The overall goal of this project is to provide a core facility for use by all members of the Program. Signal transduction via protein phosphorylation is a complex, interdigitated network of protein kinase (protein phosphorylating) enzymes, and protein phosphatase (phosphoprotein dephosphorylating) enzymes, and protein phosphatase inhibitors. Each enzyme-substrate interaction is uniquely regulated. In order to analyze this network in vitro, the components of each interaction must be purified, permitting the reconstitution of each individual interaction in isolation. One function of this core is to provide adequate supplies of each of these components in order to enable the studies proposed by the other Projects. The Alzheimer amyloid precursor protein (APP), its Drosophila homologue APP-like (APPL), and the APP promoter-binding homeoproteins form a major focus of the Program, and purified preparations of each will be provided by this core. In addition to this array of enzymes and substrates, highly specific antibody reagents are also required in order to probe these systems in situ, either in intact cellular systems or in postmortem human tissue. This core will be responsible for formulating epitope and immunogen strategies, immunization, collection and storage of antisera, screening, characterization and purification. As these antibody reagents are used in a variety of experimental systems, immunoreactive endogenous proteins will be implicated as possibly being a protein of interest: APP or APPL, for example. This core will provide for purification of these APP-like immunoreactive proteins (APP-LIPs) and for their subsequent microsequencing, in order to definitively establish their identities. cDNA constructs for DNA transfer experiments will also be maintained by this Core. Transfected cell systems will be prepared in other projects in order to test the effects of specific changes in protein phosphorylation enzymes upon expression or catabolism of the proteins under study. Transgenic mice will be prepared by this core in order to define at a higher organizational level the biological consequences of disordered APP metabolism or disordered protein phosphorylation. Transgene constructs will include APP isoforms and mutants lacking their phosphorylation sites or their cytoplasmic domains. Other constructs will include overexpression of protein kinase C (regulator of APP expression and candidate APP kinase) and calcium/calmodulin-dependent protein kinase II (candidate APP kinase and tau kinase). The brains of these mice will be examined pathologically (Project 3) as well as used in enzymological (Project 1) and cell biological (Project 2) studies.
Showing the most recent 10 out of 115 publications
