Activation of p38- and CRM1-dependent nuclear export promotes E2F1 degradation during keratinocyte differentiation

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1 (2007) 26, & 2007 Nature Publishing Group All rights reserved /07 $ ORIGINAL ARTICLE Activation of p38- and CRM1-dependent nuclear export promotes degradation during keratinocyte differentiation IA Ivanova 1 and L Dagnino 1,2 1 Department of Physiology and Pharmacology and Regulatory Biology, University of Western Ontario, London, Ontario, Canada and 2 Department of Paediatrics, Child Health Research Institute and Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada E2F factorsmodulate a plethora of cell functions, including proliferation, differentiation, DNA repair and apoptosis. We have shown that differentiation in primary epidermal keratinocytesleadsto downregulation via activation of protein kinase C and p38 mitogen-activated protein kinase. We now demonstrate that downregulation in differentiating keratinocytesinvolvesits ubiquitination, aswell asproteasomal degradation subsequent to CRM1-dependent nuclear export. nuclear export specifically in response to differentiation requiresregionsadjacent to the cyclin A-binding domain in the N-terminusof thisprotein. Significantly, inhibition of p38 interfereswith nuclear export and degradation of during differentiation, but hasno effect on in undifferentiated cells. Thus, induction of differentiation in epidermal keratinocytesactivatesa specific program for post-transcriptional downregulation of, which involvessignaling through p38 and activation of nuclear export pathways. (2007) 26, doi: /sj.onc ; published online 21 August 2006 Keywords: keratinocyte; E2F; differentiation Introduction The epidermis is a complex stratified squamous epithelium, distinctive among adult tissues for its constant selfrenewal and regeneration properties. Normal epidermal self-renewal is achieved in part through differentiation of keratinocyte stem cells from the basal layer of the epidermis (Ito et al., 2005). Differentiation stimuli trigger a complex series of events that lead to keratinocyte migration to the suprabasal layers of the epidermis, expression of differentiation markers and permanent withdrawal from the cell cycle (Byrne et al., 2003). Key for embryonic and postnatal epidermal morphogenesis and Correspondence: Dr L Dagnino, Department of Physiology and Pharmacology, Medical Sciences Building, University of Western Ontario, London, Ontario, Canada N6A 5C1. ldagnino@uwo.ca Received 8 May 2006; revised 28 June 2006; accepted 30 June 2006; published online 21 August 2006 homeostasis are the families of the E2F transcription factors and the retinoblastoma (prb) protein. Alterations in the expression of either E2F or prb proteins disrupt epidermal formation and regeneration, and contribute to tumor formation (D Souza et al., 2002; Chang et al., 2004; Ruiz et al., 2004, 2006). E2F factors are differentially regulated during murine keratinocyte maturation (Dagnino et al., 1997b; D Souza et al., 2001). Specifically, mrna and protein are expressed in undifferentiated keratinocytes in culture and in situ, but their levels decrease in differentiated cells. is central for maintenance of the undifferentiated phenotype in keratinocytes. Indeed, inactivation of the E2f1 gene leads to marked defects in key properties of these cells, such as growth, ability to adhere to extracellular matrix substrates and migration (D Souza et al., 2002). Conversely, abnormal expression in differentiated keratinocytes leads to ectopic DNA synthesis (D Souza et al., 2001) and inhibits upregulation of differentiation markers (Jones et al., 1997). function is modulated through precise temporal control mechanisms, which involve transcriptional and post-transcriptional pathways. The latter likely ensure that timely degradation of occurs to allow proper keratinocyte differentiation. Induction of differentiation by Ca 2 þ in murine keratinocytes triggers a signaling cascade that activates protein kinase C (PKC) d and Z, followed by p38b mitogen-activated protein kinase (MAPK), and culminates in proteolysis (Ivanova et al., 2006). We have further examined the pathways that lead to Ca 2 þ -induced degradation in differentiating keratinocytes and now show that targeting of for proteasomal degradation involves multiple events, including ubiquitination and nuclear export. We further demonstrate that the latter is essential for proteolysis in response to differentiation stimuli in keratinocytes. Results Differentiation in keratinocytes induces ubiquitination and proteasomal degradation Primary murine epidermal keratinocytes remain undifferentiated by culture in medium with low extracellular

