Phosphoinositide 3-Kinase Signaling to Akt Promotes Keratinocyte Differentiation Versus Death*

Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of “late” differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.

The epidermis is a self-renewing stratified epithelium in which the loss of terminally differentiated cells from its surface is balanced by cells that leave the proliferative basal layer and enter differentiation (1,2). Because a metabolically dead cornified cell envelope is the end point of epidermal differentiation, this process may be viewed as a specialized form of programmed cell death (3). Moreover, the apoptotic program and keratinocyte differentiation share overlapping signaling effector mechanisms (4). Notably, the caspase-3 cysteine protease, an integral component of the cell death machinery, was implicated recently in embryonic keratinocyte differentiation control downstream of Notch1 (5), and activation of the related caspase-14 has been reported during adult keratinocyte differentiation (6). Nevertheless, "canonical" apoptosis and epidermal differentiation are distinct processes, with diverse execution times and biological outcomes; the former leads to the elimination of individual dead cells from tissues within hours, whereas the latter relies on the survival and synchronized maturation of whole sheets of cells over the course of weeks. Thus, an outstanding question is how keratinocytes can activate arrays of death-inducing signals during differentiation and yet remain protected from premature death.
A candidate pathway for the survival of differentiating keratinocytes is the signaling module formed by the Class IA PI3K 4 and the downstream serine-threonine kinase Akt effectors (Akt/PKB-1, -2, and -3 isoforms) (7). The PI3K family is divided into three distinct classes (Class I, II, and III) based on primary structure, substrate specificity, and mode of regulation (8). Class I PI3Ks include four distinct p110 catalytic isoforms, further divided into Class IA (-␣, -␤, -␦) and IB (-␥); among these, p110␣ and p110␤ are the isoforms expressed mostly in epithelia (9). The Class I PI3K-Akt axis has key roles in the transduction of survival and/or mitogenic signals from receptor tyrosine kinases and cell adhesion events, including those mediated by the calcium-dependent cadherin molecules (10 -12). Akt-dependent protein phosphorylation directly inhibits the activities of the FoxO and BAD pro-apoptotic proteins and promotes cell survival through indirect effects on p53 and NF-B and via stimulation of glucose metabolism and protein synthesis (13,14). Although murine knock-outs of the p110␣ and p110␤ genes result in embryonic lethality, Akt-1 and -2 double knock-out animals die perinatally and display severe atrophy and impaired differentiation of the epidermis, suggesting essential function of these molecules in the tissue homeostasis (15). Whether Akt proteins have specific roles in differentiation or simply affect cell proliferation still needs to be defined.
Class IA PI3Ks are classically activated by tyrosine phosphorylation events, with the lipid kinase being recruited to the cell membrane by interactions of the SH2 domains of the p85 regulatory subunit with tyrosine-phosphorylated YXXM amino acid motifs of receptor tyrosine kinases (16) and/or scaffolding proteins, such as IRS1-4 and GAB1-3 (17). Potential links between tyrosine kinase signaling and PI3K/Akt activation in the context of epidermal differentiation are suggested by the similar epidermal phenotypes of mice lacking the insulin-like growth factor-1 receptor, the epidermal growth factor receptor (EGFR), or Akt-1 and -2 (15,18), with impaired cell proliferation in the basal layer and differentiation abnormalities in the suprabasal keratinocyte layers. Notably, tyrosine phosphorylation of ␤and ␥-catenin molecules in complex with E-cadherin is induced during the assembly of cell-cell adhesive structures of differentiating keratinocytes, an event largely dependent on the increased activity of Fyn/Src kinases during this process (19). The tissue-specific ablation of E-cadherin impairs both the survival and the differentiation of epidermal keratinocytes and mammary epithelial cells, indicating critical roles of this molecule in orchestrating cellular signals favoring both processes in epithelia (20,21). At present, the signals downstream of E-cadherin in this context still await identification.
Thus, keratinocytes are endowed with active signaling machinery that may impinge on PI3K/Akt during differentiation; whether the endogenous PI3K/Akt axis is engaged during this process still needs to be established. We show here that the PI3K/Akt pathway is activated early on during differentiation both in cultured keratinocytes and in the epidermis in vivo and that its signaling activity is critical for the survival of differentiating cells and the proper execution of the differentiation program. We provide evidence that in this process the activation of the PI3K pathway relies on a differentiation-specific mechanism that requires both tyrosine kinase activity and the engagement of E-cadherin adhesive functions.

