Translational deregulation in PDK-1-/- embryonic stem cells.
PDK-1 is a protein kinase that is critical for the activation of many downstream protein kinases in the AGC superfamily, through phosphorylation of the activation loop site on these substrates. Cells lacking PDK-1 show decreased activity of these protein kinases, including protein kinase B (PKB) and p70S6K, whereas mTOR activity remains largely unaffected. Here we show, by assessing both association of cellular RNAs with polysomes and by metabolic labeling, that PDK-1-/- embryonic stem (ES) cells exhibit defects in mRNA translation. We identify which mRNAs are most dramatically translationally regulated in cells lacking PDK-1 expression by performing microarray analysis of total and polysomal RNA in these cells. In addition to the decreased translation of many RNAs, a smaller number of RNAs show increased association with polyribosomes in PDK-1-/- ES cells relative to PDK-1+/+ ES cells. We show that PKB activity is a critical downstream component of PDK-1 in mediating translation of cystatin C, RANKL, and Rab11a, whereas mTOR activity is less important for effective translation of these targets.
Tominaga
Yuichi
Y
Cancer Research Institute, University of California, 2340 Sutter St. N319, San Francisco, CA 94115, USA.
Tamgüney
Tanja
T
Kolesnichenko
Marina
M
Bilanges
Benoit
B
Stokoe
David
D
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D055316:Cystatin C; D015891:Cystatins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000166:cytology*; D004789:Enzyme Activation; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D053245:RANK Ligand; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D020133:Reverse Transcriptase Polymerase Chain Reaction; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology*; D013395:Sucrose / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013997:Time Factors; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D064413:3-Phosphoinositide-Dependent Protein Kinases; D002352:Carrier Proteins; D055316:Cystatin C; D015891:Cystatins; D008562:Membrane Glycoproteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D053245:RANK Ligand; D012313:RNA; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D013395:Sucrose; D058570:TOR Serine-Threonine Kinases; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C525030:CST3 protein, human; C525031:Cst3 protein, mouse; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; C546842:MTOR protein, human; C546843:mTOR protein, mouse