WIPK and SIPK. We found that NtMPK4 was activated by salicylic acid-induced protein kinase kinase (SIPKK), which has been isolated as an SIPK-interacting MAPK kinase. In NtMPK4 activity-suppressed tobacco, wound-induced expression of jasmonic acid (JA)-responsive genes was inhibited. NtMPK4-silenced plants showed enhanced sensitivity to ozone. Inversely, transgenic tobacco plants, in which SIPKK or the constitutively active type SIPKK(EE) was overexpressed, exhibited greater responsiveness to wounding with enhanced resistance to ozone. We further found that NtMPK4 was expressed preferentially in epidermis, and the enhanced sensitivity to ozone in NtMPK4-silenced plants was caused by an abnormal regulation of stomatal closure in an ABA-independent manner. These results suggest that NtMPK4 is involved in JA signaling and in stomatal movement.     

The protein-tyrosine kinase substrate p36 is also a substrate for protein kinase C in vitro and in vivo.

p36, a major in vivo substrate of protein-tyrosine kinases, is shown to be phosphorylated at serine 25, a site very close to the major site of tyrosine phosphorylation by pp60v-src, tyrosine 23 (J. R. Glenney, Jr., and B. F. Tack, Proc. Natl. Acad. Sci. USA 82:7884-7888, 1985). We present evidence suggesting that protein kinase C mediates phosphorylation of serine 25.     

Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells

A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.     

Essential protein interactions within the replisome regulate DNA replication

Comment on: Zhang H, et al. Proc Natl Acad Sci USA 2011; 108:9372-7.     

Glycogen synthase kinase (GSK)-3 and mammalian target of rapamycin complex 1 (mTORC1) cooperate to regulate protein S6 kinase 1 (S6K1)

Comment on: Shin S, et al. Proc Natl Acad Sci USA 2011; 108:1204–13     

Horizontal gene transfer in microbial genome evolution

Horizontal gene transfer is the collective name for processes that permit the exchange of DNA among organisms of different species. Only recently has it been recognized as a significant contribution to inter-organismal gene exchange. Traditionally, it was thought that microorganisms evolved clonally, passing genes from mother to daughter cells with little or no exchange of DNA among diverse species. Studies of microbial genomes, however, have shown that genomes contain genes that are closely related to a number of different prokaryotes, sometimes to phylogenetically very distantly related ones. (Doolittle et al., 1990, J. Mol. Evol. 31, 383-388; Karlin et al., 1997, J. Bacteriol. 179, 3899-3913; Karlin et al., 1998, Annu. Rev. Genet. 32, 185-225; Lawrence and Ochman, 1998, Proc. Natl. Acad. Sci. USA 95, 9413-9417; Rivera et al., 1998, Proc. Natl. Acad. Sci. USA 95, 6239-6244; Campbell, 2000, Theor. Popul. Biol. 57 71-77; Doolittle, 2000, Sci. Am. 282, 90-95; Ochman and Jones, 2000, Embo. J. 19, 6637-6643; Boucher et al. 2001, Curr. Opin., Microbiol. 4, 285-289; Wang et al., 2001, Mol. Biol. Evol. 18, 792-800). Whereas prokaryotic and eukaryotic evolution was once reconstructed from a single 16S ribosomal RNA (rRNA) gene, the analysis of complete genomes is beginning to yield a different picture of microbial evolution, one that is wrought with the lateral movement of genes across vast phylogenetic distances. (Lane et al., 1988, Methods Enzymol. 167, 138-144; Lake and Rivera, 1996, Proc. Natl. Acad. Sci. USA 91, 2880-2881; Lake et al., 1999, Science 283, 2027-2028).     

MAP kinase phosphorylation of plant profilin

Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P-X-T-P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45(Ntf4) and SIPK, when activated by the tobacco MAP kinase kinase NtMEK2, can phosphorylate the tobacco profilin NtProf2. Mutagenesis of the threonine residue in this motif identified it as the site of MAP kinase phosphorylation. Fractionation of tobacco pollen extracts showed that p45(Ntf4) is found exclusively in the high-speed pellet fraction while SIPK and profilin are predominantly cytosolic. These data identify one of the first substrates to be directly phosphorylated by MAP kinases in plants.     

