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Phosphorylation of recombinant tau by cAMP-dependent protein kinase. Identification of phosphorylation sites and
C W Scott1, R C Spreen, J L Herman
1Pharmacology Department, ICI Pharmaceuticals Group, ICI Americas Inc., Wilmington, Delaware 19897.
Abstract:
Tau protein is an integral component of paired helical filaments, a pathological feature of Alzheimer's disease. tau extracted from these filaments displays decreased electrophoretic mobility due to aberrant phosphorylation. Here we show that recombinant human tau can be phosphorylated by cAMP-dependent protein kinase resulting in decreased electrophoretic mobility. Phosphorylation of tau by cAMP-dependent protein kinase caused a 92% decrease in the maximum rate of tau-induced microtubule assembly. The sites of phosphorylation were identified by digesting phosphorylated tau with proteases, separating the peptides by reversed-phase HPLC, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase N-terminal sequencing. Five phosphorylation sites were identified, two of which were located within microtubule binding domains. One site was previously shown to be the sole phosphorylation site for CaM kinase II; phosphorylation at this site by CaM kinase II was sufficient to cause decreased electrophoretic mobility (Steiner, B., Mandelkow, E. M., Biernat, J., Gustke, N., Meyer, H. E., Schmidt, B., Mieskes, G., Soling, H. D., Drechsel, D., Kirschner, M. W., Goedert, M., and Mandelkow, E. (1990) EMBO J. 9, 3539-3544). Thus two different second messenger-dependent protein kinases can phosphorylate tau at the same site and induce a shift in tau mobility like that seen in Alzheimer's disease.
Insights
cAMP-dependent protein kinase phosphorylates tau protein, a key component in Alzheimer's disease pathology. This phosphorylation significantly reduces tau's ability to assemble microtubules, mimicking disease-related changes.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Tau protein is a major component of paired helical filaments found in Alzheimer's disease (AD).
- Phosphorylation of tau alters its properties, including decreased electrophoretic mobility, a hallmark observed in AD.
Purpose of the Study:
- To investigate the effect of cAMP-dependent protein kinase (PKA) on tau protein phosphorylation.
- To determine if PKA-mediated tau phosphorylation impacts microtubule assembly and mimics AD-related tau pathology.
Main Methods:
- Recombinant human tau was phosphorylated using PKA.
- Electrophoretic mobility shifts were analyzed.
- Tau-induced microtubule assembly rates were measured.
- Phosphorylation sites were identified using protease digestion, HPLC, mass spectrometry, and N-terminal sequencing.
Main Results:
- PKA phosphorylated tau, resulting in decreased electrophoretic mobility.
- PKA phosphorylation caused a 92% reduction in the rate of tau-induced microtubule assembly.
- Five phosphorylation sites were identified, with two within microtubule-binding domains.
- One identified site was previously shown to be phosphorylated by CaM kinase II.
Conclusions:
- cAMP-dependent protein kinase can phosphorylate tau protein, leading to reduced electrophoretic mobility.
- This phosphorylation significantly impairs tau's ability to promote microtubule assembly.
- Two distinct kinases (PKA and CaM kinase II) can phosphorylate tau at the same site, inducing mobility shifts similar to those seen in Alzheimer's disease.