Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.
The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.
Knodler
Leigh A
LA
Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT 59840, USA. lknodler@niaid.nih.gov
Winfree
Seth
S
Drecktrah
Dan
D
Ireland
Robin
R
Steele-Mortimer
Olivia
O
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D055550:Protein Stability; D012475:Salmonella / Q000472:pathogenicity*; D054875:Ubiquitination; D037521:Virulence Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D037521:Virulence Factors; C489224:SopB protein, Salmonella; D008239:Lysine