As a first step in understanding the function of the 68-kDa
Alz-50 antigen (A68) in the pathophysiology of
Alzheimer's disease (AD), we have reexamined preliminary observations in our laboratory (Wolozin and Davies, 1986) of a
protein kinase activity associated with crude preparations of the
protein. This study was undertaken to determine whether the
kinase activity is an inherent property of the
Alz-50 antigen, or is a property of an associated
protein. Phosphorylation was therefore examined by incubating A68-enriched preparations with radiolabelled
ATP. This resulted in the appearance of a labelled 68-kDa
phosphoprotein, comigrating with the Alz-50 reactive A68
protein. The labelling of this 68-kDa
protein occurred in the presence of 2% SDS, suggesting that it is more likely to represent an autophosphorylation than a transfer of
phosphate mediated by another
kinase. Upon further inspection, it was found that the autophosphorylated 68-kDa
protein was not localized to regions of AD brain where A68 was detectable, but displayed a more ubiquitous distribution. In addition, this
phosphoprotein was also observed to be present in similar preparations from normal brain, which lacked the
Alz-50 antigen (Wolozin et al, 1986). These findings indicate that the
auto-kinase activity at 68 kDa is not closely associated with the A68
protein, but with a comigrating contaminant in the preparation. Other experiments in this study indicate that A68 is not a substrate for in vitro phosphorylation. Following incubation of A68 preparations with radiolabelled
ATP, immunoprecipitation of the
antigen did not reveal any
phosphate transfer to the
protein. These results were unaffected by a prior incubation with
alkaline phosphatase, even when the subsequent phosphorylation reactions were conducted in the presence of
protein kinase activators. Incubation with
alkaline phosphatase did not produce any alterations in electrophoretic mobility of A68, nor did it affect the binding of
antibodies directed against
phosphatase-sensitive
epitopes with A68. Thus, despite the suggestion that A68 is a modified form of tau, the
antigen exhibits remarkable differences from tau with regard to its sensitivity to
kinases and to
alkaline phosphatase.