members of the p53 family mediate genotoxic stress responses in cells ( vogelstein et al , 2000 ; yang & mckeon , 2000 ; moll & slade , 2004 ; westfall & pietenpol , 2004 ) . the identification of p63 and p73 greatly expanded the appreciation of the importance of p53 family members , not just for genome surveillance but also for coordinating normal development ( yang & mckeon , 2000 ; moll & slade , 2004 ; westfall & pietenpol , 2004 ) . the p63 protein shares several conserved regions found in all p53 family members , including a trans - activating ( ta ) domain , a dna - binding domain ( dbd ) and an oligomerization domain ( yang et al , 1998 ) . the p63 gene is transcribed from two alternative transcription start sites that yield ta domain - containing isoforms and n isoforms , the latter of which lack the ta domain and might act as dominant negatives of tap63 isoforms ( yang et al , 1998 ; petitjean et al , 2008 ) . recent evidence indicates that regulation of protein stability is probably the principal mechanism by which the actions of p63 are controlled . for example , genotoxic stress - induced phosphorylation of p63 prevents its ubiquitination and degradation by the proteasome ( rossi et al , 2006 ; li et al , 2008 ; macpartlin et al , 2008 ) . however , molecular modulators underlying these events remain poorly described . in addition , genetic models demonstrating the functional importance of p63 stability in vivo are lacking . in this study , we show that cyclin - dependent kinase ( cdk ) 5 and abl enzyme substrate 1 ( cables1 ) , a cdk - interacting protein ( zukerberg et al , 2000 ; wu et al , 2001 ) , protects p63 from ubiquitin - mediated proteasomal degradation through direct physical interaction . using female germ cells lacking cables1 as an in vivo model , we show that radiation - induced oocyte loss is partly rescued through reduced accumulation of phosphorylated tap63. these results identify a new regulatory step modulating p63 function in vivo and provide further insights into mechanisms that govern female germline integrity . cables1 interacts with both p53 and p73 ( tsuji et al , 2002 ) and modulates the activity of female germline stem cells and oocyte quality ( lee et al , 2007 ) . we therefore felt it was reasonable to consider that p63 , which is expressed predominantly in female germ cells , is regulated by direct interaction with cables1 . to test this , we first co - expressed tap63 and flag - tagged cables1 in cos7 cells , and lysates were immunoprecipitated . cables1 was detected in immunocomplexes containing tap63 , indicative of an interaction between the two proteins ( fig 1a ) . to determine whether endogenous tap63 normally interacts with endogenous cables1 , we analysed lysates of neonatal ( postnatal day 10 ; p10 ) mouse ovaries , which contain a large number of meiotically arrested oocytes that express both proteins ( fig 1b ; suh et al , 2006 ; lee et al , 2007 ) . the co - immunoprecipitation analysis revealed that cables1 was present in the tap63 immunocomplex and , reciprocally , tap63 was present in the cables1 immunocomplex ( fig 1c , d ) . we next mapped the domain(s ) of tap63 that interact with cables1 by constructing tap63 deletions that removed various regions , including the ta domain , dbd and carboxyl terminus ( yang et al , 1998 ) . we observed that cables1 was lost in the immunocomplex only after both the ta domain and c terminus of tap63 were deleted ( fig 2a , b ) . this result was confirmed by immunoprecipitation using a flag - specific antibody to detect flag - tagged cables1 , followed by immunoblotting with a haemagglutinin ( ha)-specific antibody to detect ha - tagged tap63. the dbd of tap63 alone did not interact with cables1 ( fig 2c ) . the c terminus of tap63 contains an oligomerization domain , a sterile alpha motif ( sam ) and a ta - inhibitory domain ( yang et al , 1998 ) . after constructing additional deletion mutants , we observed that interaction between tap63 and cables1 was lost when the sam was removed ( fig 2e , f ) . conversely , through expression of wild - type cables1 and a cables1 mutant lacking the cyclin - interaction domain ( cid ) , we observed that tap63 immunoprecipitated with both wild - type and cid cables1 ( fig 2g i ) . together , these results indicate that the amino terminus of cables1 interacts with the ta domain and sam of tap63 to promote physical association of the two proteins . one consequence of cables1tap63 interaction was the maintenance of tap63 protein levels ( fig 3a , b ) . this interaction is required to stabilize tap63 , as cables1 has no effect on levels of the dbd of tap63 ( fig 3c ) , which does not