Characterisation of CCaMK in symbiosis signalling

Harrison, Joanna (2015) Characterisation of CCaMK in symbiosis signalling. Doctoral thesis, University of East Anglia.

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Many plants form important and beneficial symbioses with microbes including arbuscular mycorrhization and the legume-rhizobial interaction. The establishment of both these symbioses involves common genes which encode the Sym pathway proteins, of which calcium- and calmodulin-dependent protein kinase (CCaMK) is a central component. CCaMK has been genetically positioned downstream of a nuclear calcium spiking signal and is widely thought to detect and transduce this signal. In the presence of calcium CCaMK undergoes a conformational change, but the nature of this was previously unknown. Here I show that this change likely corresponds to an elongation of the visinin-like domain and this exposes a hydrophobic patch in the full-length protein. This hydrophobic patch could facilitate the binding of CCaMK to target proteins. Calcium-binding to the visinin-like domain stimulates autophosphorylation, in particular at T271 (in Medicago truncatula CCaMK). However, the molecular mechanism by which this occurs was not well-established. By contrast, the mechanism of autophosphorylation of CaMKII, a similar animal protein, has been determined to be intra-oligomeric and inter-subunit. Work presented here shows for the first time that MBP-CCaMK forms an oligomer of 16-18 subunits when purified from E. coli and experimental evidence is most consistent with an intra-oligomeric, inter-subunit mechanism of autophosphorylation. This is expected to facilitate the stabilisation of a robust “off-state” of the protein. Finally, CCaMK was assessed in planta. Preliminary data suggested the presence of potential alternative splice forms of CCaMK, and I undertook an investigation to determine if these were present during symbiosis at the RNA and protein level. Very low levels of splice variants were detected at the RNA level only, suggesting that they do not play a key role during symbiosis. A multi-faceted study is therefore presented, and this provides deeper insights into the function of CCaMK in the Sym pathway.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Nicola Veasy
Date Deposited: 03 May 2016 13:42
Last Modified: 03 May 2016 13:42

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