- Email: [email protected]
Protein phosphorylation in and around signal transduction
54
Research Update
TRENDS in Plant Science Vol.7 No.2 February 2002
9 Liu, J-W. et al. (2001) Evaluation of the seeds oils from a canola plant genetically transformed to produce high levels of γ-linolenic acid. In γ-Linolenic Acid: Recent Advance in Biotechnology and Clinical Applications (Huang, Y-S. and Ziboh, V.A., eds), pp. 61–71, American Oil Chemists’ Society Press, Champaign, IL, USA 10 Metz, J.G. et al. (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293, 290–293
11 Takeyama, H. et al. (1997) Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp. Microbiology 143, 2725–2731 12 Watanabe, K. et al. (1997) Fatty acid synthesis of an eicosapentaenoic acid-producing bacterium: de novo synthesis, chain elongation, and desaturation systems. J. Biochem. 122, 467–473 13 Bentley, R. and Bennett, J.W. (1999) Constructing polyketides: from Collie to combinatorial biosynthesis. Annu. Rev. Microbiol. 53, 411–446
14 Abbadi, A. et al. (2001) Transgenic oilseeds as sustainable source of nutritionally relevant C20 and C22 polyunsaturated fatty acids? Eur. J. Lipid Sci. Technol. 103, 106–113
Johnathan A. Napier IACR-Long Ashton Research Station, Long Ashton, Bristol, UK BS41 9AF. e-mail: [email protected]
Meeting Report
Protein phosphorylation in and around signal transduction John Mundy and Kay Schneitz Plant Protein Phosphorylation, 12–15 September 2001, Vienna, Austria.
Metabolism
The recent Plant Protein Phosphorylation meeting was exciting for two reasons. First, it was focused so that people could share results and techniques. Second, it had a broad scope because phosphorylation is involved in regulating just about everything. Cell cycle
Dénes Dudits (Hungarian Academy of Sciences, Szeged, Hungary) has identified an alfalfa cyclin-dependent kinase inhibitor (CKI) that itself becomes phosphorylated by a calmodulin-like domain kinase (CDPK), suggesting a link between calcium-based signaling and cell-cycle complexes in vivo. What is the role of individual mitogen-activated protein kinases (MAPKs), and some of their regulators, such as MAPK kinase kinase (MAPKKK), in plant development? Patrick Krysan (University of Wisconsin, USA) addressed this issue with a reversegenetics approach. He has analyzed mutants carrying T-DNA insertions in any of three Arabidopsis MAPKKK genes, ANP-1, ANP-2 and ANP-3. There appears to be some redundancy because single mutants showed no apparent phenotype. Genome-wide expression analysis of anp2 anp3 double mutants suggests a role for these genes in repressing stress responses. Cathal Wilson (University of Vienna, Austria) provided evidence that the MAP kinase p43Ntf6, the MAPK kinase NtMEK1, and NPK1, a previously known MAPKKK, constitute a MAP kinase module that functions in cytokinesis. http://plants.trends.com
Tatjana Kleinow (Max-Planck-Institut für Züchtungsforschung, Cologne, Germany) has identified Arabidopsis orthologs of the β- and γ-activator-subunit of the SNF1related kinase complex. Surprisingly plants seem to have two different kinase complex forms: one complex with an α, β and γ subunit and one with just α and the new combined βγ subunit. Several new regulatory components of the SNF1related kinase signaling cascade have been identified using the two-hybrid system and in vitro binding assays. Stress responses
Kazuo Shinozaki (RIKEN, Tsukuba, Japan) discussed the signal crosstalk and integration that probably occurs between MAPKs given their large numbers (~24 MPKs, 18 MPKKs and 30 MPKKKs in Arabidopsis) and potential redundancy and partner promiscuity. Claudia Jonak and Irute Meskiene (University of Vienna, Austria) presented work on alfalfa WIG (wound-induced glycogen synthase kinase 3 or GSK3) and SIMK (woundand salt-inducible MAPK). Further characterization of WIG is warranted because GSK3s modulate the downstream specificity of other kinase signaling pathways. Biochemical analyses indicate that activity of the SIMK MPK is attenuated by the PP2C-type phosphatase MP2C, and that MP2C interaction with SIMK is mediated by a conserved MAPK docking motif in the MP2C N-terminus. Systems to assign functions to calcium (Ca2+-) dependent protein kinases (CPDKs, including one that functions upstream of a pathway required for Avr9-induced reactive
