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Title: The molecular function of the Drosophila PAR-1 kinase in establishing cell polarity
Author: Benton, R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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Through a structure-function analysis, I demonstrated the requirement of kinase activity for Drosophila PAR-1 function. Mammalian PAR-1 homologues, the MARKs, phosphorylate microtubule-associated proteins of the tau family to inhibit their interaction with microtubules. This biochemical activity is consistent with the microtubule defects observed in par-1 mutant oocytes, and prompted me to clone, characterise and generate protein null mutants in the sole Drosophila tau homologue. Remarkably, tau mutants are viable and fertile, indicating that this conserved cytoskeletal regulator is dispensable for Drosophila development. As Tau is unlikely to be a relevant germline substrate for PAR-1, this kinase must therefore function by a distinct mechanism to that of the MARKs. To define this mechanism, I isolated PAR-1 interactors by yeast tow-hybrid screening, and recovered the Drosophila 14-3-3 homologues, 14-3-3e and leonardo (leo). 14-3-3 proteins interact with diverse targets in a phosphorylation-dependent manner to modulate their activity or localisation. The PAR-1 kinase domain binds to a region of 14-3-3 distinct from the phosphoepitope-recognition pocket and can phosphorylate a 14-3-3-binding site in Raf. PAR-1 may therefore promote 14-3-3/target interactions by generating the phosphoepitope essential for 14-3-3 binding and by directly delivering 14-3-3 to these targets. I show that mutations in 14-3-3e and leo produce phenotypes identical to par-1 mutants in both oocyte determination and A-P axis formation, indicating that 14-3-3 mediates PAR-1 function in vivo. This parallels the requirement for the C. elegans 14-3-3 protein, PAR-5, in A-P polarisation, and predicts that the binding of 14-3-3/PAR-5 to PAR-1 substrates is a conserved mechanism by which these kinases generate polarity. In computational and biochemical screens for Drosophila 14-3-3 targets, I identified the PAR-3 homologue Bazooka (BAZ), and show that PAR-1 phosphorylates BAZ on its two 14-3-3-binding sites. In epithelial cells, BAZ and PAR-1 localise to complementary apical and lateral membrane domains. I show that PAR-1 and 14-3-3 are required to polarise the follicular epithelium, and to restrict BAZ apically.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available