Lysine11-Linked Polyubiquitination of the AnkB F-Box Effector of Legionella pneumophila

Abstract

The fate of the polyubiquitinated protein is determined by the lysine linkages involved in the polymerization of the ubiquitin monomers, which has seven lysine residues (K(6), K(11), K(27), K(29), K(33), K(48), and K(63)). The translocated AnkB effector of the intravacuolar pathogen Legionella pneumophila is a bona fide F-box protein, which is localized to the cytosolic side of the Legionella-containing vacuole (LCV) and is essential for intravacuolar proliferation within macrophages and amoebae. The F-box domain of AnkB interacts with the host SCF1 E3 ubiquitin ligase that triggers the decoration of the LCV with K(48)-linked polyubiquitinated proteins that are targeted for proteasomal degradation. Here we report that AnkB becomes rapidly polyubiquitinated within the host cell, and this modification is independent of the F-box domain of AnkB, indicating host-mediated polyubiquitination. We show that the AnkB effector interacts specifically with the host E3 ubiquitin ligase Trim21. Mass spectrometry analyses have shown that AnkB is modified by K(11)-linked polyubiquitination, which has no effect on its stability. This work shows the first example of K(11)-linked polyubiquitination of a bacterial effector and its interaction with the host Trim21 ubiquitin ligase.

FIG 1

Ubiquitination of ectopically expressed AnkB. (A) HEK293T cells were transfected with Flag-tagged AnkB from L. pneumophila strain AA100/130b. Cell lysates were immunopurified with anti-Flag agarose and analyzed by immunoblotting with anti-Flag and antiubiquitin antibodies. (B) HEK293T cells were transfected with Flag-tagged AnkB from L. pneumophila strain Paris. Cell lysates were purified with anti-Flag agarose and analyzed by immunoblotting with anti-Flag and antiubiquitin antibodies. (C) HEK293T cells were transfected with AnkB variants, Flag-tagged AnkB⁹L¹⁰P or Flag-tagged AnkBΔF-box. Cell lysates were purified with anti-Flag agarose and analyzed by immunoblotting with anti-Flag antibodies.

FIG 2

AnkB directly interacts with the E3 ubiquitin ligase Trim21. (A) Flag-tagged AnkB or Flag-tagged AnkH was cotransfected with HA-tagged Trim21 in HEK293T cells. Flag-tagged AnkB or Flag-tagged AnkH was coimmunoprecipitated with anti-Flag agarose and subsequently immunoblotted with anti-HA antibodies and anti-Flag antibodies. (B) HA-tagged Trim21 was coimmunoprecipitated by using anti-HA agarose and subsequently immunoblotted with anti-Flag antibodies and anti-HA antibodies.

FIG 3

Ubiquitinated AnkB is not degraded by the proteasome. (A) HEK293T cells were untreated or treated with MG132 for 3 h. Cells were lysed, and the resulting immunoblot was probed with antiubiquitin and antiactin antibodies. (B) HEK293T cells transfected with Flag-tagged AnkB were treated with the protein synthesis inhibitor cycloheximide. To inhibit proteasomal degradation, a subset of cells was pretreated for 2 h with MG132 prior to cycloheximide treatment. At the indicated time points, cells were lysed, and equivalent amounts of protein were subjected to immunoblotting with anti-Flag and antiactin antibodies.

FIG 4

AnkB is ubiquitinated on lysine 67. (A) Flag-tagged AnkB was immunoprecipitated from transfected HEK293T cells and analyzed by SDS-PAGE and Coomassie blue staining. (B) The band at ∼74 kDa (AnkB plus 6 ubiquitin [Ub] moieties) was analyzed by mass spectrometry, and the resulting spectra within the ubiquitinated AnkB peptide (residues 63 to 75) are shown with the b ions (blue lines) and y ions (red lines). The y ions with a +2 charge are shown with the mass/charge ratio calculated to show ubiquitination on lysine 67 of AnkB.

FIG 5

AnkB is polyubiquitinated through lysine¹¹ of ubiquitin. (A) The ∼74-kDa ubiquitinated AnkB band was analyzed by mass spectrometry for ubiquitination of ubiquitin. The resulting spectrum of the modified ubiquitin peptide (residues 7 to 27) is shown, along with the b ions (blue lines) and y ions (red lines) detected. The b ions with a +1 charge and y ions with a +2 charge are shown with the mass/charge ratio calculated to show ubiquitination on lysine¹¹ of ubiquitin. (B) Predicted fragmentation pattern and mass-to-charge ratios of the +1 and +2 charges of the ions within the spectra.