oxygen species (ROS), were described by Tina Romeis (John Innes Centre, Norwich, UK). Epitope tagging has shown that tobacco NtCDPK2 is shared between pathways triggered by biotic and abiotic stresses. Jörg Kudla (University of Ulm, Germany) showed that Ca2+ regulates interactions between calcineurin B-like proteins (CBL) and NAF kinase domains. Molecular and reverse genetic approaches indicate that differential CBL–calcineurin B-like interacting protein kinase (CIPK) affinities impose specificity upon Ca2+ signals. Two research groups have linked MAP kinases to genotoxic stress responses. Johannes Stratmann (University of South Carolina, Columbia, SC, USA) showed that the systemin receptor is involved in ultraviolet B (UV-B) signaling via activation of a tomato MAP kinase. It is also possible that UV-B responses are mediated non-specifically via stress pathways, including the systemin receptor. A genetic screen described by Roman Ulm (Friedrich Miescher Institute, Basel, Germany) revealed that loss of function of an Arabidopsis MAP kinase phosphatase (AtMKP1) results in ultraviolet C (UV-C) and methyl methanesulfonate (MMS)hypersensitivity. AtMKP1 interacts with several MPKs, therefore AtMPK1 must be an important regulator of MAP kinase activities in vivo. Pathogen responses
Three research groups linked pathogen responses to MAP kinase signaling. Dierk Scheel (Institute of Plant Biochemistry, Halle, Germany) described responses to the peptide elicitor PEP-13 in parsley (Petroselinum crispum). PEP-13
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02192-6
Research Update
stimulates rapid Ca2+ influx that sustains Ca2+ levels required for ROS production and activation of three MAPKCs. This biochemical system has been used to identify numerous parsley MAPK components. Yuko Ohashi (University of Tsukuba, Japan) described hypersensitivity response (HR)-induction at permissive temperatures (23°C) by tobacco mosaic virus (TMV)infected tobacco carrying the N resistance gene. This system detected induction of calmodulins and the WRK receptor-like kinase, followed by activation of woundinginduced protein kinase (WIPK) MPK, which is thought to regulate wound- and jasmonic acid-responses. WRK plays a role in HRdependent signaling as WRK overexpression results in N gene-dependent phenotypes, including cell death. Thomas Boller (F. Miescher Institute, Basel, Switzerland) described the Arabidopsis FLS2 leucine-rich repeat (LRR)-receptor kinase that recognizes a conserved domain of bacterial flagellins, the 22 residue peptide Flg22. Catalytically ‘dead’kinase mutations impair Flg22 binding by the FLS2 extracellular LRRs, and identify FLS2 and Flg22 residues required for ligand binding. The identification of proteins phosphorylated in response to the FLS2–Flg22 interaction was discussed by Scott Peck (John Innes Centre, Norwich, UK). These targets are implicated in FLS22 signaling because overexpression of two such phosphoproteins results in Flg22hypersensitivity. These phosphoproteins might be substrates of MPK6, which is activated during elicitor responses. ‘…phosphorylation of the ABI5 Bzip factor is essential for the maintenance of embryo quiescence.’ Transport
Phosphorylation by a cell-wall-associated kinase inactivates the tobacco mosaic virus movement protein (TMV-MP) required for viral genome transport between cells (Kateryna Trutnyeva, University of Vienna, Austria). Expression of wild-type and phosphorylation mutant TMV-MP fusions to green fluorescent protein shows that phosphorylation influences targeting to and translocation through plasmodesmata. Thus, differential phosphorylation might restrict viral movement. As discussed by Hrvoje Fulgosi (University of Kiel, Germany), differential phosphorylation of import receptors in the chloroplast outer envelope complex (Toc) regulate chloroplast preprotein import. Solid-phase http://plants.trends.com
TRENDS in Plant Science Vol.7 No.2 February 2002
