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Pasteurella multocida toxin stimulates mitogen-activated protein kinase via G(q/11)-dependent transactivation of the
1Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G(q/11) family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha-thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G(q/11) signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha(q) carboxyl-terminal peptide, Galpha(q)-(305-359). Like alpha-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G(q/11) family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.
Insights
Pasteurella multocida toxin (PMT) activates Erk 1/2 in HEK-293 cells by stimulating G(q/11) proteins. This leads to Ras-dependent epidermal growth factor receptor transactivation, independent of protein kinase C.
Area of Science:
- Cell Biology
- Molecular Biology
- Toxicology
Background:
- Pasteurella multocida dermatonecrotic toxin (PMT) is a potent fibroblast mitogen.
- PMT activates phospholipase C, mobilizes calcium, and activates protein kinase C via G(q/11) proteins.
- The precise mechanism of PMT-induced mitogenesis, particularly Erk pathway activation, requires further elucidation.
Purpose of the Study:
- To investigate the mechanism by which recombinant PMT (rPMT) stimulates the Erk mitogen-activated protein kinase (MAPK) cascade.
- To determine the role of G protein pathways in rPMT-induced Erk activation.
- To identify downstream signaling components involved in rPMT's mitogenic effects.
Main Methods:
- HEK-293 cells were treated with rPMT.
- Erk 1/2 phosphorylation was measured.
- Inositol phospholipid hydrolysis was assessed.
- Inhibitors of G(q/11) signaling (GRK2(K220R), Galpha(q)-(305-359)) were used.
- Protein kinase C inhibitor (GF109203X) and epidermal growth factor receptor (EGFR) inhibitor (AG1478) were employed.
- Dominant negative mutants of mSos1 and Ha-Ras were utilized.
Main Results:
- rPMT induced a dose- and time-dependent increase in Erk 1/2 phosphorylation, mirroring inositol phospholipid hydrolysis.
- rPMT-stimulated Erk phosphorylation was blocked by G(q/11) signaling inhibitors.
- rPMT-induced Erk activation was insensitive to protein kinase C inhibition but blocked by EGFR inhibition and dominant negative Ras/mSos1 mutants.
- These findings suggest a G(q/11)-dependent, protein kinase C-independent pathway involving EGFR transactivation.
Conclusions:
- rPMT activates the Erk MAPK cascade through G(q/11) heterotrimeric G proteins.
- rPMT induces Ras-dependent Erk activation via protein kinase C-independent transactivation of the epidermal growth factor receptor.
- This study elucidates a novel signaling mechanism for PMT-induced mitogenesis.