interact with cables1 ( fig 2d  f ) . to better understand the mechanism underlying p63 stabilization by cables1 , we used hep3b cells based on earlier studies that initially characterized p63 isoform stability in this cell type ( petitjean et al , 2008 ) . the treatment of hep3b cells with the protein synthesis inhibitor cycloheximide for up to 8 h decreased transfected tap63 levels ; this was not observed in the presence of cables1 ( fig 3d ) , indicating that cables1 stabilizes tap63 protein . as cables1 also interacts with the np63 isoform ( fig 2b , c ) , we also evaluated its protein stability . in accordance with previous observations ( petitjean et al , 2008 ) , transfected np63 was less stable than tap63 and showed degradation within 6 h after cycloheximide treatment . however , co - expression of cables1 increased the half - life of np63 ( supplementary fig s1 online ) . as p63 can be degraded by the proteasome ( rossi et al , 2006 ; li et al , 2008 ; macpartlin et al , 2008 ) , we tested whether stabilization of tap63 by cables1 resulted from reduced ubiquitination and proteasomal degradation . the treatment of cos7 cells with the proteasome inhibitor mg132 resulted in a near - doubling of tap63 levels ( fig 3e ) , consistent with p63 being turned over rapidly in cells under non - stress conditions . the accumulation of tap63 was even greater in the presence of cables1 , and addition of mg132 to cables1-expressing cells did not markedly alter the extent of tap63 accumulation compared with that resulting from the presence of cables1 alone ( fig 3e ) . in the absence of cables1 , however , when cables1 was expressed together with tap63 or np63 , ubiquitination of both proteins was attenuated greatly ( fig 3f ) . these findings indicate that cables1 stabilizes p63 by blocking ubiquitin - mediated proteasomal degradation of the protein . genotoxic stress induces phosphorylation of tap63 ( suh et al , 2006 ; macpartlin et al , 2008 ) , and a tight dose response relationship exists between these events and cell death ( suh et al , 2006 ) . we therefore tested whether radiation - induced dna damage affects cables1tap63 interaction by using h1299 cells so that our outcomes could be compared with those of earlier studies of tap63 phosphorylation in this cell type ( macpartlin et al , 2008 ) . to aid in the detection of tap63 phosphorylation , cells were treated with the serine and threonine phosphatase inhibitor okadaic acid ( oa ) for 4 h before collection ( macpartlin et al , 2008 ) . similar to the response reported for tap63 phosphorylation by ib kinase- ( macpartlin et al , 2008 ) , oa caused a dose - dependent shift in tap63 mobility ( fig 4a ) . furthermore , the interaction of cables1 with tap63 was enhanced markedly by oa ( fig 4b ) , and the increased interaction between endogenous cables1 and tap63 in ovarian lysates , triggered by ionizing radiation exposure , was reduced significantly by pretreatment with -phosphatase ( fig 4c ) . although more detailed information is needed to determine the significance of these findings , it seems that phosphorylation of tap63 after a genotoxic insult might facilitate its interaction with cables1 . to test further whether dna damage enhances this interaction , we used fluorescence resonance energy transfer ( fret ) to visualize directly the interaction between cables1 and tap63 ( fig 4d  f ) . the exposure of h1299 cells to ionizing radiation increased fret efficiency between cables1cyan fluorescent protein ( cfp ) and tap63yellow fluorescent protein ( yfp ; fig 4f ) . thus , genotoxic stress - induced phosphorylation of tap63 induces its interaction with cables1 , which in turn protects p63 protein from ubiquitination and proteasomal degradation . in a final set of experiments , we used cables1-null mice to evaluate the consequences of an absence of cables1 on tap63 levels and function in vivo . the female germline of these mice was selected for analysis because : ( i ) tap63 is expressed at high levels in female germ cells during meiotic arrest ( suh et al , 2006 ) ; ( ii ) cables1 and its components are expressed in female germ cells and regulate their function ( fig 1b ; iwaoki et al , 1993 ; lee et al , 2004 , 2007 ; park et al , 2004 ) ; ( iii ) endogenous cables1 interacts with endogenous p63 in germ cells of mouse ovaries ( fig 1c ) ; and ( iv ) tap63 has an indispensable role in mediating dna damage - induced oocyte loss ( suh et al , 2006 ; livera et al , 2008 ) . under physiological conditions , tap63 levels in oocytes were lower in ovaries of cables1-null mice compared with wild - type females ( fig 5a  d ) . phosphorylated tap63 did not accumulate in oocytes