The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family.
Exoenzyme S (ExoS), which has been implicated as a virulence factor of Pseudomonas aeruginosa, catalyzes transfer of the ADP-ribose moiety of NAD+ to many eukaryotic cellular proteins. Its preferred substrates include Ras and several other 21- to 25-kDa GTP-binding proteins. ExoS absolutely requires a ubiquitous eukaryotic protein factor, termed FAS (factor activating ExoS), for enzymatic activity. Here we describe the cloning and expression of a gene encoding FAS from a bovine brain cDNA library and demonstrate that purified recombinant FAS produced in Escherichia coli activates ExoS in a defined cell-free system. The deduced amino acid sequence of FAS shows that the protein (245 residues, calculated molecular mass 27,743 Da) belongs to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins. Various functions have been reported for members of the 14-3-3 family, including phospholipase A2 activity and regulation of tyrosine hydroxylase, tryptophan hydroxylase, and, possibly, protein kinase C activities. Identification of FAS as a 14-3-3 protein establishes an additional function for this family of proteins--the activation of an exogenous ADP-ribosyltransferase. Elucidation of the precise role of FAS in activating ExoS will contribute to understanding the molecular mechanisms by which P. aeruginosa causes disease.
Fu
H
H
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
Coburn
J
J
Collier
R J
RJ
D048948:14-3-3 Proteins; D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010802:Phylogeny; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D001427:Bacterial Toxins; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D014446:Tyrosine 3-Monooxygenase; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S