E2F1 stability is regulated by a novel-pkc/p38b MAP kinase signaling pathway during keratinocyte differentiation

Transcription

1 (2006) 25, & 2006 Nture Pulishing Group All rights reserved /06 $ ORGINAL ARTICLE stility is regulted y novel-pkc/p38 MAP kinse signling pthwy during kertinoyte differentition IA Ivnov 1, SJA D Souz 1 nd L Dgnino 1,2 1 Deprtment of Physiology nd Phrmology, University of Western Ontrio, London, Ontrio, Cnd nd 2 Deprtment of Peditris, University of Western Ontrio, London, Ontrio, Cnd E2F trnsription ftors regulte prolifertion, differentition, DNA repir nd poptosis. Tight E2F regultion is ruil for epiderml formtion nd regenertion. However, virtully nothing is known out the moleulr events modulting E2F during epiderml kertinoyte differentition. Eluidtion of these events is essentil to understnd epiderml morphogenesis, trnsformtion nd repir. Here we show tht, in differentiting kertinoytes, C 2 þ -indued protein kinse C (PKC) tivtion downregultes protein levels. Further, we hve identified PKC d nd g s those isoforms speifilly involved in indution of protesoml degrdtion. We lso demonstrte tht downregultion y novel PKC isozymes requires tivtion of p38 mitogentivted protein kinse (MAPK). This is the first exmple of regultion in the E2F trnsription ftor fmily y tivtion of PKC nd MAPK in the ontext of iologilly signifint differentition stimuli in epitheli. (2006) 25, doi: /sj.on ; pulished online 22 August 2005 Keywords: kertinoyte; E2F; differentition Introdution The E2F fmily of trnsription ftors is wellestlished regultor of mny ell responses, suh s ell yle progression, differentition, poptosis nd DNA repir (Cm nd Dynlht, 2003; Bell nd Ryn, 2004). However, key issue tht remins poorly understood is, how externl stimuli tht indue hnges in prolifertion or differentition re trnsmitted intrellulrly to regulte E2F. Further, lthough it hs een estlished tht E2F proteinsn e regulted trnsriptionlly nd post-trnsltionlly, the identifition of the physiologil stimuli governing suh regultion is in its infny. E2F proteinsre differentilly modulted in epiderml kertinoytes., -2 nd -3 re undnt in Correspondene: Dr L Dgnino, Deprtment of Physiology & Phrmology, University of Western Ontrio, London, Ontrio, Cnd N6A 5C1. E-mil: ldgnino@uwo. Reeived 12 April 2005; revised 8 July 2005; epted 8 July 2005; pulished online 22 August 2005 undifferentited kertinoytes. Differentition triggers downregultion of these E2F forms nd upregultion of E2F5 (D Souz et l., 2001). The iologil importne of funtion nd regultion in epiderml homeostsis hs een mply demonstrted. For exmple, is essentil to mintin norml prolifertion in undifferentited kertinoytes, nd intivtion of the E2f1 gene in mie yieldsepiderml ellswith douling times out seven-fold longer thn wild-type ells, nd whih exhiit severely redued ility to re-epithelilize the skin during utneous regenertion (D Souz et l., 2002). Conversely, downregultion is neessry for proper kertinoyte differentition, sexogenous expression in differentiting ultured kertinoytes prevents exit from the ell yle, uses poptosis nd interfereswith expression of differentition mrkers, suh s trnsglutminse 1 nd kertin 10 (Diker et l., 2000; D Souz et l., 2001). Further, norml expression in the squmous epitheli of trnsgeni mie indueshyperprolifertion, hyperplsi nd poptosis in vivo, oth in interfolliulr nd folliulr epidermis(piere et l., 1998, 1999). In the epidermis, n extrellulr C 2 þ grdient is present from the undifferentited sl to the terminlly differentited ornified lyer. Indeed, n importnt mehnism for kertinoyte indution of terminl differentition involvesc 2 þ signling (reviewed in Koster nd Roop, 2004). In ulture, primry mouse kertinoytesn e mintined sn undifferentited ell popultion, nd n e indued to differentite y elevting the extrellulr C 2 þ onentrtion to mm (Dotto, 1999). Clium tretment of these ells tivtes protein kinse C (PKC), tyrosine kinses nd phosphtidyl inositol-3 (PI-3) kinse, mimiking epiderml differentition proesses, inluding entry into quiesene nd expression of differentition mrkers. Centrl in kertinoyte withdrwl from the ell yle is the E2F/ network (Ruiz et l., 2004). However, severl key issues remin to e resolved: Wht re the pthwystht onvey extrellulr differentition signls to downstrem E2F proteins? To whih extent do these pthwys onverge in the regultion of other differentition trgets? Wht re the mehnisms of E2F regultion in differentiting kertinoytes? We hve egun to ddress those questions, nd demonstrte tht C 2 þ -indued tivtion of PKC in differentiting kertinoytesresultsin

