b55 is a regulatory b subunit of the phosphatase complex pp2a ( sontag , 2001 ) .
pp2a participates in an immense number of pathways , and this explains the lethal phenotype associated with knockout of the catalytic c subunit in mice ( gtz et  al . , 1998 ) .
few b - subunit knockout mice have been generated , and none has shown lethality ; instead , these mice have tissue - specific pathological features ( louis et  al . , 2011 ,
, the regulatory b subunits only contribute to pathways ascribed to their specific substrates so that they act uniquely under different pathological contexts ( sablina et  al . , 2010 ) .
we recently found that pp2a / b55 promotes the growth of colorectal cancer by dephosphorylating phd2 and modifying its enzymatic properties ( di conza et  al . , 2017 ) .
phd2 ( or egln1 ) is a member of the hypoxia - inducible factor ( hif)-prolyl hydroxylase domain proteins ( phds ) family ( epstein et  al . , 2001 ) .
these enzymes are crucial for hif activation and the cellular response to hypoxia ( chan et  al . , 2005 ) .
indeed , under normoxia , phds utilize oxygen and alpha - ketoglutarate ( -kg ) to carry out a hydroxylation reaction on specific hif prolines that lead to ubiquitination and proteasomal degradation ( keith et  al . , 2011 ) . in  addition to oxygen and cosubstrates , the mtor pathway ,
when dephosphorylated by pp2a / b55 , phd2 shows impaired enzymatic activity , allowing full activation of hif1 in hypoxia ( di conza et  al . , 2017 ) . here , we investigate the molecular interaction between phd2 and b55 in response to glucose starvation in the context of breast cancer cells .
the main function of phd2 is the hydroxylation at specific proline residues within the alpha subunit of hifs . based on our previous findings showing the interaction and negative regulation of phd2 by b55/pp2a ( di conza et  al . ,
2017 ) , we wondered whether phd2 was able to degrade b55 in a feed - forward loop .
we found that co - overexpression of phd2 and b55 in hek293 t cells resulted in reduced b55 protein levels ( figure  1a ) that was linked to its increased ubiquitination ( figure  1b ) and proteasomal degradation ( figure  1c ) .
the control of b55 protein levels was specific for phd2 only , because either phd1 or phd3 was not able to induce b55 proteasomal degradation ( figure  s1a ) . mirroring the overexpression data , phd2 knockdown increased the half - life of b55 as assessed by translation blockade with  cycloheximide ( figures 1d and s1b ) .
inhibition of phd2 enzymatic activity by iox2 or dimethyloxaloylglycine ( dmog ) impaired both b55 ubiquitination and degradation ( figures 1e and 1f ) .
moreover , physiological inhibition of phd2 by exposure to hypoxia was able to induce b55 accumulation as well ( figures s1c and s1d ) .
overall , these data indicate that phd2  hydroxylase function is required for targeting b55 to the proteasome .
mass spectrometry analysis of b55 isolated from hek293 t cells , upon co - expression of phd2 , revealed three hydroxylation sites in p159 , p236 , and p319 of b55 that we singularly mutated into an alanine .
of the three b55 point mutants , only b55 exhibited increased resistance to phd2-mediated degradation ( figure  1 g ) . to confirm that this residue is hydroxylated by phd2
, we performed an in  vitro hydroxylation assay where recombinant phd2 was incubated with increasing doses of a b55 peptide and the km was determined by using a michaelis - menten curve or a lineweaver - burk ( l - b ) plot ( figure  1h ) .
the same assay was then performed by using a b55 peptide spanning from amino acid 311 to 329 , therefore containing p319 , or b55 p - oh ( where p319 is synthetically hydroxylated ) and hif1 ( 556575 ) peptides as negative and positive controls , respectively .
this assay confirmed that phd2 was able to directly hydroxylate b55 in p319 , although with a lower efficiency than for hif1 ( figure  1i ) .
however , when we tested the ubiquitination levels of this mutant , we could not detect any difference ( figure  s1e ) . because proline 319 lies in the region of b55 that allows its binding to the scaffold subunit of the pp2a complex ( li and virshup , 2002 )
, we hypothesized that the hydroxylation of this residue is important to detach b55 from the complex , therefore making it more available for degradation .
when measuring the binding of b55 and b55 to the scaffold a and the catalytic c subunits of the pp2a complex , we found that b55 had higher affinity for the complex than b55 ( figure  1j ) .
moreover , recombinant phd2 was able to reduce the binding of b55 ( but not of b55 ) to the a subunit of pp2a complex ( figure  1k ) .
