Many skin disease are caused by disruption of desmosomal cellular junction integrity either by autoimmune antibody production or aberrant expression of mutated proteins. Investigation of the regulation of desmosome formation or function would lead to insights into the cellular basis of many skin diseases. While significant progress has been made in the identification of desmosomal components, little is known about junctional assembly and/or signal transduction at these sites. This proposal investigates the possibility that tyrosine phosphorylation may be involved in the regulation of these junctions and that a protein tyrosine phosphatase may be present in epithelial desmosomes. Since desmosomes are crucial for the structure and integrity of the skin, this proposal will suggest a role for aberrant tyrosine phosphorylation in various skin diseases. I. Characterization of the desmosomal protein tyrosine phosphatase activity. Based on the structural similarity of desmosome constituents with adherens junction proteins, there was reason to suspect that the desmosomes may be regulated by tyrosine phosphorylation similar to the adherens junctions. The desmosomal cadherins, plakoglobin, and band 6 (also called plakophilin) are structurally similar to adherens junction components (cadherins, beta catenin and plakoglobin) which are known to be regulated by tyrosine phosphorylation. To investigate the possibility of desmosome regulation by phosphorylation, I will determine if there is tyrosine phosphatase enzymatic activity present in preparations enriched in desmosomal proteins. Protein tyrosine phosphatases (PTPs) are specifically inhibited by addition of vanadate. Immunoblot analysis of desmosome preps plus and minus vanadate will be performed and changes in phosphorylation will be detected by the use of anti-phosphotyrosine antibodies. II. Isolation and cloning of the desmosomal protein tyrosine phosphatase. The former aim will suggest the presence of a PTP but to understand its function in regulating desmosomes it must first be cloned and sequenced. We have found that desmosomes contain an 90kDa protein that is immunologically related to PTP-mu. Based on its homology to PTP-mu we have used an RT-PCR based strategy to isolate an 800 bp fragment of the PTP. We will use this fragment to isolate the full length protein by RT-PcR or cDNA library screening. III. Analysis of changes in tyrosine phosphorylation or desmosomal PTP expression during differentiation and disease progression in the skin. We will analyze three types of alterations in the desmosomal PTP during different stages of differentiation or disease: 1) changes in expression of the desmosomal PTP 2) changes in association of the desmosomal PTP with other components of the desmosome 3) changes in tyrosine phosphorylation at the desmosome.
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