Monoclonal antibody PHF‐1 recognizes tau protein phosphorylated at serine residues 396 and 404

Abstract

The microtubule-associated protein tau is hyperphosphorylated in the paired helical filaments (PHFs) of Alzheimer's disease. Immunological and direct chemical studies have identified Ser396 and Ser404 as two of the phosphorylated sites. Previously, we have demonstrated, using synthetic tau peptides containing phosphorylated Ser396, that this site is recognized by the monoclonal antibody PHF-1. The present study extends this observation by showing that PHF-1 recognizes tau peptides containing either individually phosphorylated Ser396 or Ser404, but that there is a > 10-fold increase in the sensitivity of detection of tau peptides by PHF-1 when both serines are phosphorylated. The recognition of singly or doubly phosphorylated Ser396 and Ser404 in tau by PHF-1 can also be demonstrated in Chinese hamster ovary cells transfected with full-length wild-type tau constructs or mutant constructs with Ala substituted for Ser396 or Ser404. We conclude that the PHF-1 epitope contains both phosphorylated Ser396 and Ser404.

Proapoptotic Effects of Tau Cleavage Product Generated by Caspase-3

“…We used an anti-PHF-1 Ab that recognizes the phosphorylated, PHF-like C-terminal of Tau (residues 387-404) in Alzheimer's patients (Otvos et al, 1994), to show that Tau is susceptible to cleavage by caspase during the process of neuronal cell death. There are multiple phosphorylation sites in the C-terminal region of Tau including Ser396 and Ser404 (Ishiguro et al, 1995;Reynolds et al, 1997a,b).…”

The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

“…Blots were probed with antibodies to phospho-Ser473-Akt, phospho-Thr308-Akt, total Akt, phospho-Ser21-GSK3α, phospho-Ser9-GSK3β, total GSK3β (Cell Signaling Technology, Beverly, MA), total GSK3α (Southern Biotech, Birmingham, AL), I-2 (BD Transduction Laboratories, Lexington, KY), myc (UAB peptide synthesis core facility), PP1 (Santa Cruz Biotechnologies, Santa Cruz, CA), SERCA2 (Affinity BioReagents, Golden, CO), and β-tubulin (Sigma, St. Louis, MO). The tau antibodies used in this study were Tau5 and 5A6 (Tau5 from Dr. L. Binder), which are phosphoindependent tau antibodies [4,23] and PHF-1 (from Dr. P. Davies), which recognizes tau phosphorylated at Ser-396/404 [36]. Immunoblots were developed using horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (Bio-Rad Laboraories, Hercules, CA), followed by detection with enhanced chemiluminescence.…”

Axin negatively affects tau phosphorylation by glycogen synthase kinase 3β

“…For immunoblotting samples were diluted directly into 2· SDS buffer and samples were electrophoresed on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes and blotted as above. To evaluate the phosphorylation of tau, the following phosphate-dependent antibodies were used: Tau-1 (a gift from Dr L. Binder), which recognizes a dephosphorylated epitope between residues 189 and 207 (Binder et al 1985;Szendrei et al 1993) and, PHF-1 (a gift from Dr P. Davies) which recognizes tau phosphorylated at Ser396/ 404 (Greenberg and Davies 1990;Otvos et al 1994) (numbering based on longest human isoform (Goedert et al 1989)). Total tau levels were determined using the combination of phospho-independent antibodies of Tau-5 (a gift from Dr L. Binder) (Carmel et al 1996) and 5A6 (Johnson et al 1997).…”