Specific Processing of Native and Phosphorylated τ Protein by Proteases

Abstract

Protein τ is a group of developmentally regulated proteins with implications in the pathogenesis of Alzheimer's Disease (AD). To study whether phosphorylation of τ by different protein kinases may affect subsequent reactivity to proteases, human recombinant τ-3 was phosphorylated with PKA or a double-stranded DNA-dependent protein kinase (referred to as DNA-PK), followed by incubation with thrombin or a double-stranded DNA (dsDNA)-stimulated protease. Quantitative degradation of τ was measured by the disappearance of the substrates or the appearance of products on SDS–PAGE. With thrombin, τ-3 phosphorylated by DNA-PK was degraded faster than the native protein which was processed at a faster rate than τ-3 phosphorylated by PKA. With the dsDNA-stimulated protease, however, τ-3 phosphorylated by PKA was processed faster than that phosphorylated by DNA-PK. Thrombin-mediated degradation of DNA-PK phosphorylated τ-3 gave a pattern different from that using the native or the PKA phosphorylated τ-3 as substrates. These results suggest that the rate and/or sequence of phosphorylation at specific sites of τ may provide “micro-environments” and/or conformations which alter their accessibility and/or reactivity to proteases.