Protein Kinase FA/Glycogen Synthase Kinase‐3α After Heparin Potentiation Phosphorylates τ on Sites Abnormally Phosphorylated in Alzheimer's Disease Brain
[[abstract]]Previously, we identified protein kinase F-A/glycogen synthase kinase-3 alpha (GSK-3 alpha) as a brain microtubule-associated tau kinase that phosphorylates Ser(235) and Ser(404) of tau and causes its electrophoretic mobility shift in gels, a unique property characteristic of paired helical filament-associated pathological tau (PHF-tau) in Alzheimer's disease brains. In this study, we found that the activity of kinase F-A/GSK-3 alpha towards phosphorylation of brain tau could be stimulated approximately fourfold by heparin. The phosphorylation molar ratio was increased simultaneously up to 9 mol of phosphates/mol of tau, resulting in a reduced mobility of tau with an apparent molecular mass shift to similar to 68 kDa in sodium dodecyl sulfate gels, which is very similar to that observed in Alzheimer-tau. Tryptic digestion of P-32-labelled tau, 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 tau. 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 tau 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-tau, the results provide initial evidence that protein kinase F-A/GSK-3 alpha after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau T in Alzheimer's disease brains.[[fileno]]2050116010053[[department]]生科