altogether , these data suggest that b55 hydroxylation is important to disassemble the pp2a complex , rendering b55 available for degradation . to analyze the degradation of endogenous b55 , we silenced phd2 in three breast cancer cell lines , mda - mb231 , mcf7 , and skbr3 .
in all cell lines , we observed an increase of b55 protein levels upon silencing of phd2 ( figure  2a ) that was not linked to an augmentation in the mrna levels ( figures 2b and 2c ) .
contrariwise , overexpression of phd2 could reduce endogenous b55 protein levels ( figure  s1f ) . because a previous report has shown that b55 plays an important role in response to nutrient deprivation ( reid et  al . ,
2013 ) , in order to investigate the biological readout of our findings , we cultured breast cancer cells in glucose - deprived medium at several time points .
we observed that b55 was progressively degraded ( figures 2d and 2e ) and that , in the same conditions , silencing of phd2 was able to rescue the degradation of b55 ( figures 2f and 2 g ) . based on the observation that glucose starvation did not affect the overall levels of phd2
accordingly , lack of glucose , by slowing down the tricarboxylic acid ( tca ) cycle , would lead to a change in the amount of -kg , a co - substrate needed for phd2 activity , as well as fumarate and succinate , two metabolites that inhibit phd2 and derive from the oxidation of -kg in the tca cycle ( isaacs et  al .
to test this hypothesis , we measured by mass spectrometry the levels of -kg , succinate , and fumarate upon glucose starvation . at all the time points
tested , in both mda - mb231 and mcf7 cells , the levels of fumarate rapidly declined , whereas succinate was not affected ( figures 2h and 2i ) . as a consequence ,
the -kg - to - fumarate ratio gradually and significantly increased , possibly playing in favor of phd2 function ( figures 2j and 2k ) . to further explain why fumarate dropped more rapidly than -kg under glucose starvation , we analyzed the levels of malate ( that generates from fumarate in the tca cycle ) and we measured the amount of -kg deriving from glutamine .
in both cell lines , malate levels were tremendously reduced to a similar extent as observed for fumarate ( figures 2h and 2i ) .
this , together with the fact that succinate levels were unaltered , suggests a block of succinate dehydrogenase ( sdh ) activity upon glucose starvation ( andreev et  al . , 2015 ) .
at least in mcf7 , the amount of -kg coming from glutamine was increasing under glucose deprivation versus fed conditions , as evidenced by u - c - glutamine fractional labeling ( figure  s2a ) .
reductive carboxylation ( rc ) , a pathway that utilizes glutamine - derived -kg to generate citrate and eventually acetyl - coa ( metallo et  al . , 2011 ) ,
was slightly but significantly decreased under glucose starvation as proven by a drop of the m  + 1 acetyl - coa isotopologue in the presence of 5-c - glutamine ( figure  s2b ) .
levels of glutamine - derived -kg were close to the detection limit in mda - mb231 cells ( not shown ) .
overall , our data indicate that , in the absence of glucose , at least in breast cancer cells , a block of sdh activity , per se or together with increased glutamine anaplerosis , results in a high -kg - to - fumarate ratio .
we transfected fed and glucose - deprived mcf7 cells with a luciferase construct fused to the oxygen degradation domain of hif1 ( oddd - luc ) .
when active , phd2 leads to the degradation of this oddd - luc , resulting in overall reduction of luciferase signal . during glucose starvation ,
the activity of both endogenous and overexpressed phd2 was strongly increased ( figure  2l ) , and addition of fumarate to the medium was able to rescue this induction ( figure  s2c ) , thus supporting that increased phd2 activation in glucose starvation leads to b55 degradation .
we then wondered whether the degradation of b55 was necessary for the activation of survival / apoptotic pathways in response to nutrient deprivation ( sun et  al . , 2015 ) .
cell - cycle analysis showed that deprivation of glucose induced a high percentage of cell death in both mda - mb231 and mcf7 cell lines ( figure  3a ) .
however , cells silenced for phd2 showed 30% of reduction in cell death ( figures 3a3c ) , whereas the cell - cycle phases were unchanged ( figures s2d and s2e ) . furthermore , this protection against cell death was b55 dependent because combined knockdown of b55 and phd2 was able to restore apoptosis in cells silenced for phd2 only ( figures 3d , 3e , s2f , and s2 g ) . similarly to phd2
silencing , overexpression of b55 or b55 was able to induce protection against cell death as well ( figure  3f ) . complementing these data , physiological expression of b55 in mcf7 silenced for phd2 was able to  reduce glucose starvation - induced cell death , whereas un - degradable b55 was ineffective ( figure  3 g ) .
