REGULATION OF PROTEIN DEPHOSPHORYLATION

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Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that is a critical component of signaling pathways regulating cell function. The heterodimeric core of PP2A associates with a variety of regulatory subunits that contribute to the specificity and subcellular localization of the enzyme. PP2A also interacts with a number of proteins that are not considered to be conventional regulatory subunits of the enzyme. The investigators anticipate that identification of additional interacting partner for PP2A will provide new insights into the cellular organization of the enzyme and that such organization dictates specificity of enzyme action. One sub-population of PP2A is associated with microtubules in neurons. Tau and MAP2 are neuronal microtubule-associated proteins (MAPs) that are highly phosphorylated in vivo and are targets for numerous protein kinases in vitro. The phosphorylation state of tau and MAP2 determines their abilities to bind to and thus stabilize microtubules. Results from investigators' laboratory indicate that PP2A dephosphorylates tau and MAP2 in vivo, suggesting the enzyme regulates the neuronal cytoskeleton. An important aspect of this work is that one of the pathological hallmarks of Alzheimer's Disease includes hyperphosphorylation of tau and disruption of axonal microtubules. The proposal has three specific aims. 1) The first aim will test the hypothesis that an anchoring protein that promotes targeting of PP2A to microtubules is required for efficient dephosphorylation of tau and MAP2. The molecular basis of PP2A interaction with the microtubule cytoskeleton will be explored. Putative anchoring protein(s) will be purified, characterized, and cDNAs cloned. The role of anchoring in regulating PP2A will be examined with the assumption that direct protein-protein interactions define the specificity of PP2A toward neuronal MAPs. 2) The underlying hypothesis of aim 2 is that suppression of PP2A in neurons of intact animals will lead to increased phosphorylation of tau and neuronal degeneration. SV40 small-t antigen, a specific inhibitor of PP2A, will be expressed in transgenic mice using neuron-specific promoters. The effects of reduced PP2A activity will be assessed by measuring tau phosphorylation and neuronal morphology. 3) The hypothesis to be tested in this aim is that two novel PP2A- interacting protein influence the activity and function of PP2A. Characterization of p31 and p17, two PP2A-interacting proteins identified in a two-hybrid screen, will be pursued with high priority. The effects of these proteins on activity, subcellular localization, and in vivo function of PP2A will be determined. Additional candidate interacting proteins, identified in the two-hybrid screen will also be characterized.