The phosphorylation state of alphaB-
crystallin accumulated in the brains of two patients with
Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific
antibodies against alphaB-
crystallin and each of three phosphorylated
serine residues. The level of mammalian
small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-
crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-
crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-
crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with
Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-
crystallin was stained in Rosenthal fibers in brains of patients with
Alexander's disease and their peripheral portions were immunostained with
antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-
crystallin in brains of patients with
Alexander's disease or
Alzheimer's disease as well as in aged controls is Ser-59.