Requirement of the mTOR kinase for the regulation of Maf1 phosphorylation and control of RNA polymerase III-dependent transcription in cancer cells.
The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1-4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors.
Discovery Oncology, Wyeth Research, Pearl River, New York 10965. Electronic address: shorb@wyeth.com.
Boris
B
Shor
Biological Technologies, Wyeth Research, Cambridge, Massachusetts 02140.
Jiang
J
Wu
Biological Technologies, Wyeth Research, Cambridge, Massachusetts 02140.
Quazi
Q
Shakey
Discovery Oncology, Wyeth Research, Pearl River, New York 10965.
Lourdes
L
Toral-Barza
Discovery Oncology, Wyeth Research, Pearl River, New York 10965.
Celine
C
Shi
Biological Technologies, Wyeth Research, Cambridge, Massachusetts 02140.
Max
M
Follettie
Discovery Oncology, Wyeth Research, Pearl River, New York 10965. Electronic address: yuker2b@gmail.com.
Ker
K
Yu
D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012320:RNA Polymerase III / Q000378:metabolism*; D034741:RNA, Small Interfering; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D058570:TOR Serine-Threonine Kinases; D053719:Tandem Mass Spectrometry; D014158:Transcription, Genetic
D047908:Intracellular Signaling Peptides and Proteins; C520535:MAF1 protein, human; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C546842:MTOR protein, human; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D012320:RNA Polymerase III