Phosphorylation of recombinant tau by cAMP-dependent protein kinase. Identification of phosphorylation sites and effect on microtubule assembly.
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.
Scott
C W
CW
Pharmacology Department, ICI Pharmaceuticals Group, ICI Americas Inc., Wilmington, Delaware 19897.
Spreen
R C
RC
Herman
J L
JL
Chow
F P
FP
Davison
M D
MD
Young
J
J
Caputo
C B
CB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D016875:tau Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D009842:Oligopeptides; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D016875:tau Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases