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Autophosphorylation-activated protein kinase phosphorylates and inactivates protein phosphatase 2A
Abstract:
Purified preparations of a distinct autophosphorylation-activated protein kinase from bovine kidney phosphorylated and inactivated purified preparations of protein phosphatase 2A2 (PP2A2) by about 80% with the autophosphorylation-activated protein kinase, protamine kinase, and 32P-labeled myelin basic protein as substrates. Analysis of incubations performed in the presence of 0.2 mM [gamma-32P]ATP by autoradiography following SDS/PAGE and by FPLC gel permeation chromatography on Superose 12 demonstrated that the catalytic subunit of PP2A2 was phosphorylated in the incubation mixtures containing the kinase and phosphatase. Up to 0.3 mol of phosphate groups was incorporated per mol of the catalytic subunit of PP2A2 following incubation with the kinase. This phosphorylation was enhanced about 5-fold in the presence of 0.4 microM microcystin-LR. In addition, up to 1 mol of phosphate groups was incorporated per mol of the PP2A2 subunit of apparent M(r) approximately 60,000 when microcystin-LR was included. Analysis by thin-layer chromatography indicated that PP2A2 catalyzed an autodephosphorylation reaction which was inhibited by microcystin-LR. Phospho amino acid analysis showed that the catalytic subunit of PP2A2 was phosphorylated on threonine residues by the autophosphorylation-activated protein kinase. Together with previous observations, the results suggest that inactivation of PP2A by phosphorylation catalyzed by the autophosphorylation-activated protein kinase could contribute to the marked increase in the phosphorylation of cellular proteins in response to insulin and other mitogens.
Insights
A novel bovine kidney protein kinase phosphorylates and inactivates protein phosphatase 2A2 (PP2A2), a key enzyme in cellular signaling. This phosphorylation, enhanced by microcystin-LR, may explain increased protein phosphorylation observed with mitogens like insulin.
Area of Science:
- Biochemistry
- Cellular Signaling
- Enzymology
Background:
- Protein phosphatases, such as protein phosphatase 2A2 (PP2A2), play crucial roles in dephosphorylating cellular proteins.
- Dysregulation of protein phosphorylation is implicated in various cellular processes and diseases.
- Understanding the regulation of protein phosphatase activity is essential for deciphering cellular signaling pathways.
Purpose of the Study:
- To investigate the regulatory mechanisms of protein phosphatase 2A2 (PP2A2) activity.
- To identify protein kinases that can modulate PP2A2 function.
- To explore the potential role of PP2A2 phosphorylation in cellular responses to mitogens.
Main Methods:
- Purification of a bovine kidney autophosphorylation-activated protein kinase.
- In vitro phosphorylation assays using purified kinase and PP2A2.
- Analysis of protein phosphorylation by SDS/PAGE, autoradiography, and FPLC gel permeation chromatography.
- Phosphoamino acid analysis and thin-layer chromatography to identify phosphorylation sites and dephosphorylation activity.
Main Results:
- A distinct bovine kidney protein kinase was found to phosphorylate and inactivate PP2A2 by approximately 80%.
- The catalytic subunit of PP2A2 was phosphorylated on threonine residues by the kinase.
- Phosphorylation of PP2A2 was significantly enhanced (about 5-fold) by microcystin-LR.
- PP2A2 exhibited autodephosphorylation activity, which was inhibited by microcystin-LR.
Conclusions:
- Phosphorylation by the identified kinase inactivates PP2A2, suggesting a novel regulatory mechanism.
- This inactivation of PP2A2 could contribute to the increased cellular protein phosphorylation observed in response to insulin and other mitogens.
- The findings provide insights into the complex interplay between protein kinases and phosphatases in signal transduction pathways.