Cann, Nansi Emma; (2000) Characterisation of α-chimaerin isoforms and α2 sh2 domain mutants expressed in neuroblastoma cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Rac is a member of the Rho family of low molecular weight GTPases (p21s) which is involved in diverse processes including regulation of the actin cytoskeleton and transcriptional activation. Chimaerin, a multidomain GTPase activating protein (GAP) downregulates Rac by increasing its intrinsic rate of GTP hydrolysis. Two splice variants of the chimaerin gene differ in tissue and developmental expression patterns and α2-chimaerin contains an N terminal SH2 domain which is absent from αl-chimaerin. The distribution and morphological effects of the chimaerins, α2-chimaerin SH2 domain mutants and potential α2-chimaerin targets in NIE 115 neuroblastoma cells were investigated. The distribution of α1-chimaerin was predominantly cytoskeletal and α2-chimaerin cytosolic. In transiently transfected NIE 115 cells, α1-chimaerin was concentrated in the perinuclear region and its expression induced cell rounding, whilst α2-chimaerin was expressed throughout flattened, neurite bearing cells. A point mutation in the SH2 domain of α2-chimaerin induced an α1-chimaerin-like protein distribution and morphology. The effects of long term chimaerin overexpression on cell morphology and potential protein interactions were also investigated. Overexpression of α2-chimaerin induced an enlarged, flattened morphology and neurite outgrowth in the presence of serum, whilst overexpression of α1-chimaerin induced a rounded morphology with multiple peripheral actin microspikes and inhibited neurite outgrowth. p35, the neuronal cdk5 regulator and also an 130 kDa tyrosine phosphorylated protein were immunoprecipitated with chimaerin from these cell lines. Similarly an 180 kDa tyrosine phosphorylated protein was identified as a potential target of the α2-chimaerin SH2 domain. Investigation into the effects of chimaerin on activation of the transcription factor NFKB demonstrated cell type specific differences in NFKB signalling pathways between HeLa and NIE 115 cells. These results suggest that functional differences in the chimaerin isoforms are specified by the divergent N terminal sequences.

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