[Cell Cycle 3:5, 634-637; May 2004]; ©2004 Landes Bioscience
Inhibition of Chk1 by Activated PKB/Akt
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Brief Report
*Correspondence to: Emma Shtivelman; Cancer Research Institute; University of California San Francisco; 2340 Sutter Street; Room S333; San Francisco, California 94115 USA; Tel.: 415.502.1985; Fax: 415.502.3179; Email:
[email protected] Received 03/17/04; Accepted 03/31/04
PKB is a central effector of the phosphatydilinositol 3-kinase (PI3K) pathway, one of the most frequently activated signal transduction pathways in tumors (reviewed in ref. 1). PKB is activated by signaling from PI3K, which is antagonized by the tumor suppressor gene PTEN mutated or deleted in a wide variety of human tumor.2 PTEN dephosphorylates phosphatidylinositol (3,4,5) triphosphate (PIP3), the primary product of PI3K.3 Activation of PKB is initiated by the binding of PIP3 to the pleckstrin homology domain. This binding results in the subsequent targeting of PKB to the plasma membrane and activating phosphorylation events at residues Thr 308 and Ser 473 by other kinases such as PDK-1 (reviewed in ref. 4). In mammals, there are three genes encoding three different isoforms of PKB that are highly conserved in terms of sequence as well as substrate specificity but have different tissue expression patterns. Double knockout mice for PKBa,β have severe defects and die soon after birth or pre-natally.5 PKB is probably best known for its critical role in cell survival though its activation has numerous consequences: it affects transcription, protein synthesis, metabolism, apoptosis and cell cycle.1,6 Recent evidence demonstrate that PKB is also involved in positive regulation of the G2/M transition. Activity of PKB is increased in the G2/M phase of cell cycle in epithelial cells and is necessary for the timely progression through mitosis.7 Inhibition of the PI3K prevents or delays the transition from G2 to mitosis and results in apoptosis of cells arrested in G2.7,8 PKB plays a key role in these events because expression of a constitutively active PKB (caPKB) that is unresponsive to phosphatidylinositol signaling alleviates all the effects of PI3K inhibition in G2/M.7 Recent results9 show that activated PKB abrogates the DNA damage induced G2 arrest. We have identified two direct targets of PKB activity that are likely to be relevant to the regulation of G2/M by PKB under normal conditions and after DNA damage: mitotic checkpoint protein CHFR10 and DNA damage effector kinase Chk1.7 Multiple lines of evidence have confirmed that Chk1 is a conserved checkpoint kinase and is critical for the S phase and G2/M checkpoints (reviewed in refs. 11,12). Various types of DNA damage, including irradiation (IR), ultraviolet, hydroxyurea and DNA damaging drugs lead to activation of Chk1. DNA damage sensor kinases ATM/ATR directly phosphorylate Chk1 on serines 345 and 31711,12 leading to its activation. Other checkpoint proteins are also necessary for Chk1 activation: Chk1 associates with a conserved protein claspin during execution of replication checkpoint,13,14 and with tumor suppressor BRCA1 during response to double-stranded DNA breaks.15 The known mammalian targets of Chk1 are Cdc25 phosphatases (reviewed in ref. 12), tumor suppressor p5316 and the Tousled kinases,17 which contribute to cell cycle arrest and possibly apoptosis after DNA damage. Chk1 is essential for embryonic development,18,19 and its dele-
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This manuscript has been published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=894
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2Department of Molecular Genetics; College of Medicine; University of Illinois at Chicago; Chicago, Illinois USA
We have shown recently that DNA damage effector kinase Chk1 is phosphorylated in vitro by protein kinase B/Akt (PKB/Akt) on serine 280. Activation of Chk1 by DNA damage in vivo is suppressed in presence of activated PKB. In this study we show that Chk1 is phosphorylated by PKB in vivo, and that increased phosphorylation by PKB on serine 280 correlates with impairment of Chk1 activation by DNA damage. Our results indicate a likely mechanism for the negative effects that phosphorylation of serine 280 has on activation of Chk1. The Chk1 protein phosphorylated by PKB on serine 280 does not enter into protein complexes after replication arrest. Moreover, Chk1 phosphorylated by PKB fails to undergo activating phosphorylation on serine 345 by ATM/ATR. Phosphorylation by ATM/ATR and association with other checkpoint proteins are essential steps in activation of Chk1. Inhibition of these steps provides a plausible explanation for the observed attenuation of Chk1 activation by activated PKB after DNA damage.