2 1148 degradation in differentiated keratinocytes Ca 2 þ concentration (p0.05 mm, Low Ca 2 þ medium ), and differentiate in medium with higher Ca 2 þ levels ( mm, High Ca 2 þ medium ) (Dlugosz et al., 1995). We have demonstrated that induction of differentiation by Ca 2 þ in these cells increases degradation through activation of PKC d and Z, as well as p38b (Ivanova et al., 2006). To address the mechanisms of turnover during keratinocyte differentiation, we first examined the degradation pathways of exogenously expressed in these cells. Treatment of undifferentiated keratinocytes with the proteasome inhibitors MG132 or MG115 increased levels, irrespective of whether protein synthesis was inhibited with cycloheximide (; Figure 1a), and in agreement with the stabilization of endogenous by proteasomal inhibitors we have reported (Ivanova et al., 2006). In contrast, lysosome inhibition by E64 had no significant effect on levels in these cells (Figure 1a), indicating that the proteasome is a primary route of degradation in undifferentiated keratinocytes. Inhibition of proteasomal, but not lysosomal activity in differentiated keratinocytes also interfered with Ca 2 þ -induced degradation (Figure 1a). Thus, proteasomal activity is a key element in the Ca 2 þ - dependent and -independent pathways that regulate turnover in keratinocytes. Proteasomal degradation is mediated through ubiquitination and other modifications of target proteins (Asher and Shaul, 2005). To determine if ubiquitination is associated with enhanced turnover in differentiated keratinocytes, we exogenously expressed and hemagglutinin (HA)-tagged ubiquitin, and assessed the levels of ubiquitinated in HA ubiquitin immunoprecipitates in the presence of MG132, to prevent degradation. We observed higher levels of ubiquitinated in differentiated keratinocytes relative to undifferentiated cells (Figure 1b). Thus, induction of differentiation by Ca 2 þ triggers an increase in ubiquitination and subsequent targeting to the proteasome. degradation in differentiating keratinocytes is associated with nuclear export via CRM1 Abundance and subcellular localization of some E2F proteins, such as E2F5, are tightly regulated by differentiation in keratinocytes (D Souza et al., 2001; Apostolova et al., 2002), which prompted us to examine whether the decrease in stability induced by Ca 2 þ is associated with changes in its subcellular distribution. Levels of endogenous markedly decrease in cultured differentiated keratinocytes (Ivanova et al., 2006) and protein is undetectable in the differentiated layers of normal, resting epidermis in situ (data not shown). Consequently, to facilitate detection, we exogenously expressed in differentiating keratinocytes. localized to the nucleus in about 90% of undifferentiated cells (Figure 2a, Table 1). Induction of differentiation triggered export of from the nucleus into the cytoplasm, causing a fourfold increase in the fraction of the cell population in which a b MG 132 MG 115 E64 Untreated kda IP: IgG HA MG 132 MG 115 E64 Untreated γ-tubulin MG 132 HA-Ub [Ub]n- IgG γ-tubulin Cell lysate Figure 1 Ubiquitination and proteasomal degradation of in differentiating keratinocytes. (a) Primary undifferentiated keratinocytes were transfected with an -encoding vector 24 h before treatment with MG132, MG115 or E64 (all 10 mm, final). (100 mg/ml) or vehicle was added 30 min later. Thirty minutes after addition, culture medium was replaced by Low Ca 2 þ medium (0.05 mm Ca 2 þ ) to maintain undifferentiated cells, or High Ca 2 þ medium (1.0 mm Ca 2 þ ), to induce differentiation. Cells were harvested 2.5 h later and analysed for or g-tubulin, as loading control. (b) Keratinocytes were transfected with vectors encoding V5-tagged and, where indicated, HA-tagged ubiquitin (HA Ub þ ). MG132 (10 mm, final) was added 4 h posttransfection and, where indicated, Low Ca 2 þ culture medium ( High Ca 2 þ ) was replaced with High Ca 2 þ medium ( þ High Ca 2 þ ). Cell lysates prepared 24 h later were immunoprecipitated with an irrelevant antibody (IgG) or anti-ha antibody, and the presence of ubiquitinated was assessed in the HA immune complexes. The species marked with () is a nonspecific band; the arrow shows, and the bracket indicates ubiquitinated. Samples of whole-cell lysate were also analysed to assess levels of exogenous and g-tubulin. was exclusively cytoplasmic ( % of the cells). The remainder of the cells exhibited in the cytoplasm with minor nuclear staining (Table 1). The net export of from the nucleus that occurs upon differentiation is mediated by CRM1, as Ca 2 þ treatment of keratinocytes in the presence of leptomycin B (LMB) prevented exit into the cytoplasm, giving rise to a cell population in which nuclear and cytoplasmic distributions were indistinguishable from those in undifferentiated cells (86% nuclear, 14% cytoplasmic; Figure 2a). Notably, this is a regulatory event specific for, as Ca 2 þ -induced differentiation has no detectable effect on the steady-state subcellular