The U(S)3 protein kinase blocks apoptosis induced by the d120 mutant of herpes simplex virus 1 at a premitochondrial stage

Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the alpha4 gene, induces caspase-3-dependent apoptosis in HEp-2 cells. Apoptosis was also induced by the alpha4 rescuant but was blocked by the complementation of rescuant with a DNA fragment encoding the U(S)3 protein kinase (R. Leopardi and B. Roizman, Proc. Natl. Acad. Sci. USA 93:9583-9587, 1996, and R. Leopardi, C. Van Sant, and B. Roizman, Proc. Natl. Acad. Sci. USA 94:7891-7896, 1997). To investigate its role in the apoptotic cascade, the U(S)3 open reading frame was cloned into a baculovirus (Bac-U(S)3) under the control of the human cytomegalovirus immediate-early promoter. We report the following. (i) Bac-U(S)3 blocks processing of procaspase-3 to active caspase. Procaspase-3 levels remained unaltered if superinfected with Bac-U(S)3 at 3 h after d120 mutant infection, but significant amounts of procaspase-3 remained in cells superinfected with Bac-Us3 at 9 h postinfection with d120 mutant. (ii) The U(S)3 protein kinase blocks the proapoptotic cascade upstream of mitochondrial involvement inasmuch as Bac-U(S)3 blocks release of cytochrome c in cells infected with the d120 mutant. (iii) Concurrent infection of HEp-2 cells with Bac-U(S)3 and the d120 mutant did not alter the pattern of accumulation or processing of ICP0, -22, or -27, and therefore U(S)3 does not appear to block apoptosis by targeting these proteins.     

Vaccinia virus morphogenesis is blocked by temperature-sensitive mutations in the F10 gene, which encodes protein kinase 2.

Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts28, ts54, ts61, and ts15) composing a single complementation group have been mapped by marker rescue to the F10 open reading frame located within the genomic HindIII F DNA fragment. Sequencing of the F10 gene from wild-type and mutant viruses revealed single-amino-acid substitutions in the F10 polypeptide responsible for thermolabile growth. Although the ts mutants displayed normal patterns of viral protein synthesis, electron microscopy revealed a profound morphogenetic defect at the nonpermissive temperature (40 degrees C). Virion assembly was arrested at an early stage, with scant formation of membrane crescents and no progression to normal spherical immature particles. The F10 gene encodes a 52-kDa serine/threonine protein kinase (S. Lin and S. S. Broyles, Proc. Natl. Acad. Sci. USA 91:7653-7657, 1994). We expressed a His-tagged version of the wild-type, ts54, and ts61 F10 polypeptides in bacteria and purified these proteins by sequential nickel affinity and phosphocellulose chromatography steps. The wild-type F10 protein kinase activity was characterized in detail by using casein as a phosphate acceptor. Whereas the wild-type and ts61 kinases displayed similar thermal inactivation profiles, the ts54 kinase was thermosensitive in vitro. These findings suggest that protein phosphorylation plays an essential role at an early stage of virion assembly.     

Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase.

We have recently shown that changes in tyrosine phosphorylation of a 130-kDa protein(s) (pp130) may be involved in integrin signaling (Kornberg, L., Earp, H.S., Turner, C., Prokop, and Juliano, R. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8392-8396). One component of the pp130 protein complex reacts with an antibody generated against p125fak, which is a focal contact-associated tyrosine kinase (Schaller, M.D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A. B., and Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5192-5196). Both antibody-mediated integrin clustering and adhesion of KB cells to fibronectin leads to increased tyrosine phosphorylation of p125fak. The phosphorylation of p125fak is coincident with adhesion of cells to fibronectin and is maximal prior to cell spreading. Tyrosine phosphorylation of p125fak is induced when KB cells are allowed to adhere to fibronectin, collagen type IV, or laminin, but is not induced on polylysine. When KB cells are subjected to indirect immunofluorescence microscopy, p125fak colocalizes with talin in focal contacts. These data provide additional evidence that tyrosine kinases are involved in integrin signaling.     