renaturation assays indicate that outer envelope proteins phosphorylate the Toc34 and Toc159 receptors. Biochemical evidence suggests that these kinases originated from two-component signaling components. Hormones
Jêrome Giraudat (CNRS, Gif-sur-Yvette, France) described a screen for Arabidopsis mutants in stomatal responses that alter transpiration and leaf temperatures detectable by video thermography. One such abscisic acid (ABA)-insensitive mutant is caused by inactivation of the OST1 protein kinase, a homolog of wheat PKABA. Genetic analyses with ost1 confirm a role for OST1 in ABA signaling. Related work on ABA by Sébastien Mongrand (Rockefeller University, New York, NY, USA) shows that phosphorylation of the ABI5 Bzip factor is essential for the maintenance of embryo quiescence. ABI5 protein accumulation, stability and activity are tightly regulated by ABA. Recent biochemical and genetic work indicates that differential phosphorylation controls ABI5 activity. Robert Fluhr (Weismann Institute, Rehovot, Israel) described studies of tobacco LAMMER kinase PK12 that phosphorylates Ser/Thr-rich splicing factors (SRPs) including Arabidopsis AtSRp34. Interestingly, a LAMMER motif PK12 mutant form exhibits aberrant subnuclear localization, and causes aberrant AtSRp34 subnuclear distribution. SRP phosphorylation was also described by Thomas Hufnagl (University of Vienna, Austria). He has studied Arabidopsis SRP kinases (SAtSRPK1-4) with different substrate specificities. Yeast 2-hybrid screens identified other AtSRPK partners and determined the binding site of AtSRPK4 to two of them. The different roles of SRPKs during development are revealed by differential expression patterns of SRPK promoter–GUS fusions. Alison DeLong (Brown University, Providence, RI, USA) showed that the rcn1 mutation in a regulatory subunit of a PP2A phosphatase leads to reduced PP2A activity and defects in cell elongation and growth responses to the auxin transport inhibitor NPA. Genetic and pharmacological experiments show that reduced PP2A activity in rcn1 affects both basipetal and acropetal auxin transport, and that the effect on basipetal transport is independent of the AGR1 and AUX1 products. These results support a role for protein phosphorylation in regulating polar auxin transport in seedling roots.
55
Light
Histone kinase activity in Arabidopsis is induced by high irradiance levels during de-etiolation (Daniel Chamovitz, Tel Aviv University, Israel). Genetic analyses show this kinase is downstream of phytochrome and cryptochrome photoreceptors and COP1. In addition, a kinase that phosphorylates the COP9 signalosome has been partially purified. Light also stimulates transcription in chloroplasts. Gerhard Link (University of Bochum, Germany) showed that a subunit of chloroplast RNA polymerase is a CK2type kinase that connects photosynthetic activity to chloroplast gene expression. Development
Hormones are central players in plant development. John Walker (University of Missouri, Columbia, MS, USA) discussed mechanisms underlying brassinosteroid (BR) signaling. BRI1 encodes a LRR receptor-like kinase and there is evidence that BRI1 is the receptor for brassinolide (BL). An activation-tagging screen for revertants of the weak bri1-5 allele identified BRS1, encoding a serine carboxy peptidase, several homologs of BRI1, and a homolog of carrot SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (AtSERK1), implicating AtSERK1 in BR signaling. Ykä Helariutta (University of Helsinki, Finland) talked about the cytokinin receptor WOODEN LEG (also known as CRE1 or AHK4), a two component molecule required for asymmetric cell divisions during vascular development in roots. He discussed suppressor screens that might identify downstream components of this cytokinin-mediated pathway. Conclusions
In this short summary, we could only detail a few highlights of the conference. It was exciting to hear so much progress. And it made us long for a functional genomics program to unravel the complexity of phospho-relay signaling. John Mundy Molecular Biology Institute, University of Copenhagen, Ø. Farimagsgade 2A, 1353-K Copenhagen, Denmark. e-mail: [email protected] Kay Schneitz Institute of Plant Biology, University of Zürich, Zollikerstr. 107, 8008 Zürich, Switzerland.