3 degradation in differentiated keratinocytes a / LMB Table 1 Subcellular localization of proteins in keratinocytes Low Ca 2þa 1149 N b,c C N C wt A A A75 100;A NLS A75 100;A NLS a Keratinocytes were transfected with the indicated constructs and cultured for 24 h in or medium before processing for immunofluorescence microscopy. b Values represent the percentage of total number of cells counted7s.d. c N, cells with both nuclear and cytoplasmic distribution; C, cells with exclusively cytoplasmic distribution. E2F3 Together, our data indicate that induction of differentiation results in export from the nucleus, followed by proteasomal degradation in the cytoplasm. b High Ca2+ LMB γ tubulin Figure 2 Nuclear export and degradation of in differentiated keratinocytes. (a) Undifferentiated keratinocytes were transfected with vectors encoding or E2F3. Growth medium was replaced 4 h after transfection with either Low Ca 2 þ or High Ca 2 þ medium, in the presence or absence of LMB (5 ng/ml). The cells were cultured for further 24 h, before processing for indirect immunofluorescence with antibodies against the indicated tagged E2F proteins. Nuclei were visualized with Bar: 15 mm. (b) Undifferentiated keratinocytes were transfected with a vector encoding V5-tagged and, 24 h after transfection, LMB (5 ng/ml) was added. Thirty minutes later, (100 mg/ml) was also added. Culture medium was replaced with Low Ca 2 þ or High Ca 2 þ medium containing inhibitors 30 min later, and cells were harvested and lysed after an additional incubation of 2.5 h at 371C. Lysates were resolved and analysed by immunoblotting with antibodies against V5 (to detect ) or g-tubulin, used as a loading control. distribution of E2F3 (Figure 2a), whereas it triggers nuclear accumulation of E2F5 (Apostolova et al., 2002). To determine the role that CRM1-dependent export plays in the decreased stability of, we compared the abundance of in the presence and absence of LMB. Treatment of undifferentiated cells with LMB had no significant effect on protein levels (Figure 2b). In stark contrast, LMB inhibition of CRM1 precluded degradation in differentiated keratinocytes, irrespective of whether protein synthesis was inhibited with or allowed to proceed (Figure 2b). domains involved in differentiation-induced changes in subcellular localization and degradation To begin to define the regions in involved in Ca 2 þ - induced nuclear export and degradation, we exogenously expressed a series of mutants in differentiating keratinocytes (Figure 3a) and compared their subcellular distribution and abundance with those observed in undifferentiated cells. Exogenously expressed wild-type (wt) is exported from the nucleus (Figure 3b, Table 1) and its levels decrease in cells cultured in High Ca 2 þ medium, an effect more clearly observed in the absence of de novo protein synthesis (Figure 4). To map important domains for differentiation-associated regulation, we first tested (86 437), a N-terminus deletion mutant that lacks binding sequences for the ubiquitin ligase SCF p45 Skp2, implicated in degradation (Marti et al., 1999), but which still contains cyclin A-binding sequences. (86 437) was not exported from the nucleus upon induction of differentiation (Figure 3b, Table 1). Significantly, the abundance of (86 437) was not altered by Ca 2 þ treatment (Figure 4). We next examined D75 100, which contains the SCF p45 Skp2 -binding domain, but lacks cyclin A-interacting amino acids, as well as sequences previously described to direct nuclear import in other cell types (Muller et al., 1997). Notably, D efficiently localized to the nucleus in undifferentiated cells, was not exported upon Ca 2 þ treatment (Figure 3b, Table 1) and was stable in the presence of Ca 2 þ (Figure 4). Several conclusions may be drawn from these experiments. First, Ca 2 þ -induced degradation and nuclear export require an intact N-terminal domain, but not necessarily the ability to bind either SCF p45 Skp2 or cyclin A. Second, degradation of upon differentiation requires exit from the nucleus into the cytoplasm, consistent with the stabilization of in LMB-treated cells described above.

