Anthrax toxin triggers the activation of src-like kinases to mediate its own uptake

Abstract

AB-type toxins, like other bacterial toxins, are notably opportunistic molecules. They rely on target cell receptors to reach the appropriate location within the target cell where translocation of their enzymatic subunits occurs. The anthrax toxin, however, times its own uptake, suggesting that toxin binding triggers specific signaling events. Here we show that the anthrax toxin triggers tyrosine phosphorylation of its own receptors, capillary morphogenesis gene 2 and tumor endothelial marker 8, which are not endowed with intrinsic kinase activity. This is required for efficient toxin uptake because endocytosis of the mutant receptor lacking the cytoplasmic tyrosine residues is strongly delayed. Phosphorylation of the receptors was dependent on src-like kinases, which where activated upon toxin binding. Importantly, src-dependent phosphorylation of the receptor was required for its subsequent ubiquitination, which in turn was required for clathrin-mediated endocytosis. Consistently, we found that uptake of the anthrax toxin and processing of the lethal factor substrate MEK1 are inhibited by silencing of src and fyn, as well as in src and fyn knockout cells.

Fig. 1.

Anthrax toxin triggers tyrosine phosphorylation events. HeLa cells were treated with 500 ng/mL of PA63 for 1 h at 4 °C and incubated for the indicated time at 37 °C. (A) Cell extracts (40 μg of proteins) were analyzed by SDS/PAGE and Western blotting to reveal phospho-tyrosine of total proteins. (B) Immunoprecipitates against clathrin heavy chain (CHC) were analyzed by SDS/PAGE and Western blotting against phospho-tyrosine proteins and CHC, and corresponding total cell extracts were blotted against PA. The plot represents the mean of three independent experiments. Error bars represent standard deviations.

Fig. 2.

Genistein treatment delays anthrax toxin endocytosis. HeLa cells were incubated for 2 h at 37 °C or not with genistein, then treated in the presence or absence of genistein with 500 ng/mL of PA63 and 50 ng/mL LF for 1 h at 4 °C, and incubated for different times at 37 °C. Cell extracts (40 μg of proteins) were analyzed by SDS/PAGE and Western blotting to reveal LF and the N-terminal part of MEK1 [MEK1(N)], PA63, and SDS-resistant intracellular heptamer. To measure total cellular heptamers, extracts (40 μg of proteins) were submitted for 10 min at room temperature to an acid treatment, to convert SDS-sensitive PA^7mer to an SDS-resistant form detectable by SDS/PAGE and Western blotting. (B) HeLa cells were treated for 2 h at 37 °C or not with genistein. Cells were then treated in the presence or absence of genistein with 1 μg/mL of PA63 for 1 h at 4 °C (red) and incubated for 40 min at 37 °C (blue). After three washes at 4 °C with cold PBS, cells were trypsinized at 4 °C and stained for 30 min on ice with anti-PA antibodies, followed by staining for 30 min on ice with secondary fluorescent antibodies, washing in PBS + 1% FCS, and then evaluation on a FACSCalibur.

Fig. 3.

