Abstract
The product of the von Hippel-Lindau (VHL) tumor suppressor gene, pVHL, functions as a ubiquitin-protein isopeptide ligase in regulating HIF-1 protein turnover, thus accounting for the increased transcription of hypoxia-inducible genes that accompanies VHL mutations. The increased vascular endothelial growth factor mRNA stability in cells lacking pVHL has been hypothesized to be due to a similar regulation of an RNA-binding protein. We report the expression of the GLUT-1 3'-untranslated region RNA-binding protein, heteronuclear ribonucleoprotein (hnRNP) A2, is specifically increased in pVHL-deficient cell lines. Enhanced hnRNP A2 expression was apparent in all cell fractions, including polysomes, where a similar modest effect on hnRNP L (a GLUT-1 and VEGF 3'-untranslated region-binding protein), was seen. Steady state levels of hnRNP A2 mRNA were unaffected. Regulation of hnRNP A2 levels correlated with the ability of pVHL to bind elongin C. Proteasome inhibition of cells expressing wild type pVHL selectively increased cytoplasmic hnRNP A2 levels to that seen in pVHL-deficient cells. Finally, an in vivo interaction between pVHL and hnRNP A2 was demonstrated in both the nucleus and the cytoplasm. Collectively, these data indicate that hnRNP A2 expression is regulated by pVHL in a manner that is dependent on elongin C interactions as well as functioning proteasomes.
Highlights
Cell carcinomas are highly associated with mutation or transcriptional silencing of the von Hippel-Lindau (VHL) gene and subsequent loss or inactivation of the remaining VHL allele [6, 7]
We demonstrate that heteronuclear ribonucleoprotein (hnRNP) A2 levels in RCC cell lines vary inversely as a function of pVHL expression
The effects of pVHL on hnRNP A2 levels were not due to altered sequestration, as equivalent effects were seen with cells directly lysed in SDSPAGE loading buffer
Summary
Vol 276, No 43, Issue of October 26, pp. 40346 –40352, 2001 Printed in U.S.A. The Von Hippel-Lindau Protein Interacts with Heteronuclear Ribonucleoprotein A2 and Regulates Its Expression*. It has been hypothesized that a similar mechanism accounts for the stabilization of GLUT-1 and VEGF mRNA observed in pVHL-deficient cells [45, 47] In this model, an RNA-binding protein, instead of a transcription factor such as HIF-1␣, constitutes the target by which pVHL regulates the stability of these mRNA. An in vivo interaction between pVHL and hnRNP A2 is demonstrated in both proteasomally inhibited cytosolic and untreated nuclear extracts These data indicate that hnRNP A2 expression is regulated by pVHL, and suggest the possibility that hnRNP A2 is a target of the pVHL/elongin B/C/Cul2/Rbx ubiquitin degradation machinery
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