title: Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells.
abstract: Mouse embryonic stem cells (ES cells) can proliferate indefinitely. To identify potential signals involved in suppression of self-renewal, we previously screened a kinase/phosphatase expression library in ES cells, and observed that inhibition of Dual Leucine zipper-bearing Kinase (DLK) increased relative cell numbers. DLK protein was detected in both the pluripotent and differentiated states of mouse ES cells while DLK kinase activity increased upon differentiation. Overexpression of DLK in mouse ES cells displayed reductions in relative cell/colony numbers and Nanog expression, suggesting a suppressive role of DLK in self-renewal. By examining protein sequences of DLK, we identified 2 putative Akt phosphorylation sites at S584 and T659. Blocking PI3K/Akt signaling with LY-294002 enhanced DLK kinase activity dramatically. We found that Akt interacts with and phosphorylates DLK. Mutations of DLK amino acid residues at putative Akt phosphorylation sites (S584A, T659A, or S584A and T659A) diminished the level of DLK phosphorylation. While the mutated DLKs (S584A, T659A, or S584A and T659A) were expressed, a further reduction in cell/colony numbers and Nanog expression appeared in mouse ES cells. In addition, these mutant DLKs (S584A, T659A, or S584A and T659A) exhibited more robust kinase activity and cell death compared to wild type DLK or green fluorescence (GFP) controls. In summary, our results show that DLK functions to suppress self-renewal of mouse ES cells and is restrained by Akt phosphorylation.
authors: a Graduate Institute of Life Sciences; National Defense Medical Center ; Taipei , Taiwan.
authors: Cheng-Chung
authors: CC
authors: Wu
authors: Hong-Jin
authors: HJ
authors: Wu
authors: Chia-Hui
authors: CH
authors: Wang
authors: Chia-Hua
authors: CH
authors: Lin
authors: Shu-Ching
authors: SC
authors: Hsu
authors: Yi-Rong
authors: YR
authors: Chen
authors: Michael
authors: M
authors: Hsiao
authors: Scott C
authors: SC
authors: Schuyler
authors: Frank Leigh
authors: FL
authors: Lu
authors: Nianhan
authors: N
authors: Ma
authors: Jean
authors: J
authors: Lu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D002867:Chromones; D018398:Homeodomain Proteins; D020930:MAP Kinase Kinase Kinases; D009025:Morpholines; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; C475540:Nanog protein, mouse; C494174:Pou5f1 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C090757:mitogen-activated protein kinase kinase kinase 12
keywords: Akt; Dual Leucine zipper-bearing Kinase (DLK); Nanog; mouse embryonic stem cells; self-renewal