Tyrosine phosphorylation of CMG2 promotes endocytosis of the anthrax toxin. (A) HeLa cells were transfected for 48 h with CMG2-HA, treated or not (Ctrl) with 1 μg/mL of PA83 for 1 h at 4 °C, and incubated for the indicated time at 37 °C. Immunoprecipitates against HA were analyzed by SDS/PAGE and Western blotting against phospho-tyrosine proteins, CMG2-HA, and PA. (B) HeLa cells were transfected 48 h with CMG2-WT-HA or CMG2^∆Y-HA mutated on four tyrosines: Y380A, Y381A, Y445A, and Y463A. Cells were treated (+) or not (−) with 1 μg/mL of PA83 for 1 h at 4 °C and for 20 min at 37 °C. Immunoprecipitates against HA were analyzed by SDS/PAGE and Western blotting against phospho-tyrosine proteins, CMG2-HA, and PA. (C) CHO^ΔATR cells were transfected for 48 h with CMG2-WT-HA or CMG2^∆Y-HA mutated on four tyrosines: Y380A, Y381A, Y445A, and Y463A. Cells were then treated with 500 g/mL of PA83 and 50 ng/mL of LF for 1 h at 4 °C and an indicated time at 37 °C. Cell extracts (40 μg of proteins) were analyzed by SDS/PAGE and Western blotting to reveal different forms of PA: PA83, PA63, and the intracellular heptamer, SDS-resistant, N-terminal part of MEK1 and CMG2-HA. Total cellular PA^7mer was visualized as in Fig. 2A through acid treatment of cell extracts. (D) MEK1 N-terminal part levels were quantified using the Typhoon scanner and normalized to 100% at time 0 (1 h at 4 °C). The plot represents the mean of three independent experiments described in C. Errors represent standard deviations.

Fig. 4.

PA triggers activation of src-like kinases. (A) HeLa cells were treated with 500 ng/mL of PA63 for 1 h at 4 °C and incubated for the indicated time at 37 °C. Cell extracts (40 μg of proteins) were analyzed by SDS/PAGE and Western blotting to reveal phospho-tyrosine src-like kinases (Tyr-416), PA, src, yes, and fyn. (B) HeLa cells were transfected for 72 h with siRNAs against src, yes, or fyn. The efficiency of siRNas was analyzed on cell extracts (40 μg of proteins) by SDS/PAGE and Western blotting. Cells were incubated with 500 ng/mL PA63 and 50 ng/mL LF for 1 h at 4 °C and for different times at 37 °C, and cell extracts (40 μg of proteins) were analyzed by SDS/PAGE and Western blotting to reveal a PA SDS-resistant heptamer and N-terminal part of MEK1 [MEK1(N)].

Fig. 5.

Endocytosis of the anthrax toxin is inhibited in SYF cells. (A) SYF–MEFs, deficient for src, yes, and fyn, were treated with 500 ng/mL of PA63 for 1 h at 4 °C and with 50 ng/mL LF for 1 h at 4 °C and for different times at 37 °C. Cell extracts (40 μg of proteins) were analyzed by SDS/PAGE and Western blotting to reveal PA SDS-resistant heptamer (intracellular), N-terminal part of MEK1, and tubulin as equal loading. Extracts (40 μg of proteins) previously described were treated for 10 min at room temperature with acid buffer and analyzed by SDS/PAGE and Western blotting to reveal all PA heptamers (at the surface and intracellular). (B) MEF cells or SYF–MEF cells were treated with 1 μg/mL of PA63 for 1 h at 4 °C (red) and incubated for 20 min at 37 °C (blue). After three washes at 4 °C with cold PBS, cells were trypsinized at 4 °C and stained for 30 min on ice with anti-PA antibodies, followed by staining for 30 min on ice with secondary fluorescent antibodies, washing in PBS + 1% FCS, and then evaluation on a FACSCalibur. (C) SYF–MEF cells were transfected or not for 48 h with src–GFP, fyn–GFP, or yes–GFP, and treated with 500 ng/mL of PA63 for 1 h at 4 °C and for different times at 37 °C. Cell extracts (40 μg of proteins) were analyzed by SDS/PAGE and Western blotting to reveal a PA SDS-resistant heptamer, PA63, N-terminal part of MEK1 [MEK1(N)], and kinase–GFP.

Fig. 6.

Phosphorylationof CMG2 is required for its ubiquitination. HeLa cells were transfected for 48 h with CMG2-WT-HA or CMG2^∆Y-HA mutated on four tyrosines: Y380A, Y381A, Y445A, and Y463A. Cells were then treated with 500 g/mL of PA83 for indicated times at 37 °C. Immunoprecipitations were performed against HA, and the samples were analyzed by Western blotting both against the HA-tagged receptor and against ubiquitin.