INOSITOL PHOSPHOLIPID-BASED SIGNAL TRANSDUCTION IN PLANTS Phosphoinositide-Specific Phospholipase C and 3-Phosphoinositide Dependent Protein Kinase-1 in Arabidopsis thaliana
Sammanfattning: Phosphoinositides (PIs) play major roles in cell signal transduction. Although it is clear that PIs are important components also in plant signalling, little is known about their roles in plant cells, compared to animal cells. This thesis describes work on two PI-dependent enzymes involved in plant intracellular signalling, phosphoinositide-specific phospholipase C (PI-PLC) and 3-phosphoinositide dependent protein kinase-1 (PDK1) in the model plant Arabidopsis. Plant genomes encode numerous PI-PLC isoforms. However, while animal PI-PLCs fall into five distinct subfamilies, all known plant isoforms form one single family. Based on sequence comparison, plant PI-PLCs are closely related to the animal PI-PLCdelta subfamily, but with a domain structure more similar to the PI-PLCzeta subfamily. Plant PI-PLCs contain three well conserved domains, X, Y and C2, and a less conserved N-terminal region. Using sequence-specific antibodies, we have localised a plant PI-PLC, AtPLC2, to the plasma membrane. We have shown that the AtPLC2 N-terminal region, containing an EF-hand domain, is required for enzyme activity, but not for lipid binding. All cloned Arabidopsis PI-PLCs (AtPLC1-5) have an absolute calcium requirement for activity, but show differences in calcium activation profiles. For example, AtPLC2, AtPLC4 and AtPLC5 reach their maximal activity at 1 micromolar, while AtPLC1 and AtPLC3 clearly require higher concentrations of calcium for maximal activity. These differences may allow differential regulation of these enzymes. The complete set of Arabidopsis PI-PLC genes was investigated. All genes are expressed throughout the plant during normal growth conditions, whereas the expression of three genes, AtPLC1, AtPLC4 and AtPLC5 is increased by exposure to stresses, such as cold, drought, and salt. To date, very little is known about PDK1 and its physiological role in plant cells. The plant PDK1 homologue studied, AtPDK1, contains a PH domain, in similarity with animal PDK1. We have shown that AtPDK1 is autophosphorylated, and three phosphorylation sites, Thr-176, Ser-276, and Ser-382, have been identified. Mutational analysis revealed that Thr-167 and Ser-276 are essential for enzyme autophosphorylation and downstream activity. Neither of these sites are conserved in animal PDK1, suggesting different mechanisms of PDK1 regulation between plants and animals. Mutational analysis also revealed that Asp-167 is critical for AtPDK1 autophosphorylation and enzyme activity. Furthermore, we identified a potential AtPDK1 substrate, AtS6k2, a homologue to the S6 kinases, known to be involved in mammalian cell growth and proliferation.
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