Suppression of autophagy impedes glioblastoma development and induces senescence

The function of macroautophagy/autophagy during tumor initiation or in established tumors can be highly distinct and context-dependent. To investigate the role of autophagy in gliomagenesis, we utilized a KRAS-driven glioblastoma mouse model in which autophagy is specifically disrupted via RNAi against Atg7, Atg13 or Ulk1. Inhibition of autophagy strongly reduced glioblastoma development, demonstrating its critical role in promoting tumor formation. Further supporting this finding is the observation that tumors originating from Atg7-shRNA injections escaped the knockdown effect and thereby still underwent functional autophagy. In vitro, autophagy inhibition suppressed the capacity of KRAS-expressing glial cells to form oncogenic colonies or to survive low serum conditions. Molecular analyses revealed that autophagy-inhibited glial cells were unable to maintain active growth signaling under growth-restrictive conditions and were prone to undergo senescence. Overall, these results demonstrate that autophagy is crucial for glioma initiation and growth, and is a promising therapeutic target for glioblastoma treatment

Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of beta-actin and gamma-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.Publisher PDFPeer reviewe

Mps1Mph1 kinase phosphorylates Mad3 to inhibit Cdc20Slp1-APC/C and maintain spindle checkpoint arrests

<div><p>The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1^Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1^Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors <i>in vitro</i>, demonstrating that Mad3p modification can directly influence Cdc20^Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1^Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.</p></div

Kinase Activity of Fission Yeast Mph1 Is Required for Mad2 and Mad3 to Stably Bind the Anaphase Promoting Complex

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6–8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest

Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of beta-actin and gamma-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.</p

<i>mad3</i> and <i>mad2</i> phospho-mutants fail to maintain stable MCC-APC/C complexes.

<p>A. The <i>cdc25</i> strains indicated were pre-synchronised in G2 by shifting to 36°C for 3.5 hours. They were then released at 25°C and time points taken every 15 minutes. Carbendazim (CBZ) was added after 20 minutes. Extracts were made, Apc4-TAP pulled down with IgG Dynabeads, separated by SDS-PAGE and immunoblotted for associated Mad3p and Mad2p. B. The immunoblots were quantitated for Mad3p and Mad2p levels, normalised to Apc4p levels, and then plotted relative to the wild-type levels at the 45 and 60 minute time points (after release into mitosis). Plotted as mean with standard deviation (n = 2 experiments). C. Methanol-fixed cells were stained with calcofluor and scored for septation, indicating a failure to maintain spindle checkpoint arrest. This experiment was repeated twice and >100 cells scored for each strain at each time point. Plotted as mean with SEM.</p

Mad3p modification <i>directly</i> affects its ability to inhibit APC/C activity.

<p>A. Mad3p C-terminal sequence highlighting the residues mutated in the 3, 4 and 7D/E mutants. B. Fission yeast APC/C <i>in vitro</i> activity reactions showing the ubiquitination of securin^Cut2. Half of each reaction (10 μl) was taken immediately after mixing all the components and added to 4xSDS gel buffer (0 min). The remaining 10 μl were incubated at 23°C for 45 min. Control reactions, one lacking the activator Cdc20^Slp1 (-Cdc20), one lacking the APC/C (-APC/C) and one containing both Cdc20^Slp1 and APC/C (+APC/C), are shown together with wild-type Mad3p (WT), the double KEN box mutant (<i>mad3-KEN1</i>,<i>2-AAA)</i> and the corresponding phospho-mimic mutants at 1:20 and 1:100 APC/C:Mad3p molar ratios respectively. Purified cMad2p was added at 1:12.5 APC/C:Mad2p molar ratio in all reactions. A phosphoimager and Imagequant software were used to quantify the amount of radioactivity left in the un-modified securin band (*). C. Quantification of APC/C inhibition by wild-type (WT) Mad3p and mutants. Results from three independent experiments were combined and plotted (as mean with SEM). Data were normalised against APC/C activity containing no Mad2p or Mad3p (+APC/C).</p

MCC assembly defects are rather minor in the <i>mad3-C9A</i> and <i>mad2 mad3</i> double phospho-mutants when compared to <i>mps1-kd</i>.

<p>A. The <i>cdc25</i> strains indicated were pre-synchronised in G2 by shifting to 36°C for 3.5 hours. They were then released at 25°C and time points taken every 15 minutes. Native extracts were made and Cdc20^Slp1 was immunoprecipitated with anti-Flag antibodies. Immunopreciptates were separated by SDS-PAGE and immunoblotted for Cdc20^Slp1, Mad3p (anti-GFP) and Mad2p. B. These immunoblots were quantitated and plotted as the intensity of Mad3 or Mad2 relative to the Cdc20^Slp1 level at each time point (45 and 60 minutes after release into mitosis). All values were then compared to wild-type–see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005834#sec011" target="_blank">Materials and Methods</a> for details. C. Cell cycle progression was scored by DAPI staining methanol fixed cells and quantifying the % of bi-nucleate cells in the population at each time point (200 cells were scored at each time point).</p