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Doctor of Philosophy




Percival-Smith, Anthony


During formation of the anterior-posterior axis, Homeotic selector (HOX) proteins determine the identity of Drosophila body segments. HOX proteins are transcription factors that regulate gene expression during development. Besides a highly conserved DNA-binding homeodomain (HD), HOX proteins also contain functionally important, evolutionarily conserved small motifs. These short motifs found in HOX proteins may be Short Linear Motifs (SLiMs). SLiMs are proposed to be sites of phosphorylation and this may regulate the activity of HOX proteins. The primary aim of this work was to develop a comprehensive catalogue of the sites of phosphorylation and other post-translational modifications (PTMs) for Fushi tarazu (FTZ) and 8 HOX proteins extracted from developing Drosophila melanogaster embryos. Drosophila were transformed with constructs that express FTZ or HOX proteins fused to a triple tag (TT) from a heat-shock promoter. The HOXTT proteins are biologically active during embryogenesis. Triple tagged Sex combs reduced (SCRTT) protein was extracted from developing embryos and purified using Ni-NTA beads under denaturing conditions. Multiple sites of PTMs were identified in purified SCRTT by tandem mass spectrometry (MS/MS). The identified PTMs include phosphorylation at S185, S201 and T324, acetylation at K218, K434 and K439, formylation at K218, K309, K325, K369, K434 and K439, methylation at S19, S166, K168 and T364, carboxylation at W307, K309 and E323 and hydroxylation at P22, P107, D108, D111, P269 and P306. In testing the hypothesis that HOX SLiMs are preferential sites of phosphorylation, I found that the distribution of phosphorylatable residues, S, T and Y was biased to SLiMs, but there was no support for the hypothesis that SLiMs are preferentially phosphorylated.

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