GSBS Dissertations and Theses

Title

Cloning, Characterization and Functional Analysis of TPR, an Oncogene-Activating Protein of the Nuclear Pore Complex: A Dissertation

Approval Date

March 1998

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Molecular Cell Biology

Subjects

Nuclear Pore Complex Proteins; Proto-Oncogene Proteins; Nuclear Envelope; Oncogene Proteins, Fusion; Cloning, Molecular; Academic Dissertations; Dissertations, UMMS

Abstract

A monoclonal antibody, mAb 203.37, raised against purified nuclear matrix proteins identified a single ~270 kDa protein that localized to the nuclear envelope. Double-label immunofluorescent microscopy using differential permeabilization protocols showed that this protein was present exclusively on the nucleoplasmic side of the nuclear envelope and that it co-localized with components of the nuclear pore complex. The nucleotide sequence of clones isolated using mAb 203.37 identified this protein as Tpr, a protein previously shown to be involved in oncogenic fusions with a number of protein kinases. Sequence analysis showed Tpr to be a 2348 amino acid protein with a predicted molecular weight of 265 kDa protein and a bipartite structure consisting of an ~1600 amino acid N-terminal domain that is almost entirely an α-helical coiled-coil followed by a highly acidic non-coiled carboxy-terminus. Ectopic expression of epitope-tagged Tpr constructs revealed two functional domains for Tpr: a nuclear pore complex binding domain and a nuclear localization sequence. The amino-terminus of Tpr, the portion of the protein shown to activate protein kinase oncogenes, did not localize to the nuclear pore complex indicating that the transforming activity of Tpr-protein kinase chimeras did not involve interactions with the nuclear pore complex. Ectopic expression of Tpr and a number of Tpr constructs resulted in the accumulation of poly (A)+ RNA in the nuclear interior but did not effect the import of a reporter protein into the nucleus indicating a role for Tpr in the export of mRNA from the nucleus.

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