these data demonstrate that phd2-mediated b55 degradation is responsible , at least in part , for cell apoptosis induced by glucose deprivation . unlike mda - mb231 and mcf7 , skbr3 cells were partially resistant to starvation - induced cell death .
when skbr3 cells were cultured in glucose - deprived medium , the protein levels of b55 were unchanged and silencing of phd2 did not significantly affect cell death ( figures 3h and 3i ) .
depletion of b55 rendered these cells responsive to glucose deprivation and ultimately able to activate the apoptotic pathway ( figure  3j ) . in a focus - forming assay , glucose starvation partly decreased the number of colonies formed by control skbr3 cells , but combined b55 silencing resulted in a further and stronger reduction ( figures 4a and 4b ) , confirming the relevance of b55 degradation in response to glucose starvation . in order to test whether the molecular interaction between phd2 and b55 had relevance in an in  vivo context
, we injected subcutaneously mda - mb231 cells stably silenced for ctrl ( shctrl ) or phd2 ( shphd2 ) in nude mice .
when the tumors reached 50100  mm , we treated the mice with glycolysis inhibitor 2-dg ( 2-deoxy - glucose ) ( in order to mimic glucose starvation ) or a vehicle control .
in the presence of phd2 ( shctrl ) , treatment with 2-dg blocks tumor growth with strong efficiency . however , when phd2 is silenced ( shphd2 ) , the tumors showed complete resistance to the treatment ( figures 4c and 4d ) .
consistent with our in  vitro findings , the levels of b55 were reduced in 2-dg - treated tumors , whereas this degradation was rescued in tumors lacking phd2 ( figure  4e ) .
histological analysis of tumor sections revealed a strong increase of tunel positivity in 2-dg - treated cells that was completely canceled by silencing of phd2 , confirming the relevance of its activity in the response to glucose starvation ( figure  4f ) .
altogether , these data prove that phd2-mediated b55 degradation allows breast cancer cell death in response to chronic glucose deprivation .
in this study , we uncover two molecular players in response to  nutrient restriction : the phosphatase b55/pp2a and the  prolyl - hydroxylase phd2 . the pathways involved in the response to nutrient deprivation have been widely investigated . in the absence of nutrients ,
the stress sensor ampk is activated , leading to the inhibition of mtor and the induction of a metabolic response able to initiate adaptation to nutrient restriction in order to allow the cell to survive in this harsh situation ( laplante and sabatini , 2012 ) . although all of the kinases of this pathway have been studied for decades ( brown et  al . , 1994 ) , the relevance of phosphatases in this matter has only recently received more attention ( reid et  al .
, 2013 , wong et  al . , 2015 ) . upon nutrient starvation ,
pp2a activity is liberated as a consequence of mtor repression , and this event leads to a pp2a - mediated block of c - myc that thus causes inhibition of proliferation ( cianfanelli et  al . , 2015 ,
moreover , the inactivation of mtor and the simultaneous activation of pp2a / b55 are necessary to switch on the autophagic pathway in a very early response to amino acid deprivation ( wong et  al . , 2015 ) .
in this study , we have further investigated the link between pp2a / b55 and nutrient shortage , unravelling how a late response to glucose restriction is accomplished by degradation of pp2a / b55. induced prevention of its degradation ( by silencing of phd2 ) or impaired control of its levels , cause a resistance to cell death .
it is thus possible that pp2a / b55 participates in a first - wave reaction in response to nutrient stress by controlling the phosphorylation cascade responsible for the growth arrest / survival of the  cells .
however , prolonged cell stress leads to the activation of apoptosis , signaled by a reduction in b55 levels that is mediated by phd2 .
the role of pp2a in response to nutrient restriction has been highlighted by several studies ( cianfanelli et  al .
, 2015 , wong et  al . , 2015 ) , and what is mostly emerging from all of them is the tight and reverse relationship between pp2a and mtor signaling under starved conditions .
similarly , we have shown that hypoxic blockade of mtor ( via the upregulation of redd1 ) releases pp2a / b55 activity , allowing dephosphorylation of phd2 and full hif1 stabilization ( di conza et  al . , 2017 ) . in the current report
, we demonstrate that , in a feed - forward loop , phd2 is able to harness its own inhibitor pp2a / b55 by inducing its hydroxylation , ubiquitination , and degradation through the proteasome .
these data indicate that b55 is a substrate of the oxygen - sensing enzyme phd2 .
however , different from what occurs for hif1 , the b55 hydroxylation is not a signal for ubiquitin ligase , but it rather facilitates the detachment of b55 from the main complex pp2a .
indeed , it has been demonstrated that the domain where the proline 319 lies , is involved in the binding with the scaffold a subunit ( li and virshup , 2002 ) .
therefore , the hydroxylation in proline 319 by inhibiting this binding favors b55 ubiquitination and degradation .
several reports have highlighted the pro - tumoral activity of b55 ( gilan et  al .
