Teo, Mabel; (1994) Purification and functional characterization of brain and recombinant chimaerin, a p21rac GTPase activating protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

n-chimaerin is a GAP for the ras-related p21rac. A 45 kDa brain protein (p45) immune reactive to anti-n-chimaerin polyclonal antibodies had selective p21 rac GAP activity, using an overlay assay. p45-chimaerin was purified 400- fold from rat brain by column chromatography. Tryptic peptides contained sequences identical to that predicted from α2-chimaerin cDNA, a splice variant encoding a divergent N-terminal with a SH2 domain. Thus, p45-chimaerin probably corresponds to SH2-containing (α2) chimaerin. A 35 kDa p21rac GAP (p35) detected in detergent soluble membrane fractions, immunoreactive to chimaerin antiserum was likely to represent n-chimaerin (α1-chimaerin). Diverse GAPs for the rho/rac family were present in the brain; p45-chimaerin was widely distributed in brain regions except cerebellum, and was present in both membrane and cytosolic fractions. Both native and recombinant a2-chimaerin exhibited p21rac GAP activity in solution, which was stimulated by phosphatidylserine with a synergistic effect by phorbol esters. GAP activity of α2-chimaerin was unaffected by its SH2 domain. In contrast to α1-chimaerin, α2-chimaerin bound more phorbol ester in the presence of phosphatidylinositol than of phosphatidylserine. α2-Chimaerin was phosphorylated by PKC and PKA in vitro. Brain proteins interacting with α2-chimaerin were detected using 32P-labelled PKC phosphorylated chimaerin. A 60 kDa protein interacting with the SH2 domain of α2-chimaerin was purified; peptide sequences showed it to be novel. Two peptides had similarity to the consensus sequences of a MAP kinase substrate and a SH2 binding domain. These data suggest that α2-chimaerin plays a physiological role in neuronal signal transduction involving SH2-linked receptor/tyrosine kinase and p21rac signalling pathways.

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