A synaptojanin-homologous region of Salmonella typhimurium SigD is essential for inositol phosphatase activity and Akt activation.
The Ser-Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium. The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. Here, we investigated the inositol phospholipid substrate preferences of SigD. Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5-biphosphate and phosphatidylinositol 3,4,5-triphosphate over other phosphatidylinositol lipids. Phosphatidylinositol 3-phosphate was not a substrate, suggesting the 5' phosphate moiety is one of the preferred substrates. Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5-phosphatase synaptojanin. Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively. The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a DeltasigD Salmonella strain. A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt. Thus, we have characterized a region near the carboxyl-terminus of SigD which is important for phosphatase activity. We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.
Marcus
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Biotechnology Laboratory, University of British Columbia, Wesbrook Building 237, 6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3.
Wenk
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Steele-Mortimer
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D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics; D004789:Enzyme Activation; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010716:Phosphatidylinositols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D012763:Shigella flexneri / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009419:Nerve Tissue Proteins; D010716:Phosphatidylinositols; D011518:Proto-Oncogene Proteins; C060972:SopB protein, Bacteria; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C425774:IpgD protein, Shigella flexneri; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; C489224:SopB protein, Salmonella; D008239:Lysine