Molecular analysis of the LHX3 neuroendocrine transcription factor protein

Gretchen Elizabeth Parker, Purdue University

Abstract

The LIM homeodomain family of transcription factors consists of many crucial regulators of development in complex organisms. The studies described here were undertaken to better comprehend the molecular features necessary for the subcellular localization, post-translational modification, and transcriptional activity of the LHX3 LIM homeodomain protein. LHX3 is a transcription factor that is essential for development of the anterior and intermediate pituitary lobes and for motor neuron specification in mammals. Using functional fluorescent derivatives of LHX3 and confocal microscopy, it was demonstrated that the LHX3 protein is found within both the nucleoplasm and nuclear matrix cellular compartments. Four nuclear localization signals were mapped within the homeodomain and the carboxyl terminus. Specific combinations of these signals can transfer nuclear localization to cytoplasmic proteins. Mutation of the signals within the homeodomain inhibited LHX3 function. By contrast, mutation of the carboxyl terminal nuclear localization signal activated LHX3 function, indicating that this region is critical to transcriptional activity and may be a target of repressive pathways. Conservation of the identified nuclear localization signals and of the potential sites for post-translational modification by phosphorylation in both LHX3-class and LIM homeodomain family regulators suggests both class-specific and common mechanisms by which these factors may be regulated. Protein biochemistry and proteomic analyses were used to monitor post-translational modification(s) of the LHX3 protein. Two-dimensional gel electrophoresis revealed a separated protein of the predicted molecular weight and isoelectric point, consistent with post-translational modification of LHX3. On-line capillary liquid chromatography electrospray tandem ion trap mass spectrometry confirmed post-translational modification of the molecule by phosphorylation on five residues within important functional domains of the protein. Further, an analysis of abnormal LHX3 proteins associated with human pituitary disease demonstrated that mutations in LHX3 do not prevent interaction with partner proteins or DNA binding, but do impair LHX3 transactivation function. In additional experiments, the role of LHX3 in FSHβ gene activation was investigated. Together, these studies illustrate that the LHX3 protein has modular components that allow the appropriate localization and activity of the protein.

Degree

Ph.D.

Advisors

Rhodes, Purdue University.

Subject Area

Molecular biology

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