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- Hunt, L, et al.
(author)
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Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms
- 2004
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In: New Phytologist. - : Wiley. - 1469-8137 .- 0028-646X. ; 162:3, s. 643-654
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Journal article (peer-reviewed)abstract
- PI-PLCs synthesise the calcium releasing second messenger IP3. We investigated the expression patterns of the Arabidopsis PI-PLC gene family and measured in vitro activity of encoded enzymes. Gene specific RT-PCR and promoter-GUS fusions were used to analyse AtPLC gene expression patterns. The five available AtPLC cDNAs were expressed as fusion proteins in Escherichia coli. All members of the AtPLC gene family were expressed in multiple organs of the plant. AtPLC1, and AtPLC5 expression was localized to the vascular cells of roots and leaves with AtPLC5::GUS also detected in the guard cells. AtPLC4::GUS was detected in pollen and cells of the stigma surface. In seedlings, AtPLC2 and AtPLC3 were constitutively expressed, while AtPLCs 1, 4 and 5 were induced by abiotic stresses. AtPLC1-5 were all shown to have phospholipase C activity in the presence of calcium ions. AtPLCs showed limited tissue specific expression and expression of at least three genes was increased by abiotic stress. The differing calcium sensitivities of recombinant AtPLC protein activities may provide a mechanism for generating calcium signatures.
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| 2. |
- Otterhag, L, et al.
(author)
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Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase
- 2006
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In: Biochimie. - : Elsevier BV. - 1638-6183 .- 0300-9084. ; 88:1, s. 11-21
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Journal article (peer-reviewed)abstract
- The A rabidopsis 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 tar et 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. (C) 2005 Elsevier SAS. All rights reserved.
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