Abstract
Signal transduction allows cells to respond to signals from their environment and is therefore important for most biological processes. The binding of an extracellular signalling molecule to a cell-surface receptor is the first step in most signal transduction pathways. Cell-surface receptors transduce the extracellular signals by generating a cascade of
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intracellular signals that alter the behaviour of the cell. The proteins involved in signal transduction include, besides the receptors, GTP-binding proteins, protein kinases and ion channels, and these proteins are conserved between multicellular organisms. In this study, we used the nematode Caenorhabditis elegans, a simple and well-described organism that is very well suited to perform genetic experiments, to analyse conserved signal transduction pathways.
Chapter two of this thesis describes the genetic analysis of an important signaltransducing molecule, the adenylyl cyclase SGS-1. Adenylyl cyclases act downstream of certain heterotrimeric G proteins and convert ATP into the second messenger cAMP. In C. elegans, SGS-1 can be activated by the homologue of mammalian Gas, GSA-1, since mutations in sgs-1 suppress the neuronal degeneration induced by expression of constitutively active GSA-1 from its own or heat-shock promoter. We show that SGS-1 is essential for viability and that it is involved in behaviours as diverse as pharyngeal pumping and locomotion. To regulate this latter behaviour SGS-1 needs to be activated by GSA-1.
In chapter three, nxf-1, another suppressor of the neuronal degeneration induced by expression of constitutively active GSA-1 from a heat-shock promoter, is characterized. nxf-1 encodes a nuclear export factor that is involved in transport of mRNA out of the nucleus. Mutations in nxf-1 also suppress other heat-shock promoter-induced phenotypes, indicating that this locus is not a specific downstream target of Gas, but rather a general suppressor of heat-shock promoter-induced phenotypes. We postulate that the mutations in nxf-1 make the protein inactive during heat-shock, resulting in reduced transport of the activated Gas and other mRNAs out of the nucleus and thus the absence of the activated Gas phenotype after heat-shock. Chapter four reports the analysis of the C. elegans homologue of Ga12/13, GPA- 12. Loss of GPA-12 does not result in any obvious defect in development or behaviour. However, overexpression of constitutively active GPA-12 from its own or heat-shock promoter results in a developmental growth arrest that is caused by a feeding defect. Mutations in tpa-1, which encodes two PKC isoforms, suppress the developmental growth arrest induced by activated GPA-12, indicating that TPA-1 acts downstream of GPA-12. Activation of TPA-1 by the tumour-promoting phorbol ester TPA (or PMA) results in an identical developmental growth arrest, showing that activated GPA-12 and PMA use the same PKC signalling pathway.
In chapter five of this thesis, the DYRK family of protein kinases is described. A human DYRK kinase is implicated in the cognitive defects caused by trisomy of chromosome 21, and we show that in Caenorhabditis elegans overexpression of a DYRK family member, mbk-1, results in signal transduction defects in olfactory neurons. mbk-1 knockout animals do not show any obvious defects. Loss of hpk-1, another member of the DYRK family in C. elegans, does also not result in any obvious phenotype. However, a third member, mbk-2, is essential for viability.
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