Adenosine triphosphatase pontin is overexpressed in hepatocellular carcinoma and coregulated with reptin through a new posttranslational mechanism.
Reptin and Pontin are related ATPases associated with stoichiometric amounts in several complexes involved in chromatin remodeling, transcriptional regulation, and telomerase activity. We found that Reptin was up-regulated in hepatocellular carcinoma (HCC) and that down-regulation of Reptin led to growth arrest. We show here that Pontin messenger RNA (mRNA) is also up-regulated in human HCC 3.9-fold as compared to nontumor liver (P = 0.0004). Pontin expression was a strong independent factor of poor prognosis in a multivariate analysis. As for Reptin, depletion of Pontin in HuH7 cells with small interfering RNAs (siRNAs) led to growth arrest. Remarkably, Pontin depletion led to down-regulation of Reptin as shown with western blot, and vice versa. Whereas siRNAs induced a decrease of their cognate mRNA targets, they did not affect the transcripts of the partner protein. Translation of Pontin or Reptin was not altered when the partner protein was silenced. However, pulse-chase experiments demonstrated that newly synthesized Pontin or Reptin stability was reduced in Reptin- or Pontin-depleted cells, respectively. This phenomenon was reversed upon inhibition of proteasome or ubiquitin-activating enzyme (E1). In addition, proteasome inhibition could partly restore Pontin steady-state levels in Reptin-depleted cells, as shown by western blot. This restoration was not observed when cells were also treated with cycloheximide, thus confirming that proteasomal degradation in this setting was restricted to newly synthesized Pontin.


CONCLUSIONS
Reptin and Pontin protein levels are strictly controlled by a posttranslational mechanism involving proteasomal degradation of newly synthesized proteins. These data demonstrate a tight regulatory and reciprocal interaction between Reptin and Pontin, which may in turn lead to the maintenance of their 1:1 stoichiometry.
Haurie
Valérie
V
Institut National de la Santé et de la Recherche Médicale , U889 Bordeaux, France.
Ménard
Ludovic
L
Nicou
Alexandra
A
Touriol
Christian
C
Metzler
Philippe
P
Fernandez
Jérémy
J
Taras
Danièle
D
Lestienne
Patrick
P
Balabaud
Charles
C
Bioulac-Sage
Paulette
P
Prats
Hervé
H
Zucman-Rossi
Jessica
J
Rosenbaum
Jean
J
D000074183:ATPases Associated with Diverse Cellular Activities; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D049109:Cell Proliferation; D004265:DNA Helicases / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology*; D061988:Proteasome Inhibitors; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis
D002352:Carrier Proteins; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C486549:RUVBL1 protein, human; C122079:RUVBL2 protein, human