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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Record number 109477
Title Characterization of the Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 protein
Author(s) Shah, K.
Source Wageningen University. Promotor(en): S.C. de Vries. - S.l. : S.n. - ISBN 9789058084279 - 162
Department(s) Laboratory of Molecular Biology
Publication type Dissertation, internally prepared
Publication year 2001
Keyword(s) arabidopsis thaliana - somatische embryogenese - kinasen - genexpressie - signaaltransductie - arabidopsis thaliana - somatic embryogenesis - kinases - gene expression - signal transduction
Categories Genetics (General) / Nucleic Acids / Plant Development

Transmembrane receptors are prime components of cellular signalling pathways and thus help determine cell fate, growth, differentiation, migration and death. Somatic embryogenesis is the process where by somatic cells develop into plants via the same morphological stages. The Somatic Embryogenesis Receptor Kinase(SERK) gene was isolated from Daucus carota somatic cells as a marker to monitor the transition of somatic into embryogenic cells. The Arabidopsis thalianaSomatic Embryogenesis Receptor Kinase 1 (AtSERK1) gene is the most closely related Arabidopsis SERK gene and is expressed in developing ovules, early embryos and in the vascular tissues of seedlings and adult plants. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper (LZ) motif followed by five leucine-rich repeats (LRRs), a proline-rich region, a single transmembrane region and an intracellular kinase domain. The major goal of the study presented in this thesis is the biochemical characterization of the AtSERK1 protein in order to reveal some of the early steps in the signal transduction cascade mediated by AtSERK1.

The experiments to reveal the biochemical properties were performed employing the comparative modelleing of the AtSERK1 kinase domain using the insulin receptor kinase domain as a template and the phosphorylation assays of affinity purified AtSERK1 and AtSERK1 mutant proteins produced in vitro . Based on the computer modeling studies, threonine residues in the AtSERK1 activation loop of catalytic sub-domain VIII were postulated to be targets for autophosphorylation activity. Single amino acid mutations replacing threonine residues in the AtSERK1 A-loop reveal the importance of Thr-468 in both AtSERK1 auto- and transphosphorylation. The ability of the AtSERK1 protein to transphosphorylate a catalytically inactive AtSERK1 protein shows that AtSERK1 catalyzes an intermolecular mechanism of autophosphorylation. Based on these observations, similar to the mechanism employed by animal receptors, the presence of a minor population of catalytically active AtSERK1 molecules in vitro , or ligand induced conformational changes in vivo , the activation of AtSERK1 requires intermolecular autophosphorylation. Activation of the receptor may require the movement of the AtSERK1 threonine containing A-loop followed by release of transphosphorylation activity and protein substrate binding. In this model, phosphorylation of Thr-468 in the A-loop of one AtSERK1 monomer is also essential for releasing catalytic activity of that same monomer.

The experiments to reveal the sub-cellular localization and dimerization of the AtSERK1 protein were performed using transient transfections in cowpea mesophyll protoplasts. In addition, the potential for dimerization of the AtSERK1 protein was determined by fluorescence resonance energy transfer (FRET) between GFP-derivative-AtSERK1 fusion in plant cells and in the yeast two hybrid based protein interaction system. The AtSERK1 protein is targeted to plasma membranes of plant cells. The extracellular LRRs, and in particular the N-linked oligosaccharides that are present on them are essential for correct localization of the AtSERK1 protein. The AtSERK1 protein exists as a monomer in the membrane. Only a minority of the AtSERK1 receptor molecules on the plasma membrane is in a predimerized state. In the absence of the LZ domain, the small population of predimerized AtSERK1 receptors is not detectable anymore, which shows the importance of the LZ domain in dimerization. While identification of the ligand for AtSERK1 is clearly essential for determining its precise mode of action, it is proposed that AtSERK1 exhibits ligand-induced homodimerization.

To demonstrate the interaction of the AtSERK1 protein with one of the potential downstream proteins, the Arabidopsis kinase associated protein phosphatase (KAPP) in vitro , phosphorylation experiments and transient transfections in cowpea mesophyll protoplasts were performed. It was shown that the kinase domain of AtSERK1 interacts with the kinase interaction domain of KAPP in a phosphorylation-dependant manner. The kinase interaction domain of KAPP does not interact with the catalytically inactive kinase mutant or the mutants lacking the essential threonines in the AtSERK1 A-loop for phosphorylation of AtSERK1 kinase. Using GFP-derivative fusions, we also show that the KAPP and AtSERK1 proteins are colocalized in plant cells at the plasma membrane and in intracellular vesicles, but only interact physically intracellularly. It is proposed that KAPP plays an essential role in internalization and inactivation of the AtSERK1 protein. The studies on the phosphorylation and dephosphorylation of these fusion proteins in live plant cells would therefore give more insight into the internalization mechanisms of the AtSERK1 protein.

The research described in this thesis provides ample information regarding the molecular nature of the AtSERK1 mediated signal transduction. It has formed the basis for in vivo experiments to study receptor dimerization and activation, and subsequent binding of downstream partners of AtSERK1. The identification of ligand(s) for AtSERK1 is now of crucial importance for future studies on AtSERK1 signal transduction.

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