Pertussis toxin directly activates endothelial cell p42/p44 MAP kinases via a novel signaling pathway

Bordetella pertussis generates a bacterial toxin utilized in signal transdu ction investigation because of its ability to ADP ribosylate specific G pro teins. We previously noted that pertussis toxin (PTX) directly activates en dothelial cells, resulting in disruption of monolayer integrity and interce llular gap formation via a signaling pathway that involves protein kinase C (PKC). We studied the effect of PTX on the activity of the 42- and 44-kDa extracellular signal-regulated kinases (ERK), members of a kinase family kn own to be activated by PKC. PTX caused a rapid time-dependent increase in b ovine pulmonary artery endothelial cell ERK activity that was significantly attenuated by 1) pharmacological inhibition of MEK, the upstream ERK activ ating kinase, 2) an MEK dominant-negative construct, and 3) PKC inhibition with bisindolylmaleimide. There was little evidence for the involvement of either G beta gamma -subunits, Ras GTPases, Raf-1, p60 src, or phosphatidyl inositol 3'-kinases in PTX-mediated ERK activation. Both the purified beta -oligomer binding subunit of the PTX holotoxin and a PTX holotoxin mutant g enetically engineered to eliminate intrinsic ADP ribosyltransferase activit y completely reproduced PTX effects on ERK activation, suggesting that PTX- induced ERK activation involves a novel PKC-dependent signaling mechanism t hat is independent of either Ras or Raf-1 activities and does not require G protein ADP ribosylation.