, 2015 , hein et  al . , 2016 , reid et  al . ,
the role of phd2 in cancer is more controversial , having shown even opposite effects in different tumor contexts ( chan et  al . , 2009 ,
if , on one hand , we show that oxygen shortage enables b55 accumulation , on the other hand , we speculate that in breast cancer cells glucose deprivation reduces the production of fumarate mainly because of a block of sdh ( andreev et  al . , 2015 )
, tilting the balance toward an excess of -kg at the expense of the phd2 inhibitor fumarate , overall resulting in enhanced phd2 activity and increased b55 degradation . in parallel ,
, unpublished data ) that further boosts phd2 function and therefore b55 degradation ( di conza et  al . , 2017 ) .
yet , it remains to be defined how glucose starvation causes this sdh block in this specific cell context .
altogether , this study shows that phd2 takes part in the network of nutrient - sensing proteins by regulating pp2a in breast cancer cells .
hek293 t , mcf7 , mda - mb231 , and skbr3 cell lines were cultured in dmem ( gibco ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) ( gibco ) , 2  mm glutamine ( life technologies ) , and 100 units / ml penicillin/100  g / ml streptomycin ( life technologies ) .
cells were maintained in a humidified incubator at 37c and 5% co2 . for glucose starvation experiments ,
dmem glucose - free ( life technologies ) has been supplemented with heat - inactivated  fbs previously dialyzed with slide a lyzer cassette ( thermo scientific ) .
transfections were performed with lipofectamine 2000 transfection reagent or lipofectamine rnaimax ( life technologies ) , according to the manufacturer s instructions .
polar metabolites were extracted using a methanol - water extraction ( fendt et  al . , 2013 ) . following extraction ,
the extract was centrifuged for 10  min at 4c at 20,000   g , and the supernatant was transferred to ms vials .
measurements by gas chromatography ( gc ) or liquid chromatography ( lc ) were performed as described in supplemental experimental procedures .
an in  vitro decarboxylation assay was performed as previously published  ( zheng et  al . , 2014 ) to assess the hydroxylation of a specific peptide by recombinant phd2 .
the sequences of the peptides used are as follows :  b55 peptide , wdlnmenrpvetyqvheyl ; b55 p - oh peptide , wdlnmenrp#vetyqvheyl ( # denotes hydroxylation ) ; hif1 ( 556575 ) , dldlemlapyipmdddfqlr .
mda - mb231 cells were harvested and single - cell suspensions of 10   10 cells in 100  l of pbs and matrigel solution ( 1:1 ) were injected subcutaneously into the flank of cd1 mice .
tumor volumes were measured three times a week with a caliper and calculated using the formula : v  =   [ d  d]/6 , where d is the minor tumor axis and d is the major tumor axis .
statistical significance was calculated by two - tailed unpaired t test with p  < 0.05 considered statistically significant .
g.d.c . performed experimental design , experiments , acquisition of data , analysis and interpretation of data and wrote the manuscript .
 summaryb55 is a regulatory subunit of the pp2a phosphatase . 
 we have recently found that b55-associated pp2a promotes partial deactivation of the hif - prolyl - hydroxylase enzyme phd2 . here 
 , we show that , in turn , phd2 triggers degradation of b55 by hydroxylating it at proline 319 . in the context of glucose starvation 
 , phd2 reduces b55 protein levels , which correlates with mda - mb231 and mcf7 breast cancer cell death . under these conditions , 
 phd2 silencing rescues b55 degradation , overcoming apoptosis , whereas in skbr3 breast cancer cells showing resistance to glucose starvation , b55 knockdown restores cell death and prevents neoplastic growth in  vitro . 
 treatment of mda - mb231-derived xenografts with the glucose competitor 2-deoxy - glucose leads to tumor regression in the presence of phd2 .  
 knockdown of phd2 induces b55 accumulation and treatment resistance by preventing cell apoptosis . 
 overall , our data unravel b55 as a phd2 substrate and highlight a role for phd2-b55 in the response to nutrient deprivation . 