Activation of Akt/Protein Kinase B in Epithelial Cells by theSalmonella typhimurium Effector SigD

The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival. Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser473 and Thr308. We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. In HeLa cells, wild typeS. typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr308 and Ser473 and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt. The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival. Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser473 and Thr308. We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. In HeLa cells, wild typeS. typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr308 and Ser473 and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt. protein kinase B phosphatidylinositol 3-kinase phosphatidylinositol 3,4,5-triphosphate Salmonellapathogenicity island 4)P2, phosphatidylinositol 3,4-bisphosphate type III secretion system epidermal growth factor base pair(s) phosphate-buffered saline green fluorescent protein The serine-threonine kinase Akt (also known as PKBα)1 is a key regulator of cellular survival that is activated by a variety of extracellular signals including mitogens (for review see Ref. 1Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1399) Google Scholar). In the current model for Akt activation, Akt is first recruited from the cytoplasm to cellular membranes by interaction of its pleckstrin homology domain with the phosphatidylinositol 3-kinase (PI3-K) lipid products phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 3,4-biphosphate (PI(3,4)P2) (2Marte B.M. Downward J. Trends Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (647) Google Scholar). Akt activity is not stimulated by translocation itself but is dependent upon phosphorylation at two residues (Ser473 and Thr308). Thr308 is phosphorylated by the phosphoinositide-dependent kinase 1, which also contains a pleckstrin homology domain (3Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1048) Google Scholar, 4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar). The mechanism of phosphorylation of Ser473 remains elusive; although phosphoinositide-dependent kinase 1 is a possible candidate, the integrin-linked kinase and most recently autophosphorylation have also been implicated (5Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (947) Google Scholar, 6Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 7Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). The facultative intracellular pathogen Salmonella entericaserovar Typhimurium (S. typhimurium) is an important causative agent of food-borne gastroenteritis in humans. Penetration of the intestinal epithelium and survival within a variety of mammalian cell types is essential for pathogenicity and is dependent on a number of virulence factors. The genes encoding these factors are located within five discrete S almonella pathogenicity islands (SPIs) on the bacterial chromosome (reviewed in Ref. 8Marcus S.L. Brumell J.H. Pfeifer C.G. Finlay B.B. Microbes Infect. 2000; 2: 145-156Crossref PubMed Scopus (303) Google Scholar). Encoded in SPI1 is a type III secretion system (TTSS), which translocates effectors directly into the cytoplasm of host cells and is required for bacterial invasion of nonphagocytic cells (9Galan J.E. Curr. Opin. Microbiol. 1999; 2: 46-50Crossref PubMed Scopus (156) Google Scholar). This remarkable process is instigated by interactions between bacterial effectors, components of eukaryotic signaling pathways, and the actin cytoskeleton, resulting in membrane ruffling on the cell surface and subsequent bacterial internalization (10Fu Y. Galan J.E. Mol. Microbiol. 1998; 27: 359-368Crossref PubMed Scopus (200) Google Scholar, 11Hardt W.D. Chen L.M. Schuebel K.E. Bustelo X.R. Galan J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 12Hersh D. Monack D.M. Smith M.R. Ghori N. Falkow S. Zychlinsky A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2396-2401Crossref PubMed Scopus (601) Google Scholar, 13Kaniga K. Tucker S. Trollinger D. Galan J.E. J. Bacteriol. 