Research Update
TRENDS in Plant Science Vol.7 No.2 February 2002
9 Liu, J-W. et al. (2001) Evaluation of the seeds oils from a canola plant genetically transformed to produce high levels of γ-linolenic acid. In γ-Linolenic Acid: Recent Advance in Biotechnology and Clinical Applications (Huang, Y-S. and Ziboh, V.A., eds), pp. 61–71, American Oil Chemists’ Society Press, Champaign, IL, USA 10 Metz, J.G. et al. (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293, 290–293
11 Takeyama, H. et al. (1997) Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp. Microbiology 143, 2725–2731 12 Watanabe, K. et al. (1997) Fatty acid synthesis of an eicosapentaenoic acid-producing bacterium: de novo synthesis, chain elongation, and desaturation systems. J. Biochem. 122, 467–473 13 Bentley, R. and Bennett, J.W. (1999) Constructing polyketides: from Collie to combinatorial biosynthesis. Annu. Rev. Microbiol. 53, 411–446
14 Abbadi, A. et al. (2001) Transgenic oilseeds as sustainable source of nutritionally relevant C20 and C22 polyunsaturated fatty acids? Eur. J. Lipid Sci. Technol. 103, 106–113
Johnathan A. Napier IACR-Long Ashton Research Station, Long Ashton, Bristol, UK BS41 9AF. e-mail: [email protected]
Meeting Report
Protein phosphorylation in and around signal transduction John Mundy and Kay Schneitz Plant Protein Phosphorylation, 12–15 September 2001, Vienna, Austria.
Metabolism
The recent Plant Protein Phosphorylation meeting was exciting for two reasons. First, it was focused so that people could share results and techniques. Second, it had a broad scope because phosphorylation is involved in regulating just about everything. Cell cycle
Dénes Dudits (Hungarian Academy of Sciences, Szeged, Hungary) has identified an alfalfa cyclin-dependent kinase inhibitor (CKI) that itself becomes phosphorylated by a calmodulin-like domain kinase (CDPK), suggesting a link between calcium-based signaling and cell-cycle complexes in vivo. What is the role of individual mitogen-activated protein kinases (MAPKs), and some of their regulators, such as MAPK kinase kinase (MAPKKK), in plant development? Patrick Krysan (University of Wisconsin, USA) addressed this issue with a reversegenetics approach. He has analyzed mutants carrying T-DNA insertions in any of three Arabidopsis MAPKKK genes, ANP-1, ANP-2 and ANP-3. There appears to be some redundancy because single mutants showed no apparent phenotype. Genome-wide expression analysis of anp2 anp3 double mutants suggests a role for these genes in repressing stress responses. Cathal Wilson (University of Vienna, Austria) provided evidence that the MAP kinase p43Ntf6, the MAPK kinase NtMEK1, and NPK1, a previously known MAPKKK, constitute a MAP kinase module that functions in cytokinesis. http://plants.trends.com
Tatjana Kleinow (Max-Planck-Institut für Züchtungsforschung, Cologne, Germany) has identified Arabidopsis orthologs of the β- and γ-activator-subunit of the SNF1related kinase complex. Surprisingly plants seem to have two different kinase complex forms: one complex with an α, β and γ subunit and one with just α and the new combined βγ subunit. Several new regulatory components of the SNF1related kinase signaling cascade have been identified using the two-hybrid system and in vitro binding assays. Stress responses
Kazuo Shinozaki (RIKEN, Tsukuba, Japan) discussed the signal crosstalk and integration that probably occurs between MAPKs given their large numbers (~24 MPKs, 18 MPKKs and 30 MPKKKs in Arabidopsis) and potential redundancy and partner promiscuity. Claudia Jonak and Irute Meskiene (University of Vienna, Austria) presented work on alfalfa WIG (wound-induced glycogen synthase kinase 3 or GSK3) and SIMK (woundand salt-inducible MAPK). Further characterization of WIG is warranted because GSK3s modulate the downstream specificity of other kinase signaling pathways. Biochemical analyses indicate that activity of the SIMK MPK is attenuated by the PP2C-type phosphatase MP2C, and that MP2C interaction with SIMK is mediated by a conserved MAPK docking motif in the MP2C N-terminus. Systems to assign functions to calcium (Ca2+-) dependent protein kinases (CPDKs, including one that functions upstream of a pathway required for Avr9-induced reactive
