Lee, Joel; (1994) Regulation and structure/function relationships of the Rac1 GTPase-activating protein, n-chimaerin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Brain specific n-chimaerin cDNA encodes a protein with two functional regions, an amino-terminal phospholipid-dependent phorbol ester binding domain related to the C1-regulatory sequence of protein kinase C (PKC) and a carboxyl-terminal Rac1 GTPase-activating protein (Rac1 GAP) domain related to the breakpoint cluster region gene product, Bcr. Recombinant n-chimaerin expressed in Escherichia coli as glutathione S-transferase, maltose binding protein and TrpE fusion proteins was purified and used in the structure/function studies. A radiolabelled 65Zn2+-blotting technique was used to demonstrate that zinc bound with high affinity specifically at the amino-terminal region. Phorbol ester binding by n-chimaerin was found to be zinc-dependent. The n-chimaerin carboxyl-terminal region had a higher specific Rac1 GAP activity than the full-length protein. This suggested a regulatory role for the amino-terminal region. Various lipids including second messengers known to modulate PKC and GAP activities for Ras and Rho were tested; phosphatidylserine (PS) and phosphatidic acid (PA) stimulated n-chimaerin while lysophosphatidic acid inhibited. Phorbol esters synergized with PS or PA in stimulating n-chimaerin. In addition, phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, sphingosine as well as the cis-unsaturated fatty acids, arachidonate, oleate and linolenate inhibited n-chimaerin. In contrast, the saturated fatty acids, arachidate and palmitate were significantly less inhibitory to n-chimaerin. Finally, mutational analysis of the Rac1 GAP domain of n-chimaerin allowed the identification of amino acids essential for GAP activity. Mutants which had no detectable GAP activity but retained the ability to bind Rac1 were also obtained and characterized. The data presented suggest that n-chimaerin is a novel functional target for phorbol esters different from PKC which may allow its substrate, Rac1, to sense changes in the levels of lipids including second messengers.

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