Uric acid might herald new hopes for multiple sclerosis patients

Uric acid: a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosisHooper, D.C. et al. (1998)Proc. Natl. Acad. Sci. U. S. A. 95, 675–680     

Phosphorylation of hormone-sensitive lipase by cyclic GMP-dependent protein kinase

In intact rat adipocytes hormone-sensitive lipase has been shown to be phosphorylated on serine residues in two different phosphorylation sites: a regulatory site phosphorylated by cyclic AMP-dependent protein kinase and a basal site, which does not directly affect the enzyme activity, phosphorylated by cyclic AMP-independent protein kinase(s) [(1984) Proc. Natl. Acad. Sci USA 81, 3317-3321]. Cyclic GMP-dependent protein kinase catalyzed the phosphorylation of the same two phosphorylation sites on the isolated enzyme, at serine residues. Both sites were phosphorylated at about the same rate, with the hormone-sensitive lipase activity concomitantly enhanced.     

Developmentally expressed myosin heavy-chain kinase possesses a diacylglycerol kinase domain.

In Dictyostelium, an ordered actin and myosin assembly-disassembly process is necessary for proper development, differentiation, and motility (Yumura S, Fukui F, 1985, Nature 314(6007): 194-196; Ravid S, Spudich JA, 1989, J Biol Chem 264(25): 15144-15150), and phosphorylation of myosin heavy chains has been implicated in the myosin assembly-disassembly process (Egelhoff TT, Lee RJ, Spudich JA, 1993, Cell 75(2):363-371). The developmentally expressed 84-kDa myosin heavy-chain kinase (MHCK) from Dictyostelium (Ravid S, Spudich JA, 1992, Proc Natl Acad Sci USA 89(13):5877-5881) is known to be a member of the protein kinase C (PKC) family. We have observed a rather striking homology between the large central domain of MHCK and the catalytic domain of diacylglycerol kinase (DGK), indicating that MHCK is in fact a gene fusion between a DGK and a PKC, possessing two separate kinase domains. The combined diacylglycerol kinase/myosin heavy-chain kinase (DGK/MHCK) may therefore have dual functionality, possessing the ability to phosphorylate both protein and lipid. We present a hypothesis that DGK/MHCK can antagonize both actin and myosin assembly, as well as other cellular processes, by coordinated down regulation of signaling via myosin heavy-chain kinase activity and diacylglycerol kinase activity.     

Reading the three-dimensional structure of lattice model-designed proteins from their amino acid sequence.

While all the information required for the folding of a protein is contained in its amino acid sequence, one has not yet learned how to extract this information to predict the detailed, biological active, three-dimensional structure of a protein whose sequence is known. Using insight obtained from lattice model simulations of the folding of small proteins (fewer than 100 residues), in particular of the fact that this phenomenon is essentially controlled by conserved contacts (Mirny et al., Proc Natl Acad Sci USA 1995;92:1282) among (few) strongly interacting ("hot") amino acids (Tiana et al., J Chem Phys 1998;108:757-761), which also stabilize local elementary structures formed early in the folding process and leading to the (postcritical) folding core when they assemble together (Broglia et al., Proc Natl Acad Sci USA 1998;95:12930, Broglia & Tiana, J Chem Phys 2001;114:7267), we have worked out a successful strategy for reading the three-dimensional structure of lattice model-designed proteins from the knowledge of only their amino acid sequence and of the contact energies among the amino acids.     

Differentiation of varicella-zoster virus ORF47 protein kinase and IE62 protein binding domains and their contributions to replication in human skin xenografts in the SCID-hu mouse

To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo.