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Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength
G Alonso1, C Ambrosino, M Jones
1European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Abstract:
We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38alpha but not p38gamma. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38alpha by MKK6 correlated with more efficient binding of MKK6 to p38alpha than to p38gamma. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38alpha alone (low MKK6 activity) or both p38alpha and p38gamma (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.
Insights
Mitogen-activated protein kinase kinase 6 (MKK6) activity level dictates p38 MAPK pathway activation. MKK6 mutants preferentially activate p38alpha, while higher activity activates both p38alpha and p38gamma.
Area of Science:
- Cellular signaling pathways
- Mitogen-activated protein kinase (MAPK) cascades
Background:
- The p38 MAPK subfamily plays crucial roles in cellular responses to stress and inflammation.
- MKK6 is a key upstream activator of p38 MAPKs, but its isoform-specific activation patterns are not fully understood.
Purpose of the Study:
- To investigate the ability of MKK6 to activate different members of the p38 MAPK subfamily.
- To identify the factors determining MKK6's selectivity in activating p38alpha versus p38gamma.
Main Methods:
- Co-expression of MKK6 and p38 MAPK isoforms in Xenopus oocytes.
- In vitro activation assays using MKK6 immunoprecipitates and recombinant proteins.
- Analysis of MKK6 binding affinity to p38alpha and p38gamma.
- Site-directed mutagenesis to map selectivity determinants.
Main Results:
- MKK6 mutants showed preferential activation of p38alpha over p38gamma, correlating with binding affinity.
- A constitutively active MKK6 mutant activated both p38alpha and p38gamma.
- Increasing MKK6 activity levels differentially activated p38alpha alone or both isoforms.
- Selectivity determinants reside in the carboxyl-terminal lobe of p38 MAPKs, excluding the activation loop and common docking sites.
- Endogenous MKK6 activity levels correlated with differential p38 MAPK activation in response to various stimuli.
Conclusions:
- The level of MKK6 activity is a critical determinant for the differential activation of p38alpha and p38gamma.
- Stimulus-induced MKK6 activity levels dictate downstream p38 MAPK pathway engagement.
- Understanding MKK6-p38 MAPK interactions provides insights into specific cellular signaling outcomes.