1995; 177: 3965-3971Crossref PubMed Scopus (257) Google Scholar). Two GTPases, Cdc42 and Rac-1, which are key regulators of eukaryotic actin dynamics, are directly targeted bySalmonella. For example, the SPI1 effector protein SopE, when microinjected into eukaryotic cells, catalyzes the exchange of GDP for GTP on Cdc42 and Rac-1, thus activating them, to induce membrane ruffling (11Hardt W.D. Chen L.M. Schuebel K.E. Bustelo X.R. Galan J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar). Although Salmonella-induced membrane ruffling is growth factor receptor independent (14Jones B.D. Paterson H.F. Hall A. Falkow S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10390-10394Crossref PubMed Scopus (89) Google Scholar) and invasion is not blocked by inhibitors of PI3-K (15Ireton K. Payrastre B. Chap H. Ogawa W. Sakaue H. Kasuga M. Cossart P. Science. 1996; 274: 780-782Crossref PubMed Scopus (289) Google Scholar), we show that S. typhimuriumrapidly activates Akt in epithelial cells. One translocated bacterial effector protein, SigD, was found to be absolutely required for both phosphorylation and activation of Akt. A single point mutation in a conserved inositol phosphatase catalytic domain of SigD (16Norris F.A. Wilson M.P. Wallis T.S. Galyov E.E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14057-14059Crossref PubMed Scopus (359) Google Scholar) abolished both Akt phosphorylation and activation. SigD was not required forSalmonella-induced membrane translocation of Akt, indicating that in this novel pathway the membrane translocation of Akt does not lead to its activation unless an exogenous bacterial factor is present. Except where indicated chemicals were obtained from Sigma. Cytochalasin D stock, 1 mg/ml in Me2SO, was stored at −20 °C. LY294002 (BioMol) stock, 100 mm in Me2SO, was stored at −80 °C. EGF (Upstate Biotechnology) stock, 100 μg/ml in water, was stored at −20 °C. HeLa cells, S. typhimurium 1344 wild type, and the SB111/pRI203, A1A1, and E12A2 mutants were as described previously (17Garcia-del Portillo F. Pucciarelli M.G. Jefferies W.A. Finlay B.B. J. Cell Sci. 1994; 107: 2005-2020Crossref PubMed Google Scholar, 18Pfeifer C.G. Marcus S.L. Steele-Mortimer O. Knodler L.A. Finlay B.B. Infect. Immun. 1999; 67: 5690-5698Crossref PubMed Google Scholar) To construct the ΔsigD mutant (missing amino acids 34–465), we used the primers D1 (5′-GCG AAT TCT ATC TGT TCA AGC ATG-3′) and D2 (5′-GTC GAC TGA GAG AAT CTG CAT TCC-3′) to amplify by polymerase chain reaction a 514-base pair fragment including the region upstream of sigD and the first 33 codons of the gene. The primers D3 (5′-GTC GAC AAA GAT CGT ACA GGG ATG-3′) and D4 (5′-CTG CAA AGT CAG GAT GTC GTC AGG-3′) were used to amplify a 484-bp fragment including the last 99 codons ofsigD and the downstream region. PCR products were confirmed by sequencing. These two PCR products were fused at a SalI site (underlined in D2 and D3) and ligated into pCRTOPO2.1 (Invitrogen). The resulting deletion cassette was released by digestion with SacI and XbaI, ligated into the corresponding sites of the positive selection suicide vector pRE112 (CmR), and transformed into SY327λpir (19Miller V.L. Mekalanos J.J. J. Bacteriol. 1988; 170: 2575-2583Crossref PubMed Scopus (1711) Google Scholar). The SL1344 sigD deletion mutant (ΔsigD) was then constructed by allelic exchange as described (20Edwards R.A. Keller L.H. Schifferli D.M. Gene (Amst. ). 1998; 207: 149-157Crossref PubMed Scopus (446) Google Scholar). The pMWDE plasmid was constructed by amplification of thesigDE operon starting 46 bp upstream of the ATG start codon (includes the ribosome binding site but not the sigDpromoter) using the primers D5 (5′-TGT GGA TCC TGT TGA ATG TTC CCA C-3′) and D6 (see above) and inserted as aBamHI-EcoRI fragment into pMW119 (AmpR) (Nippon Gene Co., Ltd.). The resulting plasmid, pMWDE, expresses the sigDE open reading frame under the control of the lacZ promoter. The C462S SigD mutant was constructed by site-directed mutagenesis converting TGT (Cys462) to TCG (Ser462) and creating anEcoRI site. Primer SIGD-EV (5′-AAA AGA TAT CTT TCC CAG TGC TTA TG-3′) and primer C462S-Rev (5′-ATT CGA ATT CCA GGC GGG TAC CG-3′) were used to amplify a 780-bp fragment including the Cys462codon. Primer C462S-For (5′-CTG GAA TTC GAA AAG CGG CAA AGA TCG-3′) and primer SIGD-StuI (5′-TCG AGG CCT AAC GCG TCA TAT AAA C-3′) were used to amplify a 300-bp fragment including the Cys462codon and the downstream region. These two fragments were inserted into pBluescript SKII(+) (Stratagene) and ligated at the newEcoRI site. The resulting plasmid contains part of thesigD ORF from the EcoRV site to theStuI site in sigE. The corresponding