oxygen species (ROS), were described by Tina Romeis (John Innes Centre, Norwich, UK). Epitope tagging has shown that tobacco NtCDPK2 is shared between pathways triggered by biotic and abiotic stresses. Jörg Kudla (University of Ulm, Germany) showed that Ca2+ regulates interactions between calcineurin B-like proteins (CBL) and NAF kinase domains. Molecular and reverse genetic approaches indicate that differential CBL–calcineurin B-like interacting protein kinase (CIPK) affinities impose specificity upon Ca2+ signals. Two research groups have linked MAP kinases to genotoxic stress responses. Johannes Stratmann (University of South Carolina, Columbia, SC, USA) showed that the systemin receptor is involved in ultraviolet B (UV-B) signaling via activation of a tomato MAP kinase. It is also possible that UV-B responses are mediated non-specifically via stress pathways, including the systemin receptor. A genetic screen described by Roman Ulm (Friedrich Miescher Institute, Basel, Germany) revealed that loss of function of an Arabidopsis MAP kinase phosphatase (AtMKP1) results in ultraviolet C (UV-C) and methyl methanesulfonate (MMS)hypersensitivity. AtMKP1 interacts with several MPKs, therefore AtMPK1 must be an important regulator of MAP kinase activities in vivo. Pathogen responses
Three research groups linked pathogen responses to MAP kinase signaling. Dierk Scheel (Institute of Plant Biochemistry, Halle, Germany) described responses to the peptide elicitor PEP-13 in parsley (Petroselinum crispum). PEP-13
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02192-6
Research Update
stimulates rapid Ca2+ influx that sustains Ca2+ levels required for ROS production and activation of three MAPKCs. This biochemical system has been used to identify numerous parsley MAPK components. Yuko Ohashi (University of Tsukuba, Japan) described hypersensitivity response (HR)-induction at permissive temperatures (23°C) by tobacco mosaic virus (TMV)infected tobacco carrying the N resistance gene. This system detected induction of calmodulins and the WRK receptor-like kinase, followed by activation of woundinginduced protein kinase (WIPK) MPK, which is thought to regulate wound- and jasmonic acid-responses. WRK plays a role in HRdependent signaling as WRK overexpression results in N gene-dependent phenotypes, including cell death. Thomas Boller (F. Miescher Institute, Basel, Switzerland) described the Arabidopsis FLS2 leucine-rich repeat (LRR)-receptor kinase that recognizes a conserved domain of bacterial flagellins, the 22 residue peptide Flg22. Catalytically ‘dead’kinase mutations impair Flg22 binding by the FLS2 extracellular LRRs, and identify FLS2 and Flg22 residues required for ligand binding. The identification of proteins phosphorylated in response to the FLS2–Flg22 interaction was discussed by Scott Peck (John Innes Centre, Norwich, UK). These targets are implicated in FLS22 signaling because overexpression of two such phosphoproteins results in Flg22hypersensitivity. These phosphoproteins might be substrates of MPK6, which is activated during elicitor responses. ‘…phosphorylation of the ABI5 Bzip factor is essential for the maintenance of embryo quiescence.’ Transport
Phosphorylation by a cell-wall-associated kinase inactivates the tobacco mosaic virus movement protein (TMV-MP) required for viral genome transport between cells (Kateryna Trutnyeva, University of Vienna, Austria). Expression of wild-type and phosphorylation mutant TMV-MP fusions to green fluorescent protein shows that phosphorylation influences targeting to and translocation through plasmodesmata. Thus, differential phosphorylation might restrict viral movement. As discussed by Hrvoje Fulgosi (University of Kiel, Germany), differential phosphorylation of import receptors in the chloroplast outer envelope complex (Toc) regulate chloroplast preprotein import. Solid-phase http://plants.trends.com
TRENDS in Plant Science Vol.7 No.2 February 2002
renaturation assays indicate that outer envelope proteins phosphorylate the Toc34 and Toc159 receptors. Biochemical evidence suggests that these kinases originated from two-component signaling components. Hormones
