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Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase
M Camps1, A Nichols, C Gillieron
1Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., CH-1228 Plan-les-Ouates, Geneva, Switzerland.
Abstract:
MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.
Insights
MAP kinase phosphatase-3 (MKP-3) selectively inactivates ERK kinases upon binding to ERK2. This activation mechanism, independent of kinase activity, highlights substrate-induced regulation in MAP kinase signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Enzymology
Background:
- Mitogen-activated protein (MAP) kinases are crucial signaling molecules involved in various cellular processes.
- MAP kinase phosphatase-3 (MKP-3) is a key enzyme that dephosphorylates and inactivates specific MAP kinases, particularly the ERK family.
- Understanding the regulation of MKP-3 activity is vital for comprehending cellular responses to external stimuli.
Purpose of the Study:
- To investigate the mechanism of MKP-3 activation by ERK2.
- To determine the specific domains of MKP-3 involved in ERK2 binding and activation.
- To compare the substrate specificity and activation of MKP-3 with a related phosphatase, MKP-4.
Main Methods:
- Purification of ERK2 and MKP-3 proteins.
- In vitro binding assays to assess protein-protein interactions.
- Enzymatic assays to measure phosphatase activity.
- Site-directed mutagenesis to identify key binding domains.
Main Results:
- MKP-3 directly binds to ERK2, leading to its catalytic activation.
- Activation is dependent on the noncatalytic N-terminus of MKP-3 and does not require protein kinase activity.
- ERK2 mutants and other MAP kinases (JNK/SAPK, p38) did not bind or activate MKP-3.
- MKP-4, a related phosphatase, showed broader substrate binding and activation by ERK2, JNK/SAPK, and p38.
Conclusions:
- Substrate binding, specifically by ERK2 to MKP-3, is a critical mechanism for regulating phosphatase activity.
- The N-terminus of MKP-3 plays a crucial role in selective ERK binding and activation.
- These findings suggest that substrate-induced catalytic activation is a general mechanism for regulating MAP kinase phosphatases in diverse signaling pathways.