4 1150 a CycA DNA DP TA prb b wt ; E2F degradation in differentiated keratinocytes E2F ; Figure 3 Nuclear export of mutants in differentiating keratinocytes. (a) Schematic of proteins exogenously expressed in keratinocytes. The domains indicated are: CycA, cyclin A-binding domain; DNA, DNA-binding domain; DP, dimerization domain; TA, transactivation domain; prb, prbbinding region. Numbers on the right indicate residues either present (86 437, 1 380) or deleted (D75 100, D and D75 100; D ) in the mutants.(b) Vectors encoding wt or the indicated mutants were transfected in undifferentiated keratinocytes and culture medium was replaced 4 h after transfection with Low Ca 2 þ or High Ca 2 þ medium. The cells were cultured for 24 h to allow differentiation by High Ca 2 þ medium, and were then processed for indirect immunofluorescence microscopy. Nuclei were visualized with Bar: 15 mm. Third, the sequences involved in nuclear export of comprise residues around the region The C-terminal transactivation domain of contains overlapping domains that mediate degradation and binding to prb (Campanero and Flemington, 1997). Specifically, key proteolysis targeting sequences have been mapped to amino acid residues , whereas a prb-binding region involved in preventing ubiquitination and proteasomal processing of when associated with prb spans residues We examined the role of these domains in differentiationinduced degradation, by expressing an mutant lacking residues D was exported from the nucleus (Figure 3b, Table 1) and degraded upon Ca 2 þ treatment in a manner indistinguishable from the wt protein (Figure 4), indicating that the destabilization effect of residues is not essential for differentiation-induced degradation. The association of degradation with changes in subcellular localization suggested that nuclear retention of might protect it from Ca 2 þ -induced proteolysis. This prompted us to investigate whether differentiationinduced degradation of required nucleocytoplasmic movement, or it could occur in constitutively cytoplasmic forms. Some members of the E2F family can be transported to and/or retained in the nucleus through their association with prb family proteins (Gaubatz et al., 2001; Apostolova et al., 2002). Thus, we examined the regulation in differentiating keratinocytes of a double mutant, D75 100, D , which lacks N-terminal sequences, as well as sequences involved in binding to prb (Figure 3a). Mutation of these two regions generated a protein that was no longer capable of localizing to the nucleus, irrespective of the differentiation status of the cells (Figure 3b, Table 1), or the presence or absence of LMB (data not shown). Notably, Ca 2 þ treatment still induced degradation of this double mutant (Figure 4), consistent with the concept that localization of to the cytoplasm is key for its proteolysis during differentiation. The transactivation domain of includes residues , and contains sequences that mediate proteasomal degradation, both in the context of and when fused to heterologous proteins (Campanero and Flemington, 1997). We wished to determine if deletion of this domain would interfere with differentiationinduced degradation, and consequently examined (1 380), which lacks the entire transactivation domain. (1 380) was expressed at high levels and exported from the nucleus in differentiating keratinocytes (Figure 3b, Table 1). However, it was not degraded upon induction of differentiation (Figure 4), indicating that the C-terminus in also has a destabilizing effect in differentiating keratinocytes. Nuclear export of is essential for its degradation in differentiating keratinocytes The increased stability of constitutively nuclear mutants suggested that localization of to the cytoplasm is a crucial event for differentiation-induced degradation. Consequently, we reasoned that forcing sequestration of into the nucleus would prevent its degradation in differentiating keratinocytes. To test this hypothesis, we generated mutants containing amino acids encoding the nuclear localization signal (NLS) present in Simian virus 40 (SV40) large T antigen, which directs a variety of proteins into the nucleus,

