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Multiple regions of MAP kinase phosphatase 3 are involved in its recognition and activation by ERK2
1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Abstract:
Mitogen-activated protein kinase phosphatase 3 (MKP3) is a specific regulator of extracellular signal-regulated protein kinase 2 (ERK2). Association of ERK2 with MKP3 results in a powerful increase in MKP3 phosphatase activity. To determine the molecular basis of the specific ERK2 recognition by MKP3 and the ERK2-induced MKP3 activation, we have carried out a systematic mutational and deletion analysis of MKP3. Using activation-based and competition-based assays, we are able to quantitatively evaluate the contributions that residues/regions within MKP3 make to ERK2 binding and ERK2-induced MKP3 activation. Our results show that recognition and activation of MKP3 by ERK2 involves multiple regions of MKP3. Thus, the kinase interaction motif (KIM; residues 61--75) in MKP3 plays a major role (135-fold) for high affinity ERK2 binding. The most important residue in the KIM sequence of MKP3 is Arg(65), which probably interacts with Asp(319) in ERK2. In addition to KIM, a unique sequence conserved in cytosolic MKPs (residues 161--177 in MKP3) also contributes to ERK2 binding (15-fold). However, these two regions are not essential for ERK2-induced MKP3 activation. A third ERK2 binding site is localized in the C terminus of MKP3 (residues 348--381). Although deletion of this region or mutation of the putative ERK specific docking sequence (364)FTAP(367) in this region reduces MKP3's affinity for ERK2 by less than 10-fold, this region is absolutely required for ERK2-induced MKP3 activation.
Insights
Mitogen-activated protein kinase phosphatase 3 (MKP3) specifically binds and activates extracellular signal-regulated protein kinase 2 (ERK2). Multiple regions of MKP3 contribute to ERK2 binding and activation, with the C-terminal region being essential for activation.
Area of Science:
- Molecular Biology
- Enzymology
- Signal Transduction
Background:
- Mitogen-activated protein kinase phosphatase 3 (MKP3) is a key phosphatase regulating extracellular signal-regulated protein kinase 2 (ERK2).
- ERK2 association with MKP3 significantly enhances MKP3's phosphatase activity.
- Understanding the molecular mechanisms of this interaction is crucial for deciphering cellular signaling pathways.
Purpose of the Study:
- To elucidate the molecular basis for specific ERK2 recognition by MKP3.
- To investigate the structural determinants of ERK2-induced MKP3 activation.
- To quantitatively assess the contribution of different MKP3 regions to ERK2 binding and activation.
Main Methods:
- Systematic mutational and deletion analysis of MKP3.
- Utilized activation-based and competition-based biochemical assays.
- Quantified the effects of residue/region modifications on ERK2 binding affinity and activation.
Main Results:
- MKP3 recognition and activation by ERK2 involve multiple distinct regions.
- The kinase interaction motif (KIM; residues 61-75) is critical for high-affinity ERK2 binding (135-fold increase), with Arg(65) being a key residue likely interacting with ERK2 Asp(319).
- A conserved cytosolic MKP sequence (residues 161-177) also contributes to ERK2 binding (15-fold), but neither KIM nor this region is essential for ERK2-induced activation.
- A third binding site in the C-terminus (residues 348-381), including the FTAP sequence (364-367), is essential for ERK2-induced MKP3 activation, despite a less significant impact on binding affinity (<10-fold reduction upon deletion/mutation).
Conclusions:
- ERK2 binding and subsequent activation of MKP3 are complex processes mediated by multiple interaction sites.
- The C-terminal region of MKP3 is indispensable for ERK2-mediated activation, highlighting a distinct mechanism beyond simple binding.
- These findings provide critical insights into the regulation of the ERK signaling pathway by MKP3.