Extended Data Fig. 9: Phosphoryl AMPylation of Fgr and Src by LnaB during infection.
From: Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling
a, 293T cells expressing C- terminal FLAG-tagged Fgr or Src were infected with the wild-type Lp02 or ΔlnaB strain of L. pneumophila. After immunoprecipitation with the anti-FLAG antibody, AMPylation of the kinases were analysed by SDS–PAGE and immunoblotting. b, AMPylation of an amoebic Fgr homologue by LnaB. 293T cells were co-transfected with the LnaB variants and the FLAG-tagged Fgr homologue from A. castellanii. AMPylation of the amoebic Fgr homologue was analysed by immunoblotting after anti-FLAG immunoprecipitation. c, AMPylation of endogenous Src by LnaB during infection. 293T cells were infected with the indicated L. pneumophila strains. AMPylation of Src was analysed by SDS–PAGE and western blotting after immunoprecipitation with the anti-Src antibody. d, 293T cells expressing the C-terminal HA-tagged Fgr variants were infected with the wild-type Lp02 stain of L. pneumophila. AMPylation of Fgr variants was analysed by western blotting after immunoprecipitation. e, The phosphorylation of Y416 and AMPylation of Src in 293T cells transfected with the LnaB variants were analysed by western blotting with an antibody specific for pY416 and anti-AMP antibody, respectively. f, Mass spectrometric analyses of the Y416-containing peptide (LIEDNEYTARQGAK) in p-Src and the LnaB-produced ADP-Src. g, MS/MS analysis of the AMPylation site on Src during infection. The fragment ions b7, b8, b10, b13 and y8, y10, y11, y12, y13 have a mass increase of 409 Da, corresponding to the addition of one ADP group, whereas the fragment ions b2 to b5 and y6, y7 do not exhibit this mass shift, confirming LnaB-catalysed AMPylation on pY416 of Src. h, Phosphorylation of endogenous Fgr in infected cells. U937 cells were infected with the indicated L. pneumophila strains at an MOI of 20 for 1 and 2 h. After immunoprecipitation with the anti-Fgr antibody, phosphorylation of Y412 of Fgr was analysed by western blotting with an antibody specific for pY412 (48984S, CST). i, 293T cells expressing the C-terminal HA-tagged Fgr Y523F were infected with the indicated L. pneumophila strains. Phosphorylation of Y412 and AMPylation of Fgr were analysed by western blotting with an anti-pY412 antibody (70926S, CST) and anti-AMP antibody, respectively.
