Identification and characterisation of MAP4K3 as a novel cell death regulator

To identify novel regulators of apoptosis, a vector based RNAi library screen was performed with UV irradiation used to induce apoptosis. The screen revealed that a shRNA targeting MAP4K3 was the most highly expressed following UV treatment and as such this candidate cell death regulator was selected for further experimentation. MAP4K3 is a Ste20 kinase protein that is activated by UV irradiation and from overexpression experiments it was shown to induce JNK activation.;To confirm the screen results, siRNA experiments revealed that suppression of MAP4K3 conferred increased cell survival following UV and cisplatin induced apoptosis. Ectopic expression showed that MAP4K3 can induce apoptosis with the kinase activity of MAP4K3 required for the maximal induction. From deletion mutants I have determined that the N-terminus kinase domain is sufficient to induce apoptosis. The MAP4K3 induced apoptosis was found to be caspase dependent and induced a conformational change of Bax with the co-overexpression of Bclxl rescuing the cell death phenotype. This suggests that MAP4K3 could activate the intrinsic pathway of apoptosis.;Ectopic expression of MAP4K3 was used to investigate the involvement of signalling transduction pathways and revealed an increase in phosphorylation of JNK and p38 kinases. The MAP4K3DeltaC mutant revealed that the kinase domain induces a phosphorylation cascade resulting in the increase in phosphorylation of the transcription factors, Stat-1 and c-Jun. The regulation of MAP4K3 was investigated and experiments suggest that the 26S proteasome degrades MAP4K3 as a proteasome inhibitor stabilises MAP4K3 expression levels. This degradation could be ubiquitin mediated as co-overexpression studies with HA-ubiquitin revealed that MAP4K3 co-immunoprecipitates with the HA ubiquitin.