2 protein destiliztion through mehnisms tht lso involve p38 tivtion. Together, these results point to PKC- nd p38-medited turnover s ritil omponent of differentition responses in epithelil ells. PKC regultion of turnover Antiody: E2F5 E2F5 431 F Results Protein kinse C tivtion modultes E2F in differentiting epiderml mouse kertinoytes In primry murine kertinoytes, indution of differentition y ulture in medium ontining 1.0 mm C 2 þ ledsto downregultion of, -2 nd -3, upregultion of E2F5, nd hngesin DNA-inding omplexes (D Souz et l., 2001). In greement with previous reports(d Souz et l., 2001), nlysis of E2F DNAinding omplexesy eletrophoreti moility shift ssys (EMSA) using lystes from undifferentited kertinoytesultured in low C 2 þ (0.05 mm) medium reveled the ontriution of, nd, s evidened y the formtion of supershifted speies in the presene of ntiodies ginst those proteins (Figure 1). In ontrst, no hnges in nding pttern were deteted in the presene of ntiodies ginst or E2F5, inditing tht these proteins my e minor onstituents of E2F DNA-inding omplexes in these ells(figure 1). Indution of differentition y ulture in high C 2 þ (1.0 mm) medium resulted in the formtion of omplexesminly omposed of nd (Figure 1). E2F5-ontining DNA inding speies were lso deteted in differentited kertinoytes (Figure 1). To identify the eventstivted y C 2 þ upstrem from E2F, we first investigted the involvement of kinse signling sdes known to e involved in primry kertinoyte differentition in the formtion of E2F/ nd E2F/ omplexes using EMSA. These signling sdes inlude PKC, tyrosine kinses nd phosphoinositide-3 kinse (Dotto, 1999). Tretment of undifferentited kertinoyteswith the PKC tivtor 12-O-tetrdenoylphorol-13-ette () mimiked the C 2 þ -indued formtion of E2F5-, - nd -ontining omplexes(figure 1). Conversely, tretment with the PKC inhiitorshelerythrine or Ro interfered with the indution of those omplexesy C 2 þ (Figure 1). In ontrst, the presene of the tyrosine kinse inhiitors genistein nd herimyin, or the phosphoinositide-3 kinse inhiitor wortmnnin did not interfere with C 2 þ -indution of the E2F/ nd E2F/ DNA-inding speies ssoited with differentition (Figure 1). Although nd the PKC inhiitorsmy individully modulte ellulr pthwys other thn PKC, tken together, our results re onsistent with the onept tht PKC is involved in regultion of E2F DNA-inding omplex formtion in differentiting kertinoytes. Ativtion of PKC y lso mimis the redution in nd inrese in E2F5 protein levels indued y C 2 þ in differentited ells(figure 2), ut doesnot F Unt Low C 2+ Ro Chel High C Antiody Low C 2+ High C 2+ High C 2+ Antiody: E2F5 lter levelsof E2F2 or E2F3 (Figure 2). Finlly, the C 2 þ -indued derese ws olished in the presene of the PKC inhiitor Ro3179, further evidening the involvement of PKC sn upstrem regultor (Figure 2). Importntly, it hseen shown tht long-term (>18 h) tretment of kertinoytesdoesnot result in downregultion of ll PKC tivity. Speifilly, lthough these onditions result in downregultion of the nd e isoforms, PKC d, Z nd z remin tive (Reynold et l., 1994; Efimov et l., 1998). Unt Inhiitor Her+Genis Wort High C Figure 1 Formtion of differentition-speifi E2F DNA-inding omplexesrequirespkc tivtion. () Primry murine kertinoyteswere isolted nd ultured in low C 2 þ medium (0.05 mm) or high C 2 þ medium (1.0 mm) for 24 h. Kertinoyte lystes were prepred nd DNA-inding retionswere onduted using n oligonuleotide proe ontining the E2F element in the DHFR promoter. DNA-inding retionswere onduted in the presene of the indited ntiodiesfor 45 min prior to seprtion y nondenturing gel eletrophoresis. Supershifted nds in the presene of the ntiodies re indited with. Undifferentited ellsultured in low C 2 þ show multiple nds orresponding to free E2F/DP omplexes(f), nd lower moility omplexes ontining nd. () Kertinoyteswere ultured for 24 h in high C 2 þ ( þ ) or low C 2 þ ( ) medium in the presene of ( nm), helerythrine (Chel, 1 mm), or Ro (Ro, 1 mm), sindited. E2F DNA-inding retionswere onduted in the presene of the indited ntiodies, nd nlyzed y EMSA. () The formtion of nd omplexesin ellsindued to differentite y ulture in medium ontining 1.0 mm C 2 þ isnot ffeted y the presene in ulture medi of the protein tyrosine kinse inhiitors herimyin A (Her, 1 mm) nd genistein (Genis, 300 mm), or to the PI-3 kinse inhiitor wortmnnin (Wort, 50 nm).

3 PKC regultion of turnover 432 Low C 2+ High C 2+ γ-tuulin 110 CHX h 94 E2F5 + CHX h Low C 2+ High C 2+ Post-trnsriptionl regultion of y PKC in differentiting kertinoytes We next foused on the mehnisms of downregultion in response to PKC tivtion. First, we ddressed the effet of on mrna levels nd found no signifint differenes etween treted nd untreted ells (dt not shown). We lso mesured -indued protein dey y exmining hnges in levelsin ellstreted with yloheximide (CHX) (Figure 3, ). The hlf-life of in the presene of E2F2 E2F3 Ro Figure 2 Regultion of E2F protein levelsduring C 2 þ - or indued kertinoyte differentition. () Kertinoyteswere ultured for 24 h in medium ontining 0.05 mm C 2 þ without dditives(low C 2 þ ), in the presene of (0.1%, finl) or ( nm, finl), or in medium ontining 1.0 mm C 2 þ (High C 2 þ ). Whole-ell lystes were prepred, resolved y SDS PAGE nd nlysed with the indited nti-e2f ntiodies. () Primry kertinoyteswere ultured for 24 h in medium ontining 0.05 mm C 2 þ nd (0.1%) or ( nm), sindited. Whole-ell lystes were prepred, resolved y SDS PAGE nd nlysed with the indited nti-e2f ntiodies. () Cellswere ultured for 24 h in medium ontining 0.05 mm C 2 þ or 1.0 mm C 2 þ (High C 2 þ ) with (0.1%, finl) or Ro3179 (1 mm, finl), sindited. Whole-ell lystes were prepred nd nlyzed for expression y immunolot. undne (% t=0) d Time (h) MG132 High C 2+ γ-tuulin CHX CHX/ Figure 3 Inresed turnover vi protesoml tivity in differentiting kertinoytes. (, ) Primry kertinoytesultured in medium ontining 0.05 mm C 2 þ were treted with CHX mg/ml 30 min prior to ddition of, used to indue differentition ( nm). Cell lystes were prepred t the indited timesfollowing ddition of, resolved y SDS PAGE nd nlyzed y immunolotting with the indited ntiodies. () Densitometri quntifition of protein levels in kertinoytestreted with CHX or CHX þ, sdesried ove. Densitometri vues were normlized to g -tuulin. The results re expressed s men þ s.d. (n ¼ 3). indites Po0.01 (Student s t-test). (d) Kertinoyte ultureswere inuted in high C 2 þ medium for 8 h prior to the ddition of MG132. Cell lystes were prepred 16 h fter MG132 tretment, nd nlysed y imunolot, sdesried in (). CHX wsout 11.5 h, nd wsredued to out 6.5 h when ws lso present (Figure 3), inditing tht PKC tivtion inreses turnover in kertinoytes. Although the CHX tretment preludes mesurement of hlf-life in epiderml ellsunder norml growth onditions, these experiments indite tht PKC

4 tivtion is suffiient to inrese turnover, independent of de novo protein synthesis. The retinolstom protein () n ssoite with nd stilize (Hofmnn et l., 1996). Hene, we investigted if the derese in stility ws ssoited with dispperne of. We exmined levelss funtion of time nd found moderte inrese fter 10 h of inution in the presene of CHX. Inution of ellsin the presene of oth CHX nd deresed levels y out 50% fter 10 h. prb hs een shown to e sustrte for PKC (Suzum et l., 2002), nd inresed phosphoryltion n derese the stility of this protein (Pen uels et l., 2003). Whether PKC-indued phosphoryltion of used the oserved dereses in levels remins to e estlished. Nevertheless, it is noteworthy tht is still highly expressed in -treted kertinoytes t time when 90% of hslredy een degrded (Figure 3), suggesting the possiility tht modifitions in nd/or my prelude the ltter from proteting from degrdtion. turnover involvesuiquitintion followed y protesoml degrdtion (Hteoer et l., 1996; Cmpnero nd Flemington, 1997). To determine if C 2 þ -indued degrdtion oursthrough protesoml tivity, we treted differentiting kertinoyte ultureswith high C 2 þ in the presene or sene of the protesoml inhiitor MG132. The presene of MG132 in kertinoytesultured rogted downregultion indued y high C 2 þ (Figure 3d), inditing tht