Bacterial effector NleL promotes enterohemorrhagic E. coli-induced attaching and effacing lesions by ubiquitylating and inactivating JNK
Fig 2
NleL ubiquitylates human JNK1.
(A) NleL ubiquitylated endogenous JNK during EHEC O157:H7 infection. HEK293T cells were infected with EHEC strains with MOI of 100:1. 2.5 h after infection, cells were washed with PBS and cultured in the fresh DMEM medium for 2 h. Cells were lysed and subjected to IP with anti-JNK antibody and IB with anti-Ub antibody. (B) NleL ubiquitylated JNK1 in vivo. HEK293T cells were co-transfected with plasmids encoding HA-tagged Ub, Flag-tagged JNK1, His6-tagged wild-type NleL or its C753A mutant. Flag-tagged JNK1 was immunoprecipitated with anti-Flag M2 beads in denaturing RIPA buffer, followed by IB analyses with anti-HA antibody. (C) NleL induced mono- and poly-ubiquitylation of JNK1 in vivo. HEK293T cells were co-transfected with plasmids encoding HA-tagged Ub, Flag-tagged JNK1 and His6-tagged NleL. Flag-tagged JNK1 was immunoprecipitated with anti-Flag beads in denaturing RIPA buffer, followed by immunoblotting with anti-Flag antibody. (D) NleL ubiquitylated JNK1 in vitro. Flag-tagged JNK1 was expressed in HEK293T cells and purified with anti-Flag M2 affinity beads, and were subjected to in vitro ubiquitylation reaction mixture with indicated components. After 60 min incubation, the reactions were stopped and subjected to IB analysis with anti-Flag antibody. (E) NleL promoted mono- and poly-ubiquitylation of JNK1 with K29-Ub. HEK293T cells were co-transfected with plasmids encoding HA-tagged Lys 29 only Ub mutant (HA-K29-Ub), Flag-tagged JNK1, His6-tagged wild-type NleL or its C753A mutant. After immunoprecipitation, immunoblotting analyses with indicated antibodies were performed. Usp2cc, the catalytical core of human deubiquitylating enzyme USP2. (F) Mapping the sites for NleL-mediated ubiquitylation of JNK1 through mass spectrometry (MS) analyses. The immunoprecipitated JNK1 from HEK293T cells expressing NleL was subjected to MS analysis to determine the potential sites for NleL-mediated ubiquitylation. (G) A schematic view of the potential ubiquitylation sites of JNK1. The residues K68, K153, K222, and K265 were highlighted in red color to indicate the major ubiquitylation sites. (H) NleL promoted poly-Ub chains on JNK1 at multiple sites. HEK293T cells were transfected with His6-tagged NleL and Flag-tagged wild-type JNK1 or the mutants with Lys-to-Arg substitution at indicated sites. (I) NleL-mediated poly-ubiquitylation of JNK1 was almost abolished by simultaneous Lys-to-Arg substitutions at three major ubiquitylation sites (K153R/ K222R/K265R, 3KR). (J) Mapping the major site(s) for NleL-induced mono-ubiquitylation on JNK1. Flag-tagged wild-type JNK1 or its mutants bearing K-to-R substitution at each potential ubiquitylation site was individually transfected to HEK293T cells with His-NleL and HA-Ub-K29. (K) Lys-to-Arg substitution at Lys 68 (K68R) of JNK1 abolished NleL-induced mono-ubiquitylation of JNK1 in mammalian cells. (L) K68R of JNK1 abolished NleL-mediated JNK1 mono-ubiquitylation in vitro. Flag-tagged wild-type JNK1 or the indicated mutants (K68R and 3KR) were separately expressed in HEK293T cells and purified with anti-Flag M2 affinity beads. Purified JNK1 protein or its mutants was subjected to in vitro ubiquitylation reaction mixture at 37°C for 1h, followed by immunoblotting with indicated antibodies. All blots were representative of at least three independent experiments.