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In vitro inhibition of MAP kinase (ERK1/ERK2) activity by phosphorylated glia maturation factor (GMF)
1Department of Neurology (Division of Neurochemistry and Neurobiology), University of Iowa College of Medicine, Iowa City, USA.
Abstract:
We report that recombinant glia maturation factor (GMF), a 17-kDa brain protein, inhibits the activity of mitogen-activated protein (MAP) kinase in the test tube assay, in particular the ERK1/ERK2 isoforms. A preliminary phosphorylation of GMF by protein kinase A (PKA) dramatically increases its inhibitory effect by over 600-fold (Ki approximately 3 nM), making it the most potent MAP kinase inhibitor ever reported. Immunoprecipitation of GMF from cell extracts using its specific antibody coprecipitates ERK (and vice versa), suggesting the association of the two proteins in the cell. The inhibitory effect of PKA-phosphorylated GMF is specific, as it does not suppress the activity of cdc2 kinase, another proline-directed kinase. Nor does it inhibit MAP kinase kinase (MEK) and MAP kinase-activated protein (MAPKAP) kinase-2, the two enzymes immediately upstream and downstream, respectively, of ERK. Of the other three enzymes that can phosphorylate GMF, only p90 ribosomal S6 kinase (RSK) enhances the inhibitory function of GMF on ERK; protein kinase C (PKC) and casein kinase II (CKII) are without effect. The inhibition of ERK by PKA-phosphorylated GMF suggests that GMF could be one of the mediators of the suppressive effect of the PKA pathway on the MAP kinase pathway. On the other hand, that RSK-phosphorylated GMF also inhibits ERK implies a negative feedback loop in the regulation of MAP kinase activity.
Insights
Glia maturation factor (GMF), a brain protein, potently inhibits mitogen-activated protein (MAP) kinase, especially ERK1/ERK2. Phosphorylation by protein kinase A (PKA) enhances GMF
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Protein kinase regulation
Background:
- Mitogen-activated protein (MAP) kinase pathways are crucial for cellular processes.
- Glia maturation factor (GMF) is a brain protein with poorly understood functions.
- Understanding protein interactions is key to deciphering cellular signaling.
Purpose of the Study:
- To investigate the inhibitory effect of glia maturation factor (GMF) on mitogen-activated protein (MAP) kinase activity.
- To determine the role of GMF phosphorylation in modulating its inhibitory function.
- To elucidate the interaction between GMF and MAP kinase in cellular contexts.
Main Methods:
- In vitro kinase assays to measure MAP kinase activity.
- Protein phosphorylation experiments using various kinases (PKA, RSK, PKC, CKII).
- Immunoprecipitation assays to detect protein-protein interactions between GMF and ERK.
Main Results:
- Recombinant GMF inhibits ERK1/ERK2 activity in vitro.
- Phosphorylation of GMF by protein kinase A (PKA) enhances its inhibitory potency by over 600-fold.
- GMF and ERK were found to co-precipitate, indicating an association within cells.
- PKA-phosphorylated GMF specifically inhibits ERK, not cdc2 kinase, MEK, or MAPKAP kinase-2.
- p90 ribosomal S6 kinase (RSK) phosphorylation also enhances GMF's inhibitory function on ERK.
Conclusions:
- Glia maturation factor (GMF) is a potent inhibitor of MAP kinase (ERK1/ERK2).
- PKA-mediated phosphorylation of GMF significantly boosts its inhibitory capacity, suggesting a role in PKA-MAPK pathway cross-talk.
- RSK-mediated phosphorylation of GMF implies a negative feedback mechanism regulating MAP kinase activity.