p21-activated kinase (paklp) is essential in fission yeast and plays roles in cell polarity and mating. The <i>pakl-34 </i>mutant has a specific mutation that does not affect essential functions but causes highly penetrant defects in cell polarity and morphology. The <i>pakl-34 </i>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 <i>pak1 </i>mutant strains were compared in this manner and differential proteins identified by mass spectrometry. General results showed very few differences between wild type and <i>pakl-34 </i>strains and huge differences between wild type and <i>pak1 </i>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 <i>pak1</i>mutants, by SDS-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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:649213 |
| Date | January 2005 |
| Creators | Davis, Hannah E. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13592 |
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