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Okadaic acid-sensitive protein phosphatases dephosphorylate MARCKS, a major protein kinase C substrate
P R Clarke1, S R Siddhanti, P Cohen
1Department of Biochemistry, University of Dundee, Scotland, UK.
Abstract:
The myristoylated alanine-rich C kinase substrate (MARCKS) undergoes a rapid and, in certain circumstances, transient increase in phosphorylation in response to stimuli that activate protein kinase C. We have investigated the protein-serine/threonine phosphatase activity responsible for reversing the phosphorylation of MARCKS. In cell-free assays, protein phosphatases 1, 2A and 2C (PP1, PP2A and PP2C) all dephosphorylate recombinant MARCKS or a synthetic peptide based on its phosphorylation site domain. In intact Swiss 3T3 cells, okadaic acid, a specific inhibitor of PP1 and PP2A, had little effect on MARCKS phosphorylation on its own, but largely prevented the dephosphorylation of MARCKS that occurred following activation of protein kinase C by bombesin with subsequent receptor blockade. These results indicate that although the dephosphorylation of MARCKS can be mediated by PP2C in vitro, this protein is dephosphorylated by okadaic acid-sensitive phosphatases in the intact cell.
Insights
The myristoylated alanine-rich C kinase substrate (MARCKS) is dephosphorylated by okadaic acid-sensitive phosphatases in cells. Protein phosphatases 1, 2A, and 2C dephosphorylate MARCKS in cell-free assays.
Area of Science:
- Cellular signaling
- Biochemistry
- Molecular biology
Background:
- Myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation is regulated by protein kinase C.
- Understanding the phosphatases that reverse MARCKS phosphorylation is crucial for cellular signaling research.
Purpose of the Study:
- To identify the specific protein-serine/threonine phosphatase activity responsible for dephosphorylating MARCKS in intact cells.
- To compare the in vitro and in vivo dephosphorylation activities on MARCKS.
Main Methods:
- In vitro dephosphorylation assays using recombinant MARCKS and synthetic peptides with purified protein phosphatases 1, 2A, and 2C (PP1, PP2A, PP2C).
- In intact Swiss 3T3 cells, treatment with okadaic acid (a PP1 and PP2A inhibitor) and stimulation of protein kinase C activation via bombesin.
- Monitoring MARCKS phosphorylation levels under various experimental conditions.
Main Results:
- PP1, PP2A, and PP2C were all capable of dephosphorylating MARCKS and its synthetic peptide in cell-free assays.
- Okadaic acid alone had minimal impact on MARCKS phosphorylation in intact cells.
- In intact cells, okadaic acid treatment significantly inhibited the dephosphorylation of MARCKS following protein kinase C activation.
Conclusions:
- While PP2C can dephosphorylate MARCKS in vitro, okadaic acid-sensitive phosphatases (likely PP1 and/or PP2A) are primarily responsible for MARCKS dephosphorylation in intact cells.
- Cellular context significantly influences the phosphatase activity involved in regulating MARCKS phosphorylation.