of cables1-null ovaries after exposure to ionizing radiation , in contrast to the pronounced accumulation of phosphorylated tap63 observed in oocytes of irradiated wild - type ovaries ( fig 5d ; supplementary fig s2 online ) . the levels of tap63 mrna showed no significant differences in wild - type and cables1-null ovaries , confirming that changes in tap63 levels were due to changes in protein stability rather than transcriptional regulation of the p63 gene . to assess the consequence of this partial loss of p63 stability in cables1-null mice , female mice were irradiated at p5 and ovaries non - irradiated wild - type and cables1-null mice had a comparable number of oocytes at p10 ( data not shown ; lee et al , 2007 ) . however , the near - complete depletion of oocytes from wild - type ovaries after exposure to ionizing radiation was attenuated significantly in ovaries of cables1-null mice ( fig 5e  h ) . in summary , physical association of cables1 with p63 inhibits its ubiquitination and degradation through the proteasome . this process is required for maximal stabilization of tap63 in female germ cells , and the subsequent death of these cells , after exposure to a genotoxic stress in vivo . however , cables1-null oocytes retain approximately 50% of the tap63 protein content observed in wild - type oocytes , and the radioprotection conveyed in oocytes by cables1 deficiency is less than that observed in p63-null mice ( suh et al , 2006 ) . thus , although the loss of cables1 reduces tap63 levels through increased degradation , tap63 that remains is sufficient in some , but not all , cables1-null oocytes for triggering apoptosis after exposure to ionizing radiation . similar to other reports ( suh et al , 2006 ; macpartlin et al , 2008 ) , our findings also indicate that tap63 is phosphorylated rapidly in response to genotoxic stress , and this might facilitate its interaction with cables1 . previous studies with doxorubicin have reported that this phosphorylation event involves serine residues on tap63 , which then stabilizes the protein ( petitjean et al , 2008 ) . however , cisplatin triggers the phosphorylation of tap63 on tyrosine residues through induction of the c - abl kinase , which also results in the stabilization of p63 ( gonfloni et al , 2009 ) . it was further reported that inhibition of the tyrosine kinase activity of c - abl by imatinib partly prevents cisplatin - induced oocyte loss in mice through destabilization of tap63 ( gonfloni et al , 2009 ) . interestingly , cables1 promotes tyrosine phosphorylation of c - abl substrates ( zukerberg et al , 2000 ) . although the mechanisms responsible for differential phosphorylation of p63 by various genotoxic stresses remain to be elucidated , cables1 might participate as a common upstream integrator of these events . mice harbouring a targeted inactivation of the cables1 gene have been described earlier by our laboratories ( zukerberg et al , 2004 ) . all animal protocols were approved by the institutional animal care and use committee of massachusetts general hospital . full - length human cables1 complementary dna has been reported previously ( sakamoto et al , 2008 ) , whereas tap63 complementary dna was provided by g. wu ( wayne state university ) . the ha- and flag - tagged constructs , and all deletion mutants , were generated by pcr . non - atretic oocyte - containing primordial and total immature ( primordial , primary and preantral ) follicle numbers were determined by histomorphometric procedures detailed previously ( jones & krohn , 1961 ; skaznik - wikiel et al , 2007 ) . photobleaching - based fret analysis was conducted essentially as described previously ( wan et al , 2008 ) . the fusion of cfp or yfp to cables1 or tap63 , respectively , does not alter protein function ( supplementary fig s3 online ) . immunofluorescence was performed , as described previously ( matikainen et al , 2001 ; suh et al , 2006 ) , using tap63 antibody provided by f. mckeon ( harvard medical school ) . sections from ovarian tissues of wild - type and cables1-null mice were always mounted adjacent to each other on same slide to ensure identical exposure to all treatments and sample processing . ubiquitination analysis was conducted as described previously ( ohta & xiong , 2001 ) with minor modifications .
cables1 protects p63 from proteasomal degradation to ensure deletion of cells after genotoxic stresscables1 interacts with tap63 to protect it from ubiquitin - mediated degradation after exposure to genotoxic stress . 
 oocytes lacking cables1 accumulate less phosphorylated tap63 in response to genotoxic stress , which enhances the survival of these cells .