Alrubaie, Saif Aldean; (2002) Analysis of the Drosophila insulin pathway using phosphorylation state-specific antibodies to Drosophila Akt; identification and characterisation of an apoptosis-associated signal. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Studies of components of the insulin/class IA phosphatidylinositol 3-kinase (PI3K) pathway in Drosophila, and more recently in mammals, have revealed a major function in the intrinsic control of cell size and organ growth during development. However, growth and cell size can be regulated through other signalling mechanisms such as those involving the small GTPase, Ras, and the transcription factor, Myc. The possibility that these systems are part of a common growth-regulating signalling network was investigated using an immunofluorescence-based assay to monitor insulin/PI3K pathway activity in vivo. This assay was based on the use of phosphorylation state-specific antibodies to detect phosphorylation and, hence, activation of a major transducer of this pathway, Drosophila Akt (dAkt). The phosphorylation and activation of dAkt by insulin was confirmed biochemically using Drosophila Schneider cells. The control of dAkt phosphorylation in vivo by Drosophila PI3K was analysed during development by in situ immunofluorescence of imaginal discs. The phosphorylation of dAkt was then analysed in clones of cells that lacked Drosophila Ras1 (dRas1) or ectopically expressed constitutively active dRas1 (dRasv12) or Drosophila Myc (dMyc). Significantly, dAkt phosphorylation was increased in dRas1v12 clones, but was not altered in dRas1-null clones. However, the analysis was complicated by the presence of an intense 'pyknotic' cell staining pattern revealed by immunostaining with one of the phospho-specific dAkt antibodies. This unexpected and intriguing observation was characterised and found to originate from cells undergoing apoptosis. Experiments in imaginal discs and in Schneider cells demonstrated that the pyknotic signal and the induction of apoptosis were accompanied by the appearance of an 80kDa signal on phospho-dAkt western blots that co-migrated with the 80kDa isoform of dAkt. Significantly, the appearance of this signal was caspase-dependent. However, further experiments ruled out the possibility that the 80kDa protein was dAkt.

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