cyclin - dependent kinase inhibitor 1a ( p21 , cip1 ) mitochondria - associated membrane nuclear localization signal
maintenance of genome stability requires mobilization of the dna damage response ( ddr ) , both to repair the day - to - day chemical nicks and bruises and resolve acute damage following exposure to ionizing radiation ( ir ) or chemotherapeutics .
cells respond to these chemical mishaps and overtly acute genotoxic insults by inducing the tumor suppressor protein p53 ( tp53 , known as p53 ) , whose role in the regulation of the ddr is complex , involving changes in the expression of more than 3,000 genes and activation of cell cycle checkpoints to allow dna repair .
the most intensively studied p53 target genes include cyclin - dependent kinase inhibitor 1a ( cdkn1a , known as p21 ) , which promotes cell - cycle arrest or senescence , and p53 upregulated modulator of apoptosis ( puma , also known as bcl2-binding component 3 [ bbc3 ] ) and bcl2-associated x protein ( bax , also known as bcl-2-like protein-4 ) , which are activated to induce apoptosis when cellular damage is irreparable .
the transcriptional activity of p53 requires post - translational modifications , including phosphorylation and acetylation , to stabilize p53 and enhance its transactivation functions , respectively .
the class iii histone deacetylase sirtuin 1 ( sirt1 ) represses p53 transcriptional activation by deacetylating p53 following dna damage .
sirt1 can be regulated transcriptionally and post - transcriptionally and through interactions with other cellular proteins in diverse pathways .
the complexity of these functions suggests that regulation of sirt1 activity probably requires additional cellular factors .
we recently identified the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 ( pacs-2 ) as a novel mediator of p53 action in response to genotoxic insult ( fig .
pacs-2 was initially identified by its cytoplasmic roles in mediating mitochondria - associated membrane ( mam ) formation , autophagy , and protein traffic in the secretory and endocytic pathways ( fig .
1 , panel b ) . in response to apo2 ligand / tumor necrosis factor - related apoptosis - inducing ligand ( apo2l / trail ) , pacs-2 switches to a proapoptotic effector that promotes membrane permeabilization of mitochondria and lysosomes , which is required for the activation of executioner caspases and cell death ( fig .
our finding that pacs-2 is required for apo2l / trail - induced apoptosis led us to ask whether pacs-2 would similarly be required for apoptosis induced by dna damage .
surprisingly , we found that whereas sirna knockdown of pacs-2 in hct116 human colon carcinoma cells reduced apo2l / trail - induced apoptosis , it sensitized cells to doxorubicin - induced apoptosis and this effect was dependent on p53 .
thus , pacs-2 has a proapoptotic role in apo2l / trail action but an anti - apoptotic role in response to genotoxic insult ( fig .
( a ) dna damage induces nuclear pacs-2-mediated cytoprotection through inhibition of sirt1-mediated p53 deacetylation to promote p21-dependent cell cycle arrest .
( b ) phosphorylation state - dependent homeostatic function of pacs-2 to regulate either mam integrity or cargo trafficking in secretory and endocytic pathways .
( c ) signaling by death ligands triggers pacs-2 dephosphorylation to induce mitochondria and lysosome membrane permeabilization , leading to caspase-3 activation and cell death .
ac , acetylated lysine ; p , akt - phosphorylated ser437 on pacs-2 ; er , endoplasmic reticulum ; mam , mitochondria - associated membrane ; trail - r , trail receptor ; tbid , truncated bid . 
 
( a ) dna damage induces nuclear pacs-2-mediated cytoprotection through inhibition of sirt1-mediated p53 deacetylation to promote p21-dependent cell cycle arrest .
( b ) phosphorylation state - dependent homeostatic function of pacs-2 to regulate either mam integrity or cargo trafficking in secretory and endocytic pathways .
( c ) signaling by death ligands triggers pacs-2 dephosphorylation to induce mitochondria and lysosome membrane permeabilization , leading to caspase-3 activation and cell death .
ac , acetylated lysine ; p , akt - phosphorylated ser437 on pacs-2 ; er , endoplasmic reticulum ; mam , mitochondria - associated membrane ; trail - r , trail receptor ; tbid , truncated bid . to characterize this previously undescribed role for pacs-2 in ddr , we used cell cycle analyses combined with western blotting ( wb ) and in vivo enterocyte migration studies in the gastrointestinal track of pacs-2 mice .
we observed that pacs-2 knockdown reduced p53 acetylation in response to dna damage , thereby blunting the induction of p21 and cell cycle progression . this p21 repression increased apoptosis as previously described , which suggests a p21-dependent cytoprotective role for pacs-2 in ddr .
consistent with the above data , we found that the migration of bromodeoxyuridine - positive ( brdu ) enterocytes along the crypt - villus following ir was restricted in wild - type mice but not in pacs-2 mice .
together , these data describe the functional importance of pacs-2 as a specific in vivo regulator of the p53-p21 axis in ddr .
we confirmed that p53 acetylation and p21 induction levels in pacs-2 depleted cells or tissues were sirt1 dependent , as sirt1 knockdown or addition of the sirt1 inhibitor ex-527 restored p53 acetylation and p21 induction , as observed by wb , as well as p21-dependent cell cycle arrest , in pacs-2 knockdown cells .
the inhibition of sirt1 by pacs-2 was also confirmed by in vitro assays showing that pacs-2 directly binds to and inhibits sirt1-catalyzed p53 deacetylation .
together , these data support a previously undescribed role of pacs-2the inhibition of sirt1 following dna damage  as a cytoprotective component of the p53 signaling pathway , although the specific protein motifs or residues involved and the molecular mechanism by which pacs-2 inhibits sirt1 catalytic activity are still not fully understood .
the promotion of p53-dependent p21 expression by pacs-2 depended on its ability to traffic to the cell nucleus .
sequence analysis of human pacs-2 identified one nuclear localization signal ( nls ) and at least 2 putative nuclear export signals . correspondingly , mutation of the pacs-2 nls blocked its nuclear trafficking .
moreover , pacs-2 responded to dna damage by increased translocation into the cell nucleus , where it repressed sirt1-mediated p53 deacetylation .
curiously , sequence alignment of the nls sequences in pacs proteins from different species reveals that pacs-2 underwent a late evolutionary adaptation to acquire nuclear trafficking motifs .
this new , and surprising , function of pacs-2 parallels the role of p53 in directing cell cycle arrest found in higher metazoans but not in worms or flies , where p53 function is limited to apoptosis induction .
thus , these findings described a new compartment - specific function of pacs-2 following dna damage in response to the heightened need for p53 to resolve the damage .
however , the mechanism by which pacs-2 is mobilized from the cytoplasm to the nucleus in response to dna damage remains unclear .
overall , our work characterizes pacs-2 as a new inhibitor of sirt1 to specifically regulate the p53p21 axis in response to dna damage .
interestingly , the role of the nuclear pool of pacs-2 in promoting p21-dependent cell cycle arrest could correlate with the role of the cytoplasmic pool of pacs-2 in promoting autophagy .
. future work will determine how the ratio of pacs-2 pools is regulated in response to different stimuli and the crosstalk between them .
 sirt1 regulates p53 transcriptional activation in response to genotoxic insult by deacetylating key lysine residues . 
 we recently identified the multifunctional protein pacs-2 as a sirt1 inhibitor . after dna damage , pacs-2 binds and inhibits sirt1 to increase p53-dependent transactivation of the cdk inhibitor p21 ( cdkn1a ) and induce cell cycle arrest . 
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