differentition triggers sde of ellulr eventstht ulminte in inresed rtes of protesoml degrdtion. Ativtion of PKCd nd PKCZ medites downregultion in differentiting kertinoytes Five PKC isozymes re expressed in the epidermis (, d, e, Z, z ) (Denning, 2004). Of these, tivtion of the, d nd Z forms hs een ssoited with vrious kertinoyte differentition responses, inluding involurin upregultion, tivtion of Fyn tyrosine kinse, nd withdrwl from the ell yle (Oh et l., 1998; Codi et l., 2000; Alt et l., 2001; Ekert et l., 2004). To identify the PKC form(s) involved in regultion, we mesured levels in ells exogenously expressing eh isozyme using reominnt denoviruses, or ontrol -gl-enoding virus(figure 4). High levels of exogenously expressed PKC isoforms re suffiient to inrese PKC tivity sustntilly, even in the sene of or other stimuli, in vrious ell types, inluding epiderml kertinoytes (Oh et l., 1998; Li et l., 1999). Exogenous expression of PKCd or Z, ut not of or e, indued mrked derese in protein levels24 nd 48 h fter denovirl infetion (Figure 4), with no signifint hnges in mrna undne (dt not shown). Expression of PKC z inonsistently hd smll effets on protein undne. To onfirm tht PKCd nd Z re indeed upstrem regultors, we lso expressed dominnt-negtive PKC regultion of turnover Adenovirus PKC α PKC δ PKC ε PKC η PKC ζ MX17 PKCα PKCδ PKCε PKCη PKCζ β gl E2F-1 (dn) formsof these enzymesvi reominnt denoviruses (Figure 5). We then mesured levels in ells infeted with denoviruses enoding these PKC forms. We lso used severl denoviruses s ontrols, inluding the nonreominnt denovirusmx17 (whih is ontrol for the PKCd viruses), s well s n denovirusenoding -gl (whih is ontrol for PKCZ nd PKCe). We then treted ll ultureswith, nd oserved tht expression of either dn PKCd or Z, ut not of -gl or dn PKCe, rogted -indued -downregultion (Figure 5). These dt suggest tht C 2 þ tretment of kertinoytes triggers differentition signling sde tht tivtes PKC d nd Z, whih, in turn, regultes protein turnover. 40 Adenovirus Figure 4 Regultion of levelsy novel PKC forms. () Protein lystes (50 mg) from kertinoytesinfeted with reominnt denovirusenoding the indited PKC formswere prepred 48 h postinfetion. The smples were nlysed y SDS PAGE nd immunolotting. () Cellswere ultured in medium ontining 0.05 mm C 2 þ nd were infeted with the indited denoviruses. Cell lystes were prepred 48 h postinfetion nd mg were nlysed s desried for pnel. p38 MAP kinse tivtion medites downregultion in differentiting kertinoytes We next investigted the identity of PKC downstrem effetorstht medite turnover. In primry epiderml kertinoytes, PKCZ tivtesfyn tyrosine kinse nd dereses ylin E/dk2 (Codi et l., 2000; Kshiwgi et l., 2000). In these ells, tivtion of PKCd results in sequentil tivtion of rs, MEKK1, MEK3 nd p38 (Ekert et l., 2004). Thispthwy is very importnt for differentition, sit promotes expression of the differentition mrkers involurin nd trnsglutminse 1 (Oh et l., 1998; Codi 433

5 PKC regultion of turnover MX17 wt dn PKCδ wt dn PKCη dn PKCε MX17 wt PKCδ dn PKCδ wt PKCη dn PKCη wt PKCε dn PKCε Figure 5 PKC d nd Z medite -indued downregultion. () Protein lystes (50 mg) from kertinoytesinfeted with reominnt denovirusenoding wild-type (wt) or dominnt-negtive (dn) PKC d, Z or e, sindited, were prepred 48 h postinfetion. The smples were nlysed y SDS PAGE nd immunolotting with ntiodies ginst the indited PKC forms. () Kertinoyteswere infeted with the indited reominnt denovirusnd ultured in 0.05 mm C 2 þ for 24 h prior to the ddition of ( nm). Cell lystes were prepred 24 h lter, nd nlysed for expression y SDS PAGE nd immunolotting. et l., 2000; Kshiwgi et l., 2000). Given tht tyrosine kinse inhiitors did not ffet C 2 þ -indued hngesin E2F DNA-inding