EXPERIMENTAL PROCEDURES
Cells, Inhibitors, and Viruses-Primary keratinocytes were isolated from 2-3-day-old C57B mice, cultured in low calcium medium (50 M CaCl 2 ), and induced to differentiate by raising the calcium concentration to 2.0 mM (high calcium medium) as described previously (22). Genistein and PP1 were from Biomol Research Laboratories. Ly 294002, wortmannin, bisindolylmaleymide, Go6976, and AG1478 were from Calbiochem.
The functional inhibition of E-cadherin adhesion molecules was carried out essentially as described previously (11), except with the following modifications: keratinocytes in low calcium medium were pretreated with 0.2 mM EGTA. The monoclonal antibodies DECMA-1 or CH-19 (Sigma) against the E-cadherin ecto-or cytoplasmic domain, respectively, were added to this medium for 20 min. A second dose of antibodies was added, and cells were further incubated in either low or high calcium medium.
A replication-defective recombinant adenovirus expressing a myristoylated, active Akt mutant (AdAkt-Myr) was a gift from Dr. K. Walsh (Tufts University School of Medicine, Boston, MA) and has been described previously (23). A replication-defective adenovirus expressing the green fluorescent protein (AdGFP) driven by a cytomegalovirus promoter was used as a control adenovirus. Adenoviruses were amplified and purified and used as described previously (24), at the indicated multiplicity of infection.
Immunoprecipitation-Immunoprecipitation was carried out as described previously (19), except for the cell lysis buffer that was supplemented with 1.75% n-octylglucoside.
PI3K and Akt Kinase Assays-Cell extracts (200 g) were immunoprecipitated with antibodies to p85␣ or phosphotyrosine (p-Tyr) antibodies, and the PI3K assay was carried out as described previously (26). The phosphorylated reaction products were separated by thin-layer chromatography by an overnight run in a buffer containing a 34:65:1 (v/v) ratio of H 2 O, n-propyl alcohol, and glacial acetic acid. Radiolabeled phosphoinositides were visualized by autoradiography. The measurement of total Akt kinase activity was performed with a non-radioactive Akt kinase assay kit from Cell Signaling and Technology, according to the manufacturer's instructions. Substrate phosphorylation was visual-FIGURE 1. PI3K and Akt activation during mouse keratinocyte differentiation. Primary keratinocytes were either kept under proliferating conditions in low calcium medium (Ϫ) or induced to differentiate by raising the extracellular calcium to 2 mM (Ca ϩϩ ) for the indicated times. A, cell lysates were immunoprecipitated (IP) with anti-p85␣-PI3K antibody (PI3K) or anti-phosphotyrosine antibody (p-Tyr), and immune complexes were assayed for PI3K enzymatic activity in presence of PI 4-monophosphate and PI 4,5-bisphosphate and [␥-32 P]ATP. The phosphorylated lipid products phosphoinositide 3,4-bisphosphate (PIP2) and phosphoinositide 3,4,5-trisphosphate (PIP3) are indicated. B, lysates were immunoprecipitated with an antibody preferentially recognizing active Akt isoforms, and Akt in vitro kinase activity was assayed in presence of cold ATP. Phosphorylation of the GSK fusion protein exogenous substrate was visualized by blotting with antibody to Ser-21/9-phosphorylated GSK-3␣/␤ (p-GSK3, upper panel). Assay normalization was performed by immunoblotting analysis of cell lