Subcellular Localization of Apoptosis Initiator NGFI-B is Regulated by MAP Kinase ERK and Apoptosis Signals

NGFI-B (nerve growth factor-inducible clone B) is a nuclear receptor that has been implicated in both cell survival and cell death. In the nucleus, NGFI-B exerts its functions as a transcription factor that promotes survival and proliferation, while translocation of NGFI-B to the mitochondria promotes apoptosis. This unique property of NGFI-B makes it a possible therapeutic target. Many questions concerning the translocation of NGFI-B remain to be solved. This thesis will investigate several issues regarding the regulation of NGFI-B migration. Nuclear fluorescence recovery after photobleaching (FRAP)-studies in CV-1 cells suggested that the nuclear import of GFP-tagged NGFI-B was energy dependent, and therefore could be regulated by nuclear import mechanisms. The threonine 142 residue of NGFI-B was previously demonstrated to be phosphorylated by the MAP kinase ERK2, which is activated by growth factor EGF. Whether the nuclear import of NGFI-B was regulated by phosphorylation of threonine 142 of NGFI-B was investigated by FRAP studies of two NGFI-B mutants. A 142 threonine to alanine mutation in NGFI-B that blocked phosphorylation of this residue did not affect the nuclear import of NGFI-B, while a threonine to glutamate mutation that mimicked constitutive phosphorylation possibly decreased the nuclear import of NGFI-B. Additional FRAP studies showed that EGF and ERK2 reduced the nuclear import of NGFI-B. Cultured cerebellar granule neurons (CGN) which are extensively used in studies of glutamateinduced toxicity, important in stroke and neurodegenerative diseases, were used as an in vitro model for studies of neuronal apoptosis. Cell death of CGN induced by glutamate appeared to be slightly reduced by inhibition of the MEK/ERK pathway, contrarily to EGF-induced ERK activation which is reported to be involved in neuronal survival. The translocation of NGFI-B out of the nucleus demonstrated by immunostaining after glutamate exposure suggests that CGN may be used as a model for determination of neuronal NGFI-B traffic. In conclusion, the subcellular localization of apoptosis initiator NGFI-B is regulated by MAP kinase ERK and apoptosis signals