Protein Kinase F A /Glycogen Synthase Kinase‐3α After Heparin Potentiation Phosphorylates τ on Sites Abnormally Phosphorylated in Alzheimer's Disease Brain

Previously, we identified protein kinase F A /glycogen synthase kinase‐3α (GSK‐3α) as a brain microtubule‐associated τ kinase that phosphorylates Ser 235 and Ser 404 of τ and causes its electrophoretic mobility shift in gels, a unique property characteristic of paired helical filament‐associated pathological τ (PHF‐τ) in Alzheimer's disease brains. In this study, we found that the activity of kinase F A /GSK‐3α towards phosphorylation of brain τ could be stimulated approximately fourfold by heparin. The phosphorylation molar ratio was increased simultaneously up to 9 mol of phosphates/mol of τ, resulting in a reduced mobility of τ with an apparent molecular mass shift to ∼68 kDa in sodium dodecyl sulfate gels, which is very similar to that observed in Alzheimer‐τ. Tryptic digestion of 32 P‐labelled τ, followed by HPLC and two‐dimensional separation on TLC cellulose plates, revealed eight major phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and protein sequence analysis further revealed that, in addition to Ser 235 and Ser 404 , heparin generated Thr 212 , Thr 231 , Ser 262 , Ser 324 , and Ser 356 , the five extra phosphorylation sites in τ. As Ser 235 , Ser 262 , Ser 324 , Ser 356 , and Ser 404 (particularly the site of Ser 262 ) have been identified as five of the most potent sites in τ responsible for reducing microtubule binding possibly involved in neuronal degeneration, and Thr 231 , Ser 235 , Ser 262 , and Ser 404 are four of the most well documented sites abnormally phosphorylated in Alzheimer‐τ, the results provide initial evidence that protein kinase F A /GSK‐3α after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF‐τ in Alzheimer's disease brains.