Abstract
Protein kinase C (PKC) isozymes regulate different vesicular trafficking steps in the recycling or degradative pathways. However, a possible role of these kinases in the retrograde pathway from endosomes to the Golgi complex has previously not been investigated. We report here the involvement of a specific PKC isozyme, PKCdelta, in the intracellular transport of the glycolipid-binding Shiga toxin (Stx), which utilizes the retrograde pathway to intoxicate cells. Upon binding to cells, Stx was shown to specifically activate PKCdelta and not PKCalpha. The involvement of PKCdelta and PKCalpha in the retrograde transport of Stx was then monitored biochemically and by immunofluorescence after inhibition or depletion of the isozymes. PKCdelta, but not PKCalpha, was shown to selectively regulate the endosome-to-Golgi transport of StxB. Upon inhibition or knockdown of PKCdelta, StxB molecules colocalized less with giantin and more with EEA1, indicating that the molecules were accumulated in endosomes, unable to reach the Golgi complex. The inhibition of Golgi transport of Stx was reflected by a strong reduction in the toxic effect, demonstrating that transport of Stx to the cytosol is dependent on PKCdelta activity. These results are in agreement with our previous data, which show that Stx is able to stimulate its own transport.
Highlights
Shiga toxin (Stx)2 is an AB5-toxin consisting of an enzymatically active A-subunit noncovalently linked to a stable pentamer of B-chains (StxB) that binds to the glycosphingolipid Gb3 and mediates transport of the toxin [1]
Upon inhibition or knockdown of protein kinase C (PKC)␦, StxB molecules colocalized less with giantin and more with EEA1, indicating that the molecules were accumulated in endosomes, unable to reach the Golgi complex
PKC␦ Is Activated by StxB—We have recently shown that the tyrosine kinase Syk is activated by Stx and regulates toxin uptake [12]
Summary
Materials—Hepes, bovine serum albumin, MESNa (mercaptoethanesulfonic acid), n-octylglucopyranoside, and tetracycline were purchased from Sigma. HeLa cells seeded out in 24-well plates were washed twice in leucine-free Hepes medium, the cells were preincubated with the different PKC inhibitors in the same medium for 30 min, before increasing concentrations of the toxins were added. The cell lysate was incubated with the BV-TAG-labeled anti-Stx antibody (0.5 g/ml), and simultaneously, to selectively quantify biotinylated Stx, the biotin-labeled Stx was captured by streptavidin-coated magnetic beads (0.1 mg/ml, Invitrogen) by gentle shaking for 1.5 h in assay diluent (0.2% bovine serum albumin, 0.5% Tween 20 in phosphate-buffered saline). To determine the level of protein sulfation in general in cells treated with the different inhibitors or transfected with siRNA, the amount of 35S-labeled proteins in the lysate was measured by trichloroacetic acid precipitation. Images were taken of thin single plane sections, and the signal intensities were quantified by using the histogram-function in the Zeiss LSM Image Browser software (Version 3)
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