Govind, Sheila; (2000) The isolation and characterisation of protein partners for Cdc42Hs. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Cdc42Hs is a member of the Rho family of GTPases that has a role in fundamental cellular processes such as the regulation of cell morphology, JNK activation, neurite outgrowth, cell polarity and cell transformation. To investigate how Cdc42Hs can control such diverse functions, a yeast two- hybrid screen was performed to identify novel Cdc42Hs-binding partners, which may be involved in mediating pathways underlying these processes. A screen of a human brain cDNA library with constitutively active Cdc42Hs bait resulted in the isolation of 358 putative targets. Seven positive clones (Cdc42Hs-Binding Protein 1-7) were characterised. CBP2, CBP6 and CBP3 were identified to be PAK, aPAK and ACK, respectively. CBP4 and CBP7 exhibit no similarity to known sequences. CBP1 and CBP5 showed identity to the hamster cDNA, encoding the insulin receptor tyrosine kinase p53/58 substrate. Full-length CBP5 cDNA is 83% identical to the hamster IRSp53 gene suggesting CBP5 is the human homologue of the hamster cDNA and was therefore renamed as insulin receptor tyrosine kinase substrate, IRS-58. Northern analysis of IRS-58 mRNA expression identified two transcripts, one of which was ubiquitously expressed, while the other is brain-enriched and possibly brain-specific. IRS-58 encodes four protein-protein interaction motifs; an SH3 domain, an SH3-binding domain, a WW-binding domain and, a novel Cdc42Hs-binding domain that exhibits similarity to, but is distinct from, the conserved p21 binding CRIB motif. Expression of IRS-58 in Swiss 3T3 fibroblasts results in the reorganisation of the actin cytoskeleton including a loss of stress fibres, cell retraction and the formation of F-actin rich clusters. Expression of IRS-58 in N1E-115 neuroblastoma cells induces the formation of thick, highly branched neurites that exhibit numerous projections along the length of the neurite. IRS-58-induced neurite formation is dependent on active Cdc42Hs and Rac as well as direct Cdc42Hs binding. Taken together these data suggest that IRS-58 is a novel effector for Cdc42Hs involved in remodelling of the F-actin cytoskeleton and in inducing morphological differentiation.

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