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Title: The role of the pak1 protein kinase in fission yeast cell polarity
Author: Davis, Hannah E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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p21-activated kinase (paklp) is essential in fission yeast and plays roles in cell polarity and mating. The pakl-34 mutant has a specific mutation that does not affect essential functions but causes highly penetrant defects in cell polarity and morphology. The pakl-34 strain has a specific defect in bipolar growth and the potential to help dissect the role of paklp in mediating cell polarity. Tagging experiments demonstrated that both wild type and pakl-34 proteins localize to cell tips and septa. I hypothesised that pak-34p may be deficient in kinase activity. I adapted a two-dimensional gel electophoresis approach, called Difference Gel Electrophoresis (DIGE), to screen for paklp substrates. Wild type and pak1 mutant strains were compared in this manner and differential proteins identified by mass spectrometry. General results showed very few differences between wild type and pakl-34 strains and huge differences between wild type and pak1 kinase-dead strains, indicating that the kinase-dead strain may not be suitable for dissecting the paklp mechanism. Specific results identified hxklp as a potential substrate but it was generally concluded that the DIGE approach may not have sufficient sensitivity and/or scope for the screening of paklp substrates. In parallel to the hypothesis-driven DIGE approach I attempted to find paklp substrates by a candidate approach, investigating the phosphorylation state of a paklp regulator, ral3p. I found differences in ral3p phosphorylation, between wild type and pak1mutants, by SDSĀ­-polyacrylamide gel electrophoresis and lambda phosphatase treatment. I also looked for differences in the sizes of paklp and pakl-34p complexes using sucrose gradients. This study (1) describes a new way to screen for novel protein kinase substrates in fission yeast and (2) suggests that hxklp and ral3p are substrates for paklp.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available