omplexes, we first turned our ttention to the mitogen-tivted protein kinse (MAPK) sde nd p38. Kertinoytes express p38, nd d (Dshti et l., 2001). We resoned tht inhiition of p38 tivity would rogte PKC-indued downregultion if p38 tsdownstrem of PKC. Thus, we expressed PKCd, nd oserved tht downregultion ws olished in the presene of the p38 inhiitorssb or SB202190, ut not in the presene of the intive nlogue SB (Figure 6). We otined similr results when we expressed PKCZ in the presene of those sme drugs (Figure 6), wheres the inhiitorsy themselveshd no effet on levels (Figure 6). At the onentrtionsused (10 mm), these p38 inhiitorsre seletive for p38 nd p38, nd do not ffet p38d (Kumr et l., 2003). Hene, p38d plys little, if ny, role in differentition-indued hnges. To determine whether p38 or p38 medite turnover, we infeted -treted kertinoyteswith reominnt denoviruses enoding dn p38 mutnts. Expression of dn p38, ut not of dn p38, interfered with the redution in protein levelsindued y (Figure 7). Thus, we hve defined hitherto unidentified pthwy in differentiting kertinoytesin whih C 2 þ tivtespkcd nd PKCZ, whih results in tivtion of p38 nd inresed protein turnover. We lso ttempted to knokdown p38 nd p38 using sirna pprohes. Although we oserved derese in p38 mrna, thisdid not trnslte into p38 protein loss even fter severl dys of sirna tretments (dt not shown). However, the results we otined y omining phrmologil nd geneti pprohesto inhiit p38 re onsistent with the notion tht this MAPK pthwy isinvolved in regultion in kertinoytes. Although the ext mehnism of p38 modultion of reminsto e eluidted, the tivtion of this pthwy isruil for norml epiderml morphogenesis PKCδ SB PKCη SB SB SB SB SB SB SB MX17 SB Figure 6 p38 inhiition interfereswith PKC-indued downregultion. Primry kertinoyteswere ultured in 0.05 mm C 2 þ nd infeted with the indited denovirusin the presene of ( nm), or p38 inhiitors(10 mm). After 24 h, ell lystes were prepred nd nlysed for expression y immunolot. nd funtion, given tht is essentil for epiderml kertinoyte prolifertion nd epiderml regenertion (D Souz et l., 2002), nd itsdownregultion is neessry for norml expression of differentition

6 mrkersnd entry into quiesene (Diker et l., 2000; D Souz et l., 2001). Disussion dn p38 40 α 40 β dn p dn p38β dn p38α Figure 7 Expression of dn p38 interfereswith -indued downregultion. Kertinoyteswere infeted with the indited denovirusnd ultured in 0.05 mm C 2 þ for 24 h prior to ddition of. Cell lystes were prepred 24 h lter, nd nlysed for expression y immunolot. E2F trnsription ftors re fundmentl regultors of ell yle progression, differentition, DNA repir nd poptosis. During epiderml development nd kertinoyte differentition, E2F formsre modulted t multiple levels, inluding mrna nd protein undne, intertionswith fmily proteinsnd suellulr distriution (D Souz et l., 2001; Apostolov et l., 2002). E2F tivity is essentil for proper epiderml morphogenesis nd kertinoyte prolifertion, s evidened y the perturtions in these proesses used y exogenous expression of dn DP-1 mutnt in primry ultured kertinoytesnd emryoni etoderml explnts(chng et l., 2004). Further, fulfills essentil, nonredundnt funtions in epiderml kertinoytes. Intivtion of the E2f1 gene ledsto redued kertinoyte prolifertion, migrtion nd tthment to the extrellulr mtrix in ulture, swell simpired epiderml regenertion fter injury in vivo (D Souz et l., 2002). In spite of the demonstrted importne of in epiderml formtion nd homeostsis, entrl question to e ddressed is how differentition signls re trnsmitted. We estlished tht indution of differentition in epiderml kertinoytesis physiologil stimulus tht negtively modultes protein stility. Seond, we demonstrte signling pthwy linking C 2 þ -indued