Controlling transcription factor activity by regulating nuclear and cytoplasmic localization is an important and evolutionarily conserved mechanism. Members of the nuclear receptor (NR) super-family of ligand regulated transcription factors undergo cycles of nuclear import and export. We have found that nuclear export of the androgen receptor (AR) in prostate cancer cells is regulated by phosphorylation. Alanine mutations in two of the seven sites that undergo androgen-induced phosphorylation cause a defect in AR export to the cytoplasm. One site that regulates AR export (Ser308) is in the N-terminal domain. The other site (Ser650) is in the hinge region of AR, proximal to the DMA binding domain that functions as a nuclear export signal. We generated a panel of AR phosphosite-specific antibodies and found that Ser650 is phosphorylated by the stress-activated MAP kinase p38 in vitro and in vivo. We found that hyperosmolarity induces AR redistribution from the nucleus to the cytoplasm. This AR redistribution to the cytoplasm can be blocked by alanine mutations in AR phosphorylation sites, does not require new protein synthesis or the export receptor Crm1, and is reversible. The hypothesis of this proposal is that multiple signal transduction pathways activated by environmental stress regulate the process of nucleocytoplasmic transport.
In Aims 1 and 2 we will identify the stress kinase pathways (p38, JNK, ERK) that regulate AR redistribution, determine if the AR redistribution is due to stimulation of nuclear export or inhibition of import, and determine the contributions of individual kinases using inhibitors, siRNA, and knockout cells. In addition to regulating the localization of AR, stress signaling exerts global effects on the nuclear transport machinery by disrupting the Ran gradient which is essential for most nuclear transport pathways.
In Aim 3 we will determine whether Ran gradient disruption involves stress signaling to factors that control Ran transport or the Ran GTPase cycle. Our studies will provide new insight into the mechanisms that mediate crosstalk between signal transduction, nuclear transport, and NR activity.
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