fragment of pMWDE was removed and replaced with the mutated fragment, producing pMWDE*. Bacteria were grown in Luria-Bertani (LB) broth overnight at 37 °C with shaking. Overnight cultures were subcultured at a dilution of 1:33 in fresh LB and incubated at 37 °C with shaking for an additional 3 h. The culture was centrifuged at 10,000 × g for 2 min at room temperature and then directly resuspended in phosphate-buffered saline (PBS). Invasion was initiated by the addition of these bacteria directly to cultured cells. Cells were then incubated at 37 °C in 5% CO2 for 5–20 min as indicated, and free bacteria were then removed by with In infection the culture was with μg/ml at min to extracellular Invasion were as described previously O. S. Finlay B.B. Cell. Microbiol. 1999; PubMed Scopus Google Scholar). Cell were by of containing directly to the in were for and were by and to were blocked in saline mm containing and 5% were used at dilution and from were used at The system was used to the HeLa cells in were incubated in for 3 h. Bacteria were for or min as Cells were on and 1 of was mm mm mm 1 mm 1 μg/ml mm 1 μg/ml μg/ml and bacteria were removed by × 1 and were incubated with 2 of (Upstate Biotechnology) at °C for 1 with were then and the were incubated for were with with containing and with kinase mm 1 mm 1 mm 1 mm mm 1 mm μg/ml The were then resuspended in of kinase containing (Upstate of in kinase was followed by at °C for were by of followed by in of was by HeLa cells grown on were with Downward J. Curr. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar) using the to the infection with bacteria was as described Cells were then or and incubated for a min was in for min followed by with and in containing and for in was followed by at were for min and the were with were using was using a with a and a cell were obtained in a × were using The activation of Akt in epithelial cells was by its phosphorylation HeLa cells were for 3 and then with S. typhimurium for the cells were to extracellular bacteria and incubated for were with Thr308 or Ser473 phosphorylation of Akt. on both residues was induced within min of the addition of bacteria and at min and In activation of Akt by EGF a and phosphorylation 1 To the Akt phosphorylation was we used the PI3-K LY294002 Cells were for min and the was the of the Ser473 phosphorylation was not in cells that been with and then with S. typhimurium for min Akt Ser473 phosphorylation was by 2 These indicate that Akt phosphorylation by Salmonella is of for Akt phosphorylation in S. HeLa cells. Cells were with S. typhimurium as was as described in Akt phosphorylation is Cells were with for min and then with S. typhimurium for EGF of Akt. Cells were with for min and then for 2 min with EGF bacterial internalization is not required for Akt Cells were with for min and then with S. typhimurium for the SPI1 is required for Akt Cells were with wild type S. typhimurium or the mutant strain for The actin agent D was used to S. typhimurium invasion of epithelial cells B.B. S. Dedhar S. J. Cell Sci. PubMed Google Scholar). of cells for min with 1 μg/ml did not Akt phosphorylation Although the of phosphorylation with that in cells, this is not to the in bacterial these invasion was of that in cells S. Akt phosphorylation in epithelial cells does not bacterial We effector by the SPI1 of S. typhimurium were involved in Akt activation. typhimurium strain with a mutation a encoding an essential of the SPI1 was used J.E. C. P. J. Bacteriol. PubMed Scopus Google Scholar). This strain has been with the of to the interaction of bacteria with HeLa cells (17Garcia-del Portillo F. Pucciarelli M.G. Jefferies W.A. Finlay B.B. J. Cell Sci. 1994; 107: 2005-2020Crossref PubMed Google Scholar). is on the bacterial surface or in this the S. typhimurium strain and bacterial internalization by binding with to Cell. Full Text PDF PubMed Scopus Google Scholar). the the mutant was at the of the wild type the mutant did not induce Akt phosphorylation when were used 2 Akt phosphorylation is dependent on or effectors by the SPI1 To effectors involved in Akt phosphorylation we S. typhimurium These mutants were constructed by the of a cassette into the S. typhimurium chromosome and been in a for increased intracellular C.G. Marcus S.L. Steele-Mortimer O. Knodler L.A. Finlay B.B. Infect. Immun. 