kertinoyte differentition with regultion, whih involvestivtion of PKCd PKC regultion of turnover nd Z, nd of p38 MAP kinse. Other differentition responses, suh s ell yle exit nd involurin expression, re indued y those two PKC enzymes (Efimov et l., 1998; Oh et l., 1998), nd PKCZ is indispensle for epiderml homeostsis in vivo (Chid et l., 2003). The mehnisti reltionship etween tivtion of PKCd nd Z reminsto e defined. One possiility is tht they modulte eh other, similr to the regultion of PKCd y PKC in lymphom ells (Romnov et l., 1998). Alterntively, these PKC isozymes my t on ommon downstrem trget(s). protein undne isprtly determined y degrdtion vi uiquitin-dependent proteolysis nd stiliztion through ssoition with, whih interfereswith uiquitintion (Hteoer et l., 1996). Importntly, degrdtion in differentiting kertinoytesinvolvesprotesoml tivity, nd oursin spite of the ontinuous presene of. Murine kertinoyte differentition is ssoited with loss of E2F/ DNA-inding omplexes(d Souz et l., 2001), nd we hve een unle to detet / omplexesin differentited ells(ia Ivnov nd L Dgnino, unpulished oservtions). The mehnisms tht medite PKC-indued dissoition of from remin to e eluidted. However, the inility of to omplex with my ontriute to its degrdtion. Regionsin outside its-interting C- terminus n lso regulte post-trnsltionl modifitions nd stility. For exmple, phosphoryltion on S403 nd T433 uses degrdtion (Vndel nd Kouzrides, 1999). Notly, these two residues re within sequenes tht orrespond to oth dk nd MAPK onsensus sites, nd re phosphorylted y dk7 nd other, syet unidentified, kinses. We hve deteted in tivted-p38 immunopreipittesfrom differentited kertinoytes(ia Ivnov nd L Dgnino, unpulished oservtions), whih leds us to speulte tht p38 my modify, thusregulting itsstility. The inresed turnover in kertinoytes ontrsts with itsstiliztion during myogeni differentition in C2/C12 myolsts, whih involves intertions with (Mrtelli et l., 2001). downregultion my e requisite for proper differentition of vrious ell types, s exogenous expression prevents terminl differentition in hondroytes(sheijen et l., 2003), predipoytes(porse et l., 2001), nd HCt ells, humn kertinoyte ell line (Prmio et l., 2000). PKCd nd Z re tivted during C 2 þ - or indued kertinoyte differentition, resulting in sequentil tivtion of rs, MEKK1, MEK3 nd p38 (Ekert et l., 2004). Thispthwy promotesexpression of the differentition mrkersinvolurin nd trnsglutminse 1 (Oh et l., 1998; Codi et l., 2000; Kshiwgi et l., 2000). In these ells, tretment ledsto p38 tivtion oserved y 2 h. Mximum p38 tivity ismintined etween 6 nd 24 h post- (Efimov et l., 2002). levelsegin to derese 2 4 h fter tretment, nd reh their minimum y 10 h. Importntly, the kinetis of loss suggest tht 435

7 436 this event ours just downstrem from or in ssoition with p38 tivtion. It islso noteworthy tht, wherestrnsriptionl tivtion of the involurin gene minly involvesp38d (Efimov et l., 2002), downregultion oursthrough p38. Although p38 nd p38 re involved in differentition of some ell types(figle et l., 2004; Qi et l., 2004), thisis, to our knowledge, the first report of inhiition E2F trnsription ftor stility y differentition-indued tivtion of MAP kinses. Vrioussignling eventsmodulte the E2F/ growth regultory network. A well-defined signling sde involves tivtion of phosphoinositide-3 kinse nd Akt/PKB, whih results in upregultion of Cylin D, followed y phosphoryltion nd relese of E2F, indiretly tivting itstrnsriptionl tivity (Brennn et l., 1997). Our studies show novel mehnism for regultion vi PKC-MAPK pthwysduring epiderml differentition, thusdding dditionl lyersof omplexity to the pthwystht modulte the E2F/ growth regultory network. Mterils nd methods PKC regultion of turnover Regents nd