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Identification and characterization of the ATP.Mg-dependent protein phosphatase activator (FA) as a microtubule
1Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Abstract:
The activating factor of ATP.Mg-dependent protein phosphatase (FA) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates, FA could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with a Km value of 0.4 microM, and tau proteins to 4 moles of phosphates per mole of proteins with a Km value of about 3 microM. When using microtubules as substrates, FA could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca+2/phospholipid-dependent protein kinase, the FA-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced by FA. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed that FA could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca+2/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due to FA. Taken together, the results provide initial evidence that the ATP.Mg-dependent protein phosphatase activating factor (FA) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.
Insights
The activating factor (FA) of ATP.Mg-dependent protein phosphatase is a potent brain microtubule kinase. FA uniquely phosphorylates MAP-2 and tau proteins, suggesting a major role in microtubule regulation.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Microtubules are crucial for neuronal structure and function.
- Microtubule-associated proteins (MAPs) regulate microtubule dynamics.
- Phosphorylation is a key post-translational modification affecting MAP function.
Purpose of the Study:
- To identify and characterize the ATP.Mg-dependent protein phosphatase activating factor (FA) in brain microtubules.
- To investigate the substrate specificity and catalytic activity of FA.
- To compare FA's phosphorylation activity with other known MAP kinases.
Main Methods:
- Purification of FA from brain microtubules.
- In vitro phosphorylation assays using purified MAP-2 and tau proteins.
- Peptide mapping and SDS-PAGE analysis to characterize phosphorylation sites and conformational changes.
Main Results:
- FA phosphorylates MAP-2 and tau proteins at distinct sites, causing conformational changes.
- FA enhances endogenous phosphorylation of multiple MAPs in microtubules.
- FA accounts for approximately 19% of total endogenous kinase activity in brain microtubules.
Conclusions:
- FA is a potent and unique MAP kinase involved in brain microtubule phosphorylation.
- FA may play a significant role in regulating microtubule function in the brain.