1999; 67: 5690-5698Crossref PubMed Google Scholar). One of these A1A1, was to induce Akt phosphorylation in HeLa cells In this the is inserted into a previously described located in the of SigD, is translocated via the SPI1 V.L. J. Bacteriol. 1998; PubMed Google Scholar). The of the is not the E12A2 in which the cassette is inserted in a downstream did induce Akt phosphorylation A A sigD deletion S. was constructed by codons from thesigD open reading the S. typhimurium ΔsigD did not induce Akt phosphorylation 3 the used in this the invasion of the SigD mutants did not from the wild type S. typhimurium of the and ΔsigD mutants with a plasmid encoding the sigD and genes under the control of the lacZ the to Akt in 3 was in the pMWDE plasmid as a SigD that is essential for secretion of the effector V.L. J. Bacteriol. 1998; PubMed Google Scholar). the phosphorylation of Akt, we the activity of Akt was increased by S. HeLa cells were with bacteria and then in which Akt was using a that both phosphorylated and were incubated with a containing the of a known of Akt, and with wild type S. typhimurium for min increased Akt kinase activity by with cells. the infection to min lead to a in Akt activity A and B of These with the phosphorylation shown in of kinase activity was abolished by the PI3-K and was to the of obtained with Akt activity was when HeLa cells were with the SPI1 mutant or with the ΔsigD Complementation of the ΔsigD strain with pMWDE the bacterial of Akt activity These typhimurium activates Akt and that this activation is SigD and the S. and and two inositol and a conserved residue in of these is essential for the activity of mammalian inositol as as in phosphatase activity of (16Norris F.A. Wilson M.P. Wallis T.S. Galyov E.E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14057-14059Crossref PubMed Scopus (359) Google Scholar, F.A. Majerus P.W. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). To this residue is also required for Akt we constructed the mutant (C462S), pMWDE*. this plasmid was used to the ΔsigD Akt activation was and Ser473 Thr308 phosphorylation was induced a to is required for Akt phosphorylation and activation by Akt activation, the of PI3-K with was interactions lead to the activation of PI3-K by with its at the plasma membrane and A. 1997; PubMed Scopus Google Scholar). with that the of was recruited to infection with wild type or ΔsigD This that does this activation is independent of shown that SigD is essential for the phosphorylation and activation of Akt in epithelial cells, we also to the of SigD in the membrane of Akt. The of Akt in epithelial cells was using and Downward J. Curr. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). HeLa cells were with the plasmid and then with S. translocation of Akt in the of both S. type and ΔsigD mutant was within 2 min of bacteria and this point membrane of Salmonella is where the bacteria are in with the host cell B.D. Smith Falkow S. 1993; PubMed Scopus Google Scholar). translocation of Akt is min which is to the induced by or in epithelial cells Downward J. Curr. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). These show that Akt is translocated to membrane ruffles induced and that this translocation is independent of The remarkable of as with host cell signaling is key to survival. the of these interactions is not important for the of bacterial effectors but also on the signaling in eukaryotic cells. One of the regulators of eukaryotic cellular survival is the protooncogene Akt. We have shown that the S. typhimurium SPI1 translocated effector SigD Akt activation. both wild type S. typhimurium and a mutant which does not SigD both the translocation of Akt to sites of Salmonella-induced membrane ruffles and the of PI3-K with bacterial factor be involved in the activation of the pathway upstream of the of Our data indicate that SigD is required for the phosphorylation and activation of Akt has been recruited to the cellular has been that the S. amino is an inositol both and the S. effector two also found in mammalian inositol (16Norris F.A. Wilson M.P. Wallis T.S. Galyov E.E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14057-14059Crossref PubMed Scopus (359) Google Scholar). of a single conserved residue in of these inositol phosphatase activity in (16Norris F.A. Wilson M.P. Wallis T.S. Galyov E.E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. 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