? Previous studies by the applicant have focused on the regulation of the cholinergic gene locus. This locus is composed of two proteins, choline acetyltransferase (CHAT) which synthesizes acetylcholine and the vesicular acetylcholine transporter (VAChT) which transports acetylcholine into synaptic vesicles. It was found that ChAT and VAChT are coordinately regulated through a protein kinase All dependent pathway. This regulation involves repression of the locus by the zinc finger transcription factor REST/NRSF, and de-repression by a neuron specific isoform of REST/NRSF called REST4. REST4 is formed through alternative splicing of the REST/NRSF gene in a process regulated by protein kinase All. REST4 was found to be localized to the nucleus, with the nuclear localization signal mapped within zinc finger domains 2-5. This domain contained signals that not only localized REST4 to the nucleus, but also translocated REST4 into the nucleus. These findings lead to a study of the nuclear trafficking of REST4 as well as REST/NRSF. A yeast-two hybrid screen using zinc finger domains 2-5 as bait resulted in the cloning of a novel cDNA. The deduced amino acid sequence of this cDNA showed the presence of three LIM domains, a domain reported to act as a protein-protein interaction domain. This protein was named the REST/NRSF-Interacting LIM domain Protein (RILP). The long-term goal of this proposal is to reveal the physiological function of RILP using molecular genetic, biochemical and structural approaches. To achieve this goal three specific aims are proposed. The first specific aim is to characterize the importance of LIM domains, C-terminal prenylation, phosphorylation, and nuclear localization signals (NLSs) for RILP function. The second specific aim is to study the effect of RILP on REST/NRSF regulated neuronal gene expression. The third specific aim is to identify RILP interacting protein. These would be either other proteins in which RILP is involved in their translocation to the nucleus or nuclear envelope proteins that RILP interacts with. These studies mentored by Drs. Louis Hersh and Brett Spear will permit the candidate to expand his expertise into new areas and to develop a research program that should lead to independent research funding. Dr. Shimojo will have access to shared-use equipment, laboratory space, and faculty and staff, which will enhance the likelihood that the proposed objectives will be successfully accomplished. ? ?
|Bassuk, Alexander G; Wallace, Robyn H; Buhr, Aimee et al. (2008) A homozygous mutation in human PRICKLE1 causes an autosomal-recessive progressive myoclonus epilepsy-ataxia syndrome. Am J Hum Genet 83:572-81|
|Shimojo, Masahito (2008) Huntingtin regulates RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) nuclear trafficking indirectly through a complex with REST/NRSF-interacting LIM domain protein (RILP) and dynactin p150 Glued. J Biol Chem 283:34880-6|
|Shimojo, Masahito; Hersh, Louis B (2006) Characterization of the REST/NRSF-interacting LIM domain protein (RILP): localization and interaction with REST/NRSF. J Neurochem 96:1130-8|
|Shimojo, Masahito (2006) Characterization of the nuclear targeting signal of REST/NRSF. Neurosci Lett 398:161-6|