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Abstract:The protein kinase activity tightly associated with paired helical filaments (PHFs) purified from the brain tissue of individuals with Alzheimer's disease has been characterized in vitro. The activity is shown to phosphorylate casein, an exogenous substrate, with a maximal velocity of -~2nmol/min/mg, suggesting it comprises a significant component of the total protein in the PHF preparation. On the basis of substrate selectivity, isoquinoline sulfonamide inhibitor selectivity, in-gel renaturation assays, and western analysis, the activity consists of closely related members of the a branch of the casein kinase 1 family of protein kinases. Because of its tight association with PHFs and its phosphate-directed substrate selectivity, casein kinase 1 is positioned to participate in the pathological hyperphosphorylation of tau protein that is observed in neurodegenerative diseases such as Alzheimer's disease.

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The Alzheimer's disease (AD) brain contains many abnormal protein modifications. These include the abnormal processing of amyloid precursor protein (APP) to form the amyloidogenic beta/A4 peptide and the abnormal phosphorylation of tau to form A68, the major constituent of the neurofibrillary tangle. In addition, many of the biochemical alterations found in the AD brain are also found in heat-shocked or stressed cells. We used heat-shocked neuronal PC12 cells to investigate the effects of stress on APP and tau. We found that by simply exposing neuronal PC12 cells to an elevated temperature (45 degrees C) for 30 minutes, they exhibited several features characteristic of the heat shock response. These included a 45% reduction in total protein synthesis, the induction of heat shock protein (hsp) 72, and increased phosphorylation of the protein synthesis initiation factor eIF-2 alpha. The heat-shocked cells also exhibited alterations in the metabolism and phosphorylation of APP. Under heat shock conditions, we found two additional APP-like polypeptides not present in controls and a significant decrease in the phosphorylation state of APP. We also found that an A68-like protein is formed in neuronal PC12 cells when subjected to elevated temperature. This A68-like protein was formed with heat shock even in the absence of protein synthesis, suggesting that its production occurred post-translationally. The tau/A68 polypeptides were identified as phosphoproteins, and the phosphorylation of tau to form A68 was reversed with recovery of the cells from heat shock. Immunoprecipitation of lysates from heat shocked cells with antibodies to hsp72/73 resulted in co-precipitation of tau, but not A68 with hsp72 indicating a stable complex formation between these two proteins. These results suggest that heat shock proteins may play either a protective or promoting role in the formation of A68 and/or the amyloidogenic C-terminal fragment of APP.

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