LAN PROTEIN IS PHOSPHORYLATED BY CYCLIC-AMP-DEPENDENT PROTEIN-KINASE AND CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE-II WITHIN ITS MICROTUBULE-BINDING DOMAINS AT SER-262 AND SER-356/

Phosphorylation of tau protein at Ser-262 has been shown to diminish i ts ability to bind to taxol-stabilized microtubules. The paired helica l filaments (PHFs) found in Alzheimer's disease brain are composed of PHF-tau, which is hyperphosphorylated at multiple sites including Ser- 262. However, protein kinase(s) able to phosphorylate this site are st ill under investigation. In this study, the ability of cyclic AMP-depe ndent protein kinase (cAMP-PK) and calcium/calmodulin-dependent protei n kinase II (CaMKII) to phosphorylate tau at Ser-262, as well as Ser-3 56, is demonstrated by use of a monoclonal antibody (12E8) which has b een shown to recognize tau when these sites are phosphorylated. Cleava ge of cAMP-PK-phosphorylated tau at cysteine residues by 2-nitro-5-thi ocyanobenzoic acid, which cuts the protein into essentially two fragme nts and separates Ser-262 from Ser-356, revealed that cAMP-PK phosphor ylates both Ser-262 and Ser-356. In addition, phosphorylation with cAM P-PK or CaMKII of recombinant tau in which Ser-262, Ser-356 or both ha d been mutated to alanines, clearly demonstrated that cAMP-PK and CaMK II were able to phosphorylate both sites. Mitogen-activated protein ki nase or protein kinase C did not phosphorylate tau at Ser-262 and/or S er-356. Finally, evidence is presented that phosphorylation of both th ese sites occurs in cultured nerve cells under certain conditions, ind icating their potential physiological relevance.