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Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade
J Yamauchi1, G Tsujimoto, Y Kaziro
1Department of Molecular Cell Pharmacology, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-1809, the Japan.
Abstract:
Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.
Insights
Heterotrimeric G protein G(q) activates p38 mitogen-activated protein kinase (MAPK) through MKK3 and MKK6. Both Galpha(q) and Gbetagamma subunits regulate these kinases via distinct signaling pathways.
Area of Science:
- Cellular signaling
- Molecular biology
- Signal transduction pathways
Background:
- Heterotrimeric G protein G(q) is known to stimulate p38 mitogen-activated protein kinase (MAPK) activity in mammalian cells.
- The precise signaling mechanisms by which G(q) subunits activate p38 MAPK remain incompletely understood.
Purpose of the Study:
- To elucidate the signaling pathways through which G(q) alpha and betagamma subunits activate p38 MAPK.
- To identify the specific mitogen-activated protein kinase kinases (MKKs) involved in G(q)-mediated p38 MAPK activation.
Main Methods:
- Utilized kinase-deficient mutants of MKK3, MKK4, and MKK6 in human embryonal kidney 293 cells.
- Assessed the impact of Galpha(q) and Gbetagamma on p38 MAPK, MKK3, and MKK6 activities.
- Investigated the involvement of phospholipase C, c-Src, Rho, Rac, and Cdc42 in the signaling cascade.
Main Results:
- Kinase-deficient MKK3 and MKK6, but not MKK4, blocked p38 MAPK activation by Galpha(q) and Gbetagamma.
- Both Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities.
- Galpha(q)-mediated MKK activation involved phospholipase C and c-Src, with differential dependence on Rho family GTPases.
- Gbetagamma-mediated MKK activation involved a non-c-Src tyrosine kinase and differential dependence on Rho family GTPases.
Conclusions:
- Galpha(q) and Gbetagamma subunits activate p38 MAPK by regulating MKK3 and MKK6.
- Distinct parallel signaling pathways are utilized by Galpha(q) and Gbetagamma to activate these upstream kinases.
- The findings provide a detailed mechanistic insight into G(q) protein signaling towards p38 MAPK activation.