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1Cancer Research Institute; University of California San Francisco; San Francisco, California USA
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ABSTRACT
Frank. W. King1 Jennifer Skeen2 Nissim Hay2 Emma Shtivelman1,*
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PKB/Akt, Chk1, phosphorylation
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INHIBITION OF CHK1 BY ACTIVATED PKB/AKT
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by LY294002). Finally, the anti-pS2800 antibodies did not recognize the mutant Chk1S280A with serine 280 to alanine substitution, confirming their specificity (Fig. 1A). To confirm that serine 280 of Chk1 is a specific PKB phosphorylation site, we have used fibroblasts derived from mouse embryos with homozygous deletion of both PKBα and β.5 PKBγ is practically not expressed in fibroblasts, and Western blot analysis of these cells with the anti-“pan” PKB antibodies fails to detect expression of PKB (not shown). The mouse embryo fibroblasts (MEF) from both wild-type and PKBα, β deleted embryos were immortalized with a dominant-negative p53 and used for Western blot analysis at an early passage number. While levels of Chk1 protein in both types of MEF were similar, we observed that Chk1 protein in wild type MEF contained phosphorylated serine 280, while Chk1 in PKB-null MEF did not (Fig. 1B). In addition, we have noticed that Chk1 in PKB-null cells appears to have reduced electrophoretic mobility compared to the Chk1 protein from the wild type cells. In general, slow electrophoretic mobility is characteristic for the activated Chk1 kinase.21 The absence of Chk1 phosphorylation on serine 280 in cells lacking expression of PKB provides a strong evidence for the specificity of this phosphorylation by PKB. We described previously that constitutively active PKB (caPKB) prevents activation of Chk1 by hydroxyurea (HU) treatment that induces replication arrest.7 CaPKB lacks the pleckstrin homology domain and, to ensure the constitutive activity of this enzyme in the absence of membrane targeting, two residues that are phosphorylated in activated PKB—serine 473 and threonine 308—were substituted by aspartic acid residues. Treatment with HU activated Chk1 in control MCFneo cells but not in MCFcaPKB cells stably expressing caPKB.7 Similarly, we observed now that in response to γ-irradiation (IR), Chk1 was activated in MCFneo, but not significantly in MCFcaPKB cells (Fig. 2A). Phosphorylation of Chk1 on serine 280 was higher in MCFcaPKB cells than in MCFneo cells, and was noticeably increased in MCFcaPKB after IR, correlating with the lack of Chk1 activation in these cells (Fig. 2A). Treatment with HU induced a significant decrease of pS280 content in Chk1 from MCFneo cells, but not from MCFcaPKB cells (data not shown).
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Figure 2 (Left). Chk1 fails to be activated by IR in presence of activated PKB (A) MCFneo and MCFcaPKB cells (left panels) and HeLa-PKB-ER cells, incubated with or without tamoxifen (4-OHT, right panels), where treated with 8 Gy of IR and incubated for 2 hrs. Cell extracts were prepared and analyzed for Chk1 kinase activity (top panels), by Western blotting with anti-pS280 antibodies (middle panels) and anti-Chk1 protein antibodies (bottom panels). (B) Flow cytometric analysis of DNA content in neuroblastoma lines before and 8 hours after IR (10 Gy). Numbers below the histograms show percentage of cells with
