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The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate
M Muda1, A Theodosiou, C Gillieron
1Serono Pharmaceutical Research Institute, CH-1228 Plan-les-Ouates, Geneva, Switzerland.
Abstract:
We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.
Insights
Mitogen-activated protein kinase phosphatase-3 (MKP-3) selectively inactivates extracellular signal-regulated kinases (ERK) by binding to them via its N-terminal domain, explaining its specificity.
Area of Science:
- Molecular Biology
- Cell Signaling
- Enzymology
Background:
- Mitogen-activated protein kinase phosphatase-3 (MKP-3) is known to selectively inactivate extracellular signal-regulated kinases (ERK).
- The precise mechanism underlying MKP-3's specificity for ERK, compared to other MAP kinases like JNK/SAPK and p38, requires further elucidation.
Purpose of the Study:
- To investigate the molecular basis for MKP-3's substrate specificity.
- To determine which domain of MKP-3 is responsible for its selective binding and inactivation of ERK MAP kinases.
Main Methods:
- Utilized biochemical assays to assess binding interactions between MKP-3 and its variants with ERK1, ERK2, JNK/SAPK, and p38 MAP kinases.
- Employed domain deletion and chimera constructs of MKP-3 to map functional regions responsible for substrate recognition and catalytic activity.
Main Results:
- MKP-3 exhibits tight binding to ERK1 and ERK2, with minimal interaction observed for JNK/SAPK and p38 MAP kinases.
- The N-terminal noncatalytic domain of MKP-3 is crucial for binding ERK1 and ERK2, while the catalytic core alone does not bind these substrates.
- Higher concentrations of the catalytic domain (MKP-3ΔN) were required to inhibit ERK2 activity compared to full-length MKP-3, underscoring the role of the N-terminal domain in specificity.
Conclusions:
- The high specificity of MKP-3 for inactivating ERK family MAP kinases is primarily mediated by tight substrate binding interactions involving its N-terminal domain.
- These findings provide critical insights into the regulatory mechanisms of MAP kinase signaling pathways.