Jêrome Giraudat (CNRS, Gif-sur-Yvette, France) described a screen for Arabidopsis mutants in stomatal responses that alter transpiration and leaf temperatures detectable by video thermography. One such abscisic acid (ABA)-insensitive mutant is caused by inactivation of the OST1 protein kinase, a homolog of wheat PKABA. Genetic analyses with ost1 confirm a role for OST1 in ABA signaling. Related work on ABA by Sébastien Mongrand (Rockefeller University, New York, NY, USA) shows that phosphorylation of the ABI5 Bzip factor is essential for the maintenance of embryo quiescence. ABI5 protein accumulation, stability and activity are tightly regulated by ABA. Recent biochemical and genetic work indicates that differential phosphorylation controls ABI5 activity. Robert Fluhr (Weismann Institute, Rehovot, Israel) described studies of tobacco LAMMER kinase PK12 that phosphorylates Ser/Thr-rich splicing factors (SRPs) including Arabidopsis AtSRp34. Interestingly, a LAMMER motif PK12 mutant form exhibits aberrant subnuclear localization, and causes aberrant AtSRp34 subnuclear distribution. SRP phosphorylation was also described by Thomas Hufnagl (University of Vienna, Austria). He has studied Arabidopsis SRP kinases (SAtSRPK1-4) with different substrate specificities. Yeast 2-hybrid screens identified other AtSRPK partners and determined the binding site of AtSRPK4 to two of them. The different roles of SRPKs during development are revealed by differential expression patterns of SRPK promoter–GUS fusions. Alison DeLong (Brown University, Providence, RI, USA) showed that the rcn1 mutation in a regulatory subunit of a PP2A phosphatase leads to reduced PP2A activity and defects in cell elongation and growth responses to the auxin transport inhibitor NPA. Genetic and pharmacological experiments show that reduced PP2A activity in rcn1 affects both basipetal and acropetal auxin transport, and that the effect on basipetal transport is independent of the AGR1 and AUX1 products. These results support a role for protein phosphorylation in regulating polar auxin transport in seedling roots.
55
Light
Histone kinase activity in Arabidopsis is induced by high irradiance levels during de-etiolation (Daniel Chamovitz, Tel Aviv University, Israel). Genetic analyses show this kinase is downstream of phytochrome and cryptochrome photoreceptors and COP1. In addition, a kinase that phosphorylates the COP9 signalosome has been partially purified. Light also stimulates transcription in chloroplasts. Gerhard Link (University of Bochum, Germany) showed that a subunit of chloroplast RNA polymerase is a CK2type kinase that connects photosynthetic activity to chloroplast gene expression. Development
Hormones are central players in plant development. John Walker (University of Missouri, Columbia, MS, USA) discussed mechanisms underlying brassinosteroid (BR) signaling. BRI1 encodes a LRR receptor-like kinase and there is evidence that BRI1 is the receptor for brassinolide (BL). An activation-tagging screen for revertants of the weak bri1-5 allele identified BRS1, encoding a serine carboxy peptidase, several homologs of BRI1, and a homolog of carrot SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (AtSERK1), implicating AtSERK1 in BR signaling. Ykä Helariutta (University of Helsinki, Finland) talked about the cytokinin receptor WOODEN LEG (also known as CRE1 or AHK4), a two component molecule required for asymmetric cell divisions during vascular development in roots. He discussed suppressor screens that might identify downstream components of this cytokinin-mediated pathway. Conclusions
In this short summary, we could only detail a few highlights of the conference. It was exciting to hear so much progress. And it made us long for a functional genomics program to unravel the complexity of phospho-relay signaling. John Mundy Molecular Biology Institute, University of Copenhagen, Ø. Farimagsgade 2A, 1353-K Copenhagen, Denmark. e-mail: [email protected] Kay Schneitz Institute of Plant Biology, University of Zürich, Zollikerstr. 107, 8008 Zürich, Switzerland.