ntiodies The ntiodies nd their soures were s follows: PKC (610107), PKCd (610397) nd PKCe (610397) from BD Trnsdution Lortories; E2F5 (SC-999) from Snt Cruz Biotehnologies; (MS880) from Neomrkers; AU1 (MMS-130R) from Covne; PKCz from BIOMOL; glyerldehyde-3-phosphte dehydrogense (; Ct. No, ) from Biogenesis nd g-tuulin (T67) from Sigm. The p38 inhiitorssb203580, SB nd SB202474, MG132, nd the PKC inhiitorsro3179 nd Ro were from Cliohem. All other regentswere from Sigm. Adenoviruses Reominnt denovirusenoding wild type (wt) PKC, wt nd dn PKCdK 376R, nd wt nd dn PKCe (Crpenter et l., 2001; Czzoli et l., 2002) were giftsof Dr T Biden. The PKCzenoding denovirusws gift from G King (Suzum et l., 2002). The dn PKCZ DNA ontins K384A muttion (Oh et l., 1998), nd wsgenerted y polymerse hin retion. Both PKCd K376R nd PKCZ K384A mutntslk kinse tivity nd exhiit isozyme-speifi dn properties (Oh et l., 1998; Crpenter et l., 2001). Plsmids enoding mouse dn p38 nd humn dn p38 DNAs(Jing et l., 1996; Engelmn et l., 1998) were gifts, respetively, from P Sherer nd J Hn. Mutnt PKCZ, p38 or p38 DNA wsloned into pshuttlecmv (gift of B Vogelstein), whih ws used to generte reominnt denoviruses using the Ad-Esy system (He et l., 1998). The virusontrolsused to ount for the two different denovirl vetor koneswere Ad-MX17 (for PKC, nd wt nd dn PKCd) nd Ad-LZ, whih enodes -gltosidse (-gl; ontrol for PKCe, wt nd dn PKCZ, PKCz, dn p38 nd dn p38). Adenoviruses were purified on CsCl grdients nd titered prior to use. Cell ulture Primry kertinoytesisolted from 1 2-dy-old CD-1 mie were ultured in C 2 þ -free Egle s minimum essentil medium supplemented with 8% Chelex-treted fetl ovine serum nd indued to differentite y elevting the C 2 þ onentrtion to 1mM (D Souz et l., 2001). Kertinoyteswere inuted in serum-free medium with virus for 5 h. The multipliities of infetion used for the viruses resulted in 90 % effiieny, nd were: PKC, 10; wt PKCd, dnpkcd, wt nd dn PKCe, nd PKCz, 50; ll other viruses, 75. These onditions ensured tht X90% of ells were infeted, nd tht expression of exogenousenzymeswssix- to nine-fold greter thn the orresponding endogenous enzyme, s mesured y immunolot. In kertinoytes, these levels of exogenous PKC expression result in three- to five-fold inreses in PKC tivity (Oh et l., 1998; Luowei et l., 1999). Inutions nd finl drug onentrtionswere:, nm, 24h; helerythrine or Ro 3179, 1 mm; genistein, 300 mm, herimyin A, 1 mm; nd wortmnnin, 50 nm; ll dded 3 4 h prior to dding 1 mm CCl 2. For turnover experiments, yloheximide (CHX; mg/ml) wsdded 30 min prior to ddition of. In experiments using protesoml inhiitors, primry kertinoyteswere inuted with MG132 (10 mm, finl) MG132 (10 mm, finl), dded to the ulture medium 8 h following djustment of C 2 þ to 1mm. The ellswere ultured in high C 2 þ medium for 24 h prior to hrvesting nd lyste preprtion. In these experiments, ws not used in onjuntion with MG132 due to ytotoxiity of the drug omintion over the inution periodsrequired. For p38 experiments, ells infeted with denovirus-enoding PKCd or PKCZ were immeditely ultured in the presene of dimethylsulfoxide () vehile or 10 mm SB203580, SB or SB Fresh medium ontining the inhiitor ws dded to the ellsevery 12 h. Cell lystes nd immunolotting Cell lystes were prepred nd nlyzed for EMSA or immunolot sdesried (D Souz et l., 2001). Frtions ontining 50 mg protein were resolved y denturing polyrylmide gel eletrophoresis (SDS PAGE), nd trnsferred to polyvinylidene difluoride or nitroellulose memrnes. Immunolots were proed with the pproprite ntiodies. or g-tuulin wsused to normlize for protein loding. All results shown re representtive of t lest three experiments. Aknowledgements We thnk DrsF Beier, S Li nd D Lithfield for helpful ommentson