Litersky J M, Johnson G V, Jakes R, Goedert M, Lee M, Seubert P
Department of Psychiatry, University of Alabama at Birmingham 35294-0017, USA.
Biochem J. 1996 Jun 1;316 ( Pt 2)(Pt 2):655-60. doi: 10.1042/bj3160655.
Phosphorylation of tau protein at Ser-262 has been shown to diminish its ability to bind to taxol-stabilized microtubules. The paired helical 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 still under investigation. In this study, the ability of cyclic AMP-dependent protein kinase (cAMP-PK) and calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate tau at Ser-262, as well as Ser-356, is demonstrated by use of a monoclonal antibody (12E8) which has been shown to recognize tau when these sites are phosphorylated. Cleavage of cAMP-PK-phosphorylated tau at cysteine residues by 2-nitro-5-thiocyanobenzoic acid, which cuts the protein into essentially two fragments and separates Ser-262 from Ser-356, revealed that cAMP-PK phosphorylates both Ser-262 and Ser-356. In addition, phosphorylation with cAMP-PK or CaMKII of recombinant tau in which Ser-262, Ser-356 or both had been mutated to alanines, clearly demonstrated that cAMP-PK and CaMKII were able to phosphorylate both sites. Mitogen-activated protein kinase or protein kinase C did not phosphorylate tau at Ser-262 and/or Ser-356. Finally, evidence is presented that phosphorylation of both these sites occurs in cultured nerve cells under certain conditions, indicating their potential physiological relevance.
已表明tau蛋白在Ser-262位点的磷酸化会降低其与紫杉醇稳定的微管结合的能力。在阿尔茨海默病大脑中发现的双螺旋丝（PHF）由PHF-tau组成，PHF-tau在包括Ser-262在内的多个位点发生了过度磷酸化。然而，能够使该位点磷酸化的蛋白激酶仍在研究中。在本研究中，通过使用单克隆抗体（12E8）证明了环磷酸腺苷依赖性蛋白激酶（cAMP-PK）和钙/钙调蛋白依赖性蛋白激酶II（CaMKII）使tau在Ser-262以及Ser-356位点磷酸化的能力，当这些位点磷酸化时，该单克隆抗体已被证明可识别tau。用2-硝基-5-硫氰基苯甲酸切割cAMP-PK磷酸化的tau的半胱氨酸残基，该酸将蛋白质切割成基本上两个片段并将Ser-262与Ser-356分开，结果表明cAMP-PK使Ser-262和Ser-356都发生了磷酸化。此外，用cAMP-PK或CaMKII对Ser-262、Ser-356或两者已突变为丙氨酸的重组tau进行磷酸化，清楚地表明cAMP-PK和CaMKII能够使这两个位点磷酸化。丝裂原活化蛋白激酶或蛋白激酶C不会使tau在Ser-262和/或Ser-356位点磷酸化。最后，有证据表明在某些条件下培养的神经细胞中这两个位点都会发生磷酸化，表明它们具有潜在的生理相关性。