5 kda wt degradation in differentiated keratinocytes / γ tubulin 1151 levels (% untreated ) Figure 4 Stability of mutants in differentiating keratinocytes. Vectors encoding wt or the indicated mutants were transfected in undifferentiated keratinocytes, and 24 h later, the cells were treated with (100 mg/ml). The culture medium was replaced 30 min following the addition of with Low or High Ca 2 þ medium (with or without ), and cells were cultured for an additional 2.5-h period. Cell lysates were prepared and analysed by immunoblot for the indicated proteins, using g-tubulin as loading control. The histograms under each blot represent normalized densitometric quantification of protein abundance (average þ s.e.m., n ¼ 3), and are expressed as the percentage of a given protein under each treatment, relative to the abundance of that same protein in undifferentiated keratinocytes cultured in Low Ca 2 þ medium without ( untreated cells). indicates significantly different levels of cells cultured in High Ca 2 þ medium and, relative to cells cultured in Low Ca 2 þ medium and (Po0.005, two-way repeated measures analysis of variance). including E2F4 (Muller et al., 1997; Apostolova et al., 2002). The addition of the NLS to wt (-NLS) produced a protein that was constitutively nuclear in 100% of undifferentiated and differentiated keratinocytes (Figure 5a), and whose levels were unaltered by differentiation (Figure 5b). We also added the NLS to D75 100; D , which is not imported into the nucleus, but is susceptible to Ca 2 þ -induced degradation. Similar to -NLS, D75 100; D NLS was nuclear in undifferentiated and in differentiated cells (Figure 5a). Notably, this mutant no longer responded to Ca 2 þ -induced destabilization, as its abundance was unchanged in differentiated cells (Figure 5b). Hence, induction of differentiation by Ca 2 þ triggers a signaling cascade that results in nuclear export. Transit of into the cytoplasm is essential for its proteasomal degradation in response to differentiation cues. nucleocytoplasmic transit is mediated by p38 MAPK We have demonstrated that activation of p38 is necessary for degradation in differentiating keratinocytes (Ivanova et al., 2006). Thus, we investigated if activation of p38 is also a prerequisite for nuclear export of. Similar to previous experiments, exogenously expressed localized to the nucleus in X90% of undifferentiated keratinocytes. This pattern of distribution was not altered upon inhibition of p38 by SB220025, or by treatment of cells with the inactive analog SB (Figure 6), indicating that p38 activity does not significantly contribute to the regulation of subcellular localization in undifferentiated cells. In stark contrast, treatment of differentiating keratinocytes with SB220025, but not with inactive SB202474, precluded translocation to the cytoplasm (Figure 6). Thus, activation of p38 by Ca 2 þ results in export from the nucleus and subsequent degradation in the cytoplasm. Discussion Differentiation induced by Ca 2 þ in epidermal keratinocytes triggers a signaling cascade that includes activation multiple kinases, with upregulation of differentiation markers, such as involucrin, and downregulation of (Efimova et al., 1998; Ivanova et al., 2006). plays a key role in proliferation of undifferentiated keratinocytes and in their ability to regenerate the epidermis after injury (D Souza et al., 2002). However, abnormal maintenance of expression in differentiated keratinocytes leads to ectopic DNA synthesis and interferes with differentiation (Dicker et al., 2000; D Souza et al., 2001). Thus, the activation of mechanisms that lead to downregulation is crucial for keratinocyte differentiation. We previously established in keratinocytes that one of the differentiation responses triggered either by Ca 2 þ,by phorbol ester-induced activation of PKC or by exogenous expression of PKC d or Z is rapid degradation. This process is mediated by activation of PKC d and Z, followed by that of p38b, and is impaired when cells are treated with Ca 2 þ or 12-O-tetradecanoylphorbol-13- acetate in the presence of exogenously expressed dominantnegative PKC d or Z mutants, or pharmacological p38

6 degradation in differentiated keratinocytes 1152 a E2F E2F wt Untreated -NLS SB ; 423 SB ; 423- NLS b kda wt -NLS 75; ; 423- NLS γ-tubulin Figure 5 Subcellular localization and stability of NLS-containing proteins. (a) Vectors encoding wt, -NLS, D75 100; D (labeled as D75;D423) or D75 100; D NLS (labeled as D75; D423-NLS) were transfected in undifferentiated keratinocytes and culture medium was replaced 4 h after transfection with Low Ca 2 þ or High Ca 2 þ medium. The cells were then cultured for 24 h to allow differentiation by High Ca 2 þ medium, and were processed for indirect immunofluorescence microscopy to detect exogenously expressed E2F proteins. Nuclei were visualized with Bar: 15 mm. (b) Undifferentiated keratinocytes were transfected with the indicated -encoding vectors 24 h before treatment with (100 mg/ml) for 30 min, followed by replacement of the culture medium with Low or High Ca 2 þ medium supplemented with. Cell lysates were prepared 2.5 h later, and analysed for levels of exogenous E2F proteins by immunoblot. g-tubulin was used as a loading control. inhibitors (Ivanova et al., 2006). We have now added mechanistic insight to those studies, and show that Ca 2 þ induction of differentiation in epidermal keratinocytes elicits export from the nucleus mediated by CRM1 and proteasomal degradation in a p38-dependent manner. The substantial cytoplasmic localization of in mature keratinocytes contrasts with its predominantly nuclear localization in undifferentiated keratinocytes and other cell types. The existence of nucleocytoplasmic shuttling pathways for is yet to be examined. However, such pathways may exist in keratinocytes, and induction of differentiation may alter steady-state distribution, favoring its cytoplasmic localization. Alternatively, differentiation may trigger changes in and/or -associated proteins, allowing establishment of interactions with CRM1 and subsequent export from the nucleus. Although downregulation of has been observed during differentiation in the Figure 6 nuclear export in differentiated keratinocytes requires p38 activation. Undifferentiated keratinocytes were transfected with a V5-tagged -encoding vector. Four hours later, the p38 inhibitor SB or its inactive analog SB was added to the culture. The culture medium was replaced with Low or High Ca 2 þ medium supplemented with inhibitors, and keratinocytes were then cultured for 24 h. The cells were processed for indirect immunofluorescence, using anti-v5 antibodies and to visualize, respectively, exogenous and nuclei. Bar, 15 mm. epidermis and other tissues in situ, as well as in various cell lines (Dagnino et al., 1997a, b; Gill and Hamel, 2000; Fajas et al., 2002), nuclear export and proteasomal degradation have not been reported for any other primary cell type. Whether this is a keratinocyte-specific or a general mechanism to regulate in differentiating, normal tissues remains to be determined. Several important domains have been mapped in. We have demonstrated that the N-terminus is critical for nuclear export in differentiating keratinocytes, although in silico analysis of the amino-acid sequence failed to reveal any consensus nuclear export signal. This region may be modified upon differentiation, allowing direct interaction with CRM1. Alternatively, the N-terminus of may facilitate interaction with other protein(s), which in turn bind to CRM1, similar to the export of nuclear p53 via CRM1, which depends on p53 association with MDM2 and subsequent interaction of MDM2 with CRM1 (Freedman and Levine, 1998). With regards to nuclear import, our data indicate that the region comprised of residues is dispensable in keratinocytes, in contrast with the reported NLS that was previously mapped to residues (Muller et al., 1997), as evidenced by the nuclear localization of D and (86 437). Notably, we observed that nuclear import was lost in D75 100; D , in which both the region around the putative NLS and a region important for prb binding were altered. Thus, at least two regions appear to contribute to nuclear import in primary keratinocytes. Given that D and (86 437) exhibit cytoplasmic distribution in cell lines with compromised prb function, such as HeLa and C33A cells (, unpublished observations), association with prb may contribute to

7 nuclear import and/or retention of this transcription factor in primary epidermal keratinocytes. Nuclear export of is a key step in its degradation triggered by differentiation stimuli. Consistent with this concept, constitutively nuclear mutants were stable in differentiating keratinocytes, irrespective of whether they contained regions known to be important for degradation, such as the region comprising amino-acid residues 1 41, which binds SCF Skp2 ubiquitin ligase. The stability of (86 437) also suggests that SCF Skp2 does not play a major role regulating stability in keratinocytes. Another line of evidence for nuclear export as a central step in degradation is provided by the observed decrease in stability of D75 100; D , which is constitutively cytoplasmic and is also destabilized by Ca 2 þ - induced differentiation. We have previously demonstrated that p38b is implicated in regulating stability during keratinocyte differentiation (Ivanova et al., 2006). The role of p38 in ubiquitination is not clear, as simultaneous treatment of keratinocytes with p38 and proteasomal inhibitors affected cell viability (IA Ivanova and L Dagnino, unpublished observations). However, activation of p38 is also essential for the nuclear export of associated with its differentiation-induced degradation. Our data are consistent with a model in which Ca 2 þ -induced activation of PKC d and Z triggers, in turn, activation of p38 in differentiating epidermal keratinocytes. The latter is able to induce export from the nucleus, targeting it for proteasomal degradation. Downregulation of may then allow keratinocyte entry into quiescence during terminal differentiation. Several key issues remain to be defined. For example, it is unclear whether any of the putative p38 target amino-acid sequences present in serve as substrates for p38. However, other transcription factors, such as nuclear factor of activated T cells, are exported from the nucleus in a manner dependent on CRM1 following their phosphorylation by p38 (Gomez et al., 2000). Our data also suggest that degradation in response to differentiation occurs predominantly in the cytoplasm. An important future step will be the elucidation of the exact mechanisms and location of degradation in differentiated keratinocytes ubiquitination specifically activated in response to differentiation stimuli in epidermal keratinocytes. Materialsand methods Cell culture and reagents Primary keratinocytes isolated from 1 to-2-day-old CD-1 mice were cultured as described (D Souza et al., 2001), and transfected with ExGen 500 (Fermentas, Burlington, Ontario, Canada), following the manufacturer s instructions. The antibodies used were as follows: (MS880; LabVision Corp., Fremont, CA, USA), anti-ha ( ; Roche Applied Science, Indianapolis, IN, USA), and anti-v5 (R960 25; Invitrogen, Carlsbad, CA, USA). Secondary antibodies were purchased from Jackson Immuno Research Laboratories Inc. (West Grove, PA, USA). MG132 (C2211), MG115 (C6706), E64 (E3132), (C7698) and were from Sigma (St Louis, MO, USA); SB and SB were from Calbiochem. LMB was from Biomol (Plymouth Meeting, PA, USA). Indirect immunofluorescence and statistical analysis Indirect immunofluorescence images were acquired as described (Vespa et al., 2005), except that the cells were first fixed in 4% paraformaldehyde (20 min, 221C), and then permeabilized (1% Triton X-100, 10 min, 221C). Processed cells were mounted in Immu-Mount medium (Thermo Shandon, Pittsburgh, PA, USA). The fraction of cells with nuclear or cytoplasmic protein distribution was determined by evaluating subcellular localization in cells. Statistical analyses were carried out with SPSS version 11, using two-way repeated measures analysis of variance, and setting significance at The results shown are representative of experiments conducted on duplicate samples, at least three times. Acknowledgements We thank Drs M Tini and S D Souza for helpful comments on the manuscript. This work was supported by grants to LD from the Natural Science and Engineering Research Council of Canada, and the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society and the Terry Fox Foundation, raised through the Terry Fox Run. IAI holds research studentships from the Terry Fox Foundation, through an award from NCIC, and from the Canadian Institutes of Health Research Training Program in Cancer Research and Technology Transfer References Apostolova MD, Ivanova IA, Dagnino C, D Souza SJA, Dagnino L. (2002). Active import and export mechanisms regulate E2F-5 subcellular localization. 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