the mnusript, nd H Dupuisfor tehnil ssistne. This work ws supported y grnts to LD from the Ntionl Siene nd Engineering Reserh Counil of Cnd, nd the Ntionl Cner Institute of Cnd (NCIC) with fundsfrom the Cndin Cner Soiety nd the Terry Fox Foundtion, rised through the Terry Fox Run. IAI is Reserh student of the Terry Fox Foundtion, through n wrd from NCIC. SJAD is CIHR New Investigtor. During the development of thiswork, LD ws CIHR/Cner Reserh Soiety In. New Investigtor. Referenes Alt A, Oh M, Li L, Grtsein M, Belnger A, Denning MF et l. (2001). Cner Res 61: Apostolov MD, Ivnov IA, Dgnino C, D Souz SJA, Dgnino L. (2002). J Biol Chem 277:

8 Bell LA, Ryn KM. (2004). Cell Deth Differ 11: Brennn P, Bge JW, Burgering BM, Groner B, Reif K, Cntrell DA. (1997). Immunity 7: Codi S, Clutti E, Tlor C, Kuroki T, Stein PL, Dotto GP. (2000). Mol Cell 6: Cm H, Dynlht BD. (2003). Cner Cell 3: Cmpnero MR, Flemington EK. (1997). Pro Ntl Ad Si USA 94: Crpenter L, Cordery D, Biden TJ. (2001). J Biol Chem 276: Czzoli R, Crig DL, Biden TJ, Smitz-Pfeiffer C. (2002). Am J Physiol 282: Chng WY, Brye DM, D Souz SJA, Dgnino L. (2004). J Biol Chem 279: Chid K, Hr T, Hiri T, Konishi C, Nkmur K, Nko K et l. (2003). Cner Res 63: D Souz SJA, Pjk A, Blzsi K, Dgnino L. (2001). J Biol Chem 276: D Souz SJA, Vesp A, Murkherjee S, Mher A, Pjk S, Dgnino L. (2002). J Biol Chem 277: Dshti SR, Efimov T, Ekert RL. (2001). J Biol Chem 276: Denning MF. (2004). Int J Biohem Cell Biol 36: Diker AJ, Pop C, Dhler AL, Serewko MM, Hildith- Mguire PA, Frzer IH et l. (2000). 19: Dotto GP. (1999). Crit Rev Orl Biol Med 10: Ekert RL, Crish JF, Efimov T, Dshti SR, Deuher A, Bone F et l. (2004). J Invest Dermtol 123: Efimov T, Deuher A, Kuroki T, Oh M, Ekert RL. (2002). J Biol Chem 277: Efimov T, L Cell P, Welter JF, Ekert RL. (1998). J Biol Chem 38: Engelmn JA, Lisnti MP, Sherer PE. (1998). J Biol Chem 273: Figle R, Brederlu A, Elmi M, Arvidsson Y, Hmzki TS, Urmoto H et l. (2004). Mol Cell Biol 24: Hteoer G, Kerkhoven RM, ShvrtsA, BernrdsR, Beijersergen RL. (1996). Genes Dev 10: He TC, Zhou S, d Cost LT, Yu J, Kinzler KW, Vogelstein B. (1998). Pro Ntl Ad Si USA 95: PKC regultion of turnover Hofmnn F, Mrtelli F, Livingston DM, Wng Z. (1996). Genes Dev 10: Jing Y, Chen C, Li Z, Guo W, Gegner JA, LinsS et l. (1996). J Biol Chem 271: Kshiwgi M, Oh M, Wtne H, Ishino K, Kshr K, Sni Y et l. (2000). 19: Koster MI, Roop DR. (2004). Eur J Cell Biol 83: Kumr S, Boehm J, Lee JC. (2003). Nt Rev Drug Disov 2: Li L, Lorenzo PS, Bogi K, Blumerg PM, Yusp SH. (1999). Mol Cell Biol 19: Luowei LI, Lorenzo PS, Bogi K, Blumerg PM, Yusp SA. (1999). Mol Cell Biol 19: Mrtelli F, Hmilton T, Silver DP, Shrpless NE, Brdeesy N, RoksM et l. (2001). Pro Ntl Ad Si USA 98: Oh M, Ishino K, Kshiwgi M, Kwe S, Chid K, Huh NH et l. (1998). Mol Cell Biol 18: Prmio JM, SegrellesC, Csnov ML, Jorno JL. (2000). J Biol Chem 275: Pen uelss, Alemny C, Noe V, Ciudd CJ. (2003). Eur J Biohem 270: Piere AM, Fisher SM, Conti CJ, Johnson DG. (1998). 16: Piere AM, Shneider-Broussrd R, Gimenez-Conti IB, Russell JL, Conti CJ, Johnson DG. (1999). Mol Cell Biol 19: Porse BT, Pedersen TA, Xu X, Linderg B, Wewer UM, Friis-Hnsen L et l. (2001). Cell 107: Qi X, Li TG, Hu J, Wng J, SimmonsH, Miur S et l. (2004). Pro Ntl Ad Si USA 101: Reynold NJ, Bldssre JJ, Henderson PA, Shuler JL, BllsLM, BurnsDJ et l. (1994). J Invest Dermtol 103: Romnov LY, Alexndrov IA, Nordn RP, Blgosklonny MV, Mushinski JF. (1998). Biohem 37: Ruiz S, SntosM, SegrellesC, LeisH, Jorno JL, BernsA et l. (2004). Development 131: Sheijen B, Bronk M, vn der Meer T, BernrdsR. (2003). Mol Cell Biol 23: Suzum K, Tkhr N, Suzum I, Isshiki K, Ueki K, Leitges M et l. (2002). Pro Ntl Ad Si USA 99: Vndel L, KouzridesT. (1999). EMBO J 18: