Abstract
PICT-1 was originally identified as a tumor suppressor. Here, we found that PICT-1 overexpression triggered pro-death autophagy without nucleolar disruption or p53 accumulation in U251 and MCF7 cells. Truncated PICT-1 fragments 181-346 and 1-346, which partly or totally lack nucleolar localization, showed weaker autophagy-inducing effects than full-length PICT-1 and a well-defined nucleolar mutant (181-479). Furthermore, PICT-1 partly localizes to the nucleolar fibrillar center (FC) and directly binds to ribosomal DNA (rDNA) gene loci, where it interacts with upstream binding factor (UBF). Overexpression of PICT-1 or the 181-479 mutant, but not the 1-346 or 181-346 mutants, markedly inhibited the phosphorylation of UBF and the recruitment of rRNA polymerase I (Pol I) to the rDNA promoter in response to serum stimulation, thereby suppressing rRNA transcription, suggesting that rRNA transcription inhibition might be an important contributor to PICT-1-induced autophagy. This is supported by the finding that CX-5461, a specific Pol I inhibitor, also induced autophagy. In addition, both CX-5461 and PICT-1, but not the 1-346 or 181-346 mutants, significantly suppressed the activation of the Akt/mTOR/p70S6K signaling pathway. Our data show that PICT-1 triggers pro-death autophagy through inhibition of rRNA transcription and the inactivation of AKT/mTOR/p70S6K pathway, independent of nucleolar disruption and p53 activation.
Highlights
The human “protein interacting with carboxyl terminus 1” (PICT-1), known as human glioma tumor suppressor candidate region 2 gene product (GLTSCR2), was originally identified as a 60 kDa (p60) interacting partner of two viral proteins, ICP0 and ICP22 [1]
In order to investigate whether the nucleolar protein PICT-1 is involved in autophagy, human glioblastoma U251 cells were co-transfected with PICT-1 and green fluorescent protein (GFP)-LC3 plasmids, and GFP-LC3 localization was examined by confocal microscopy
To further confirm these results, U251 cells overexpressing PICT-1 were treated with two autophagy inhibitors 3-methyladenine (3MA, 10 mM) and bafilomycin (BAF, 100 nM), which are known to inhibit the initiation of autophagosome and the fusion of autophagosome with lysosome, respectively
Summary
The human “protein interacting with carboxyl terminus 1” (PICT-1), known as human glioma tumor suppressor candidate region 2 gene product (GLTSCR2), was originally identified as a 60 kDa (p60) interacting partner of two viral proteins, ICP0 and ICP22 [1]. PICT-1 is considered to be a candidate tumor suppressor gene, as its diminished expression or loss is correlated with the highly malignant progression of several cancers [3,4]. In support of this hypothesis, research has shown that knockdown of PICT-1 promoted anchorage-independent tumor cell growth and decreased www.impactjournals.com/oncotarget susceptibility to apoptotic cell death in response to apoptosis-inducing stimuli, whereas overexpression of PICT-1 significantly inhibited anchorage-independent tumor cell growth and induced mitochondria-independent cell death [5,6]. The nucleolus is best known as a site of rDNA transcription and ribosome biogenesis, but accumulating evidence has demonstrated that the nucleolus participates in a diverse array of cell functions such as stress response, the cell cycle, aging processes, cell death, and several human diseases [14,15,16]
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