Ferrari, Giovanna Maria; (1999) The interaction of the alpha-2 chimaerin SH2 domain with target proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Rac is a member of the Rho subfamily of low molecular weight GTPases (p21s) and is involved in diverse cellular processes. GTPase-activating proteins (GAPs) regulate p21 activity by increasing intrinsic GTPase activity. The chimaerins are a family of p21-Rac GAPS with distinct patterns of tissue and developmental distribution, α2 chimaerin contains an amino-terminal SH2 domain and is selectively expressed within the nervous system. SH2 domains bind specific phosphotyrosine-containing sequences and the presence of this domain may place α2 chimaerin within tyrosine kinase signalling pathways. Comparisons between SH2 domains suggest that the mechanism of target interaction of the chimaerin SH2 domain may be distinct from that of others. Affinity chromatography was used to detect potential α2 chimaerin SH2 domain target proteins in rat brain extracts; some of these proteins were tyrosine-phosphorylated. Tubulin and actin were isolated as targets and peptide sequence information was obtained for three other potential target proteins, two of which appeared to be novel sequences. Several different kinase activities bound α2 chimaerin SH2 domain affinity columns; one of these phosphorylated full length α2 chimaerin. Full length α2 chimaerin and its isolated SH2 domain bound a phosphotyrosine column. Amino acid residue substitutions were made in the α2 chimaerin SH2 domain at sites essential for function in other SH2 domains; certain point mutations affected phosphotyrosine-binding. α2 Chimaerin probes bound two previously identified putative α2 chimaerin target proteins of molecular mass 13kDa and 64kDa; these interactions were phosphotyrosine-independent. The interactions of the 13kDa and 64kDa proteins with α2 chimaerin differed in their sensitivity to point mutation of the chimaerin SH2 domain. Specific antibodies have been raised to these proteins to facilitate further studies. Results suggest that substrates of the α2 chimaerin SH2 domain may include both tyrosine-phosphorylated and non-tyrosine phosphorylated proteins.

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