Isotype-specific degradation of Rac activated by the cytotoxic necrotizing factor 1.
The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates members of the Rho family by deamidation of glutamine 61/63. Because this amino acid is crucial for GTP hydrolysis, deamidation of glutamine 61/63 results in constitutively active Rho proteins. Recently, it was shown that the level of CNF1-activated Rac is rapidly diminished in CNF1-treated cells by proteolytic degradation. Here, we studied the requirements for CNF1-induced Rac degradation. By overexpressing His-tagged activated Rac mutants we show that constitutive activation is necessary for degradation of Rac. However, permanent activation is not sufficient for degradation, because Rac that is constitutively activated by transamidation at glutamine 61 by the Bordetella dermonecrotic toxin is not degraded. Overexpression of His-tagged Rac mutants deficient in interaction with GTPase-activating protein (Rac(N92D) and Rac(Y64H)) and guanosine nucleotide dissociation inhibitor (Rac(H103E)) were degraded after activation by CNF1, whereas Rac(Y40C), which is not able to interact with CRIB domain effectors or plenty of SH3, was not degraded. Isoprenylation and the presence of a putative mitotic destruction box are essential for CNF-induced degradation. In contrast to Rac1, Rac2, and Rac3 were not degraded following constitutive activation by CNF1. Using site-directed mutagenesis, we defined the polybasic region and amino acids 90, 107, 147, and 151 as responsible for isotype-specific degradation.
Pop
Marius
M
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Albert-Strasse 25, D-79104 Freiburg, Germany.
Aktories
Klaus
K
Schmidt
Gudula
G
D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D020033:Protein Isoforms; C060296:cytotoxic necrotizing factor type 1; D020744:rac GTP-Binding Proteins