FP35-6 The tumor suppressor protein menin regulates the phosphorylation of Hlxb9, a pancreatic β-cell differentiation factor implicated in β-cell tumors

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP35-Neoplasia of Endocrine Tissues
Bench to Bedside
Monday, June 17, 2013: 10:45 AM-11:15 AM
Presentation Start Time: 11:10 AM
Room 122 (Moscone Center)

Poster Board MON-292
Shruti S Desai*1, Sita D Modali2, Vaishali I Parekh3 and Sunita K Agarwal1
1National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, 2National Institute of Diabetes and Digestive and Kidney Diseases,NIH, Bethesda, MD, 3National Institute of Diabetes and Digestive and Kidney Diseases, NIH,, Bethesda, MD
Multiple endocrine neoplasia type 1 (MEN1) is a tumor syndrome caused by heterozygous germline mutation in the MEN1 gene, which encodes for the protein menin. Biallelic loss-of-function of the MEN1 gene in affected tissues leads to tumor development. The main endocrine tissues affected in this disorder are the parathyroids, endocrine pancreas, and anterior pituitary. Men1 loss in mouse models also leads to the development of MEN1-associated endocrine tumors that also include the insulin-secreting pancreatic islet β-cell tumors. Such β-cell tumors show increased expression of Hlxb9, an embryonic β-cell differentiation factor. Although menin is shown to directly interact with Hlxb9, the mechanism by which menin regulates Hlxb9 in normal as well as in tumorigenic conditions has not been determined. Phospho-peptide analysis revealed two potential phosphorylation sites in Hlxb9 at ser78 and ser80, which are conserved in mouse and human. Therefore, we hypothesized that the phosphorylation status of Hlxb9 could play an important role in β-cell tumorigenesis. To demonstrate the existence of Hlxb9 phosphoryation at ser78 and ser80, plasmids expressing Myc-tagged Hlxb9-wt or Hlxb9-AA (ser78 and ser80 mutated to alanine) were generated. Western blot analysis of protein extracts from MIN6 cells transfected with the Hlxb9-wt plasmid showed 2 bands, and with the Hlxb9-AA plasmid showed only the lower band; indicating a secondary modification (phosphorylation) at ser78 and ser80. Also, the unphosphorylated isoform of Hlxb9 (Hlxb9-AA) showed reduced expression. Cycloheximide treatment showed that the phosphorylated isoform of Hlxb9 had an increased half-life and thus was more stable than the unphosphorylated AA isoform.  MG132 or NH4Cl treatment showed that Hlxb9 was not degraded by the proteasomal or lysosomal pathway, respectively. Therefore, it is possible that Hlxb9 is stabilized due to phosphorylation at ser78 and ser80. Interestingly, transient transfection of Hlxb9-wt in MIN6 cells in menin knockdown conditions showed that the phosphorylated isoform of Hlxb9 was increased. Therefore, phosphorylation of Hlxb9 could play an important role in endocrine tumors upon menin loss. Further studies using a phospho-specific Hlxb9 antibody would be useful to validate our findings and to determine the phosphorylation status of Hlxb9 in endocrine tumors.

Nothing to Disclose: SSD, SDM, VIP, SKA

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