Research Interest

Research Interest

Bryce M. Paschal, Ph.D.
Associate Professor of Biochemistry and Molecular Genetics
Center for Cell Signaling
Department of Biochemistry and Molecular Genetics

Research Interests:
Nuclear Transport, Signaling, and Cancer

My laboratory studies how nuclear transport and signal transduction control the compartmentalization and activity of transcription factors, particularly in the context of prostate cancer. A major focus of these studies is the androgen receptor (AR), a steroid hormone receptor that is critical for prostate cell growth. AR must undergo nuclear import in order to function as a transcription factor, a process that is normally regulated by androgen (Fig. 1). There is compelling evidence, however, that AR acquires the ability to undergo androgen-independent import in advanced prostate cancer. Defining the underlying transport mechanism might reveal new strategies to inhibit AR activity in proliferating prostate cells. AR undergoes cycles of nuclear import and export (nucleocytoplasmic shuttling). While nuclear export would be expected to provide an effective mechanism for terminating a transcriptional response to androgen, we have recently found that AR translocation to the cytoplasm is important for its activity in the nucleus. This apparent paradox may reflect an undefined step in AR maturation, or crosstalk between AR and signal transduction pathways in the cytoplasm.

 

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Fig. 1. Nuclear import of the androgen receptor (AR) is induced by androgen.
Cos7 cells expressing AR fused to green fluorescent protein (GFP-AR) were treated with synthetic androgen (R1881, 1 nM) and monitored by real-time confocal microscopy.  GFP-AR is excluded from the nucleus prior to androgen addition (t=0 min), is readily visible in the nucleus soon after androgen addition (t=15 min).

AR is the target of multiple kinases, and we have generated phosphosite antibodies to study the pathways and functions of phospho-regulation.   In the course of these studies we discovered a novel mechanism for loading protein phosphatase 2A (PP2A) onto AR. The loading mechanism requires small t antigen, a product encoded by SV40 that binds and alters the structure of a PP2A subunit. Current experiments are aimed at defining the structural basis of the PP2A loading reaction, as well as determining the cellular factors that mediate PP2A loading onto AR in untransformed cells.

Translocation of AR through the nuclear pore complex (NPC) relies on three features common to most nuclear transport pathways: (i) the use of nuclear import or export signals; (ii) the recognition of these signals by receptors that mediate translocation through the NPC; and (iii) RanGTPase-dependent assembly and disassembly of transport complexes (Fig.2). The signals that specify nuclear import and export of AR, the receptors that mediate AR translocation, and the role of the RanGTPase in AR transport are all under investigation in my laboratory. This analysis includes a Mog1, a nuclear protein that regulates the GTPase cycle of Ran.

While biochemical approaches are the cornerstone of our nuclear transport and signal transduction studies, we also employ cell biological approaches including microinjection, fluorescence microscopy and real-time imaging in live cells. Our studies benefit from animal models, and from collaborations with experts in biological mass spectrometry, pathology, and prostate cancer.

 

Fig.2. Overview of some major nuclear transport pathways in eukaryotic cells.
(A) Nuclear import of RanGDP mediated by NTF2. (B) Nuclear import of NLS cargo mediated by the importin-α:importin-β heterodimer (abbreviated Imp-α and Imp-β). Nuclear import of NLS cargo mediated by direct binding to importin-β and other importin-β family members is not shown. (C) Nuclear export pathways that mediate recycling of importin-β and importin-α the latter requires CAS as an export receptor. (D) Nuclear export of NES cargo mediated by Crm1. RanGAP is anchored to the cytoplasmic side of the NPC and RanGEF is bound to chromatin. For simplicity the model depicts the only the minimal set of components necessary to form the pre-translocation transport complexes; accessory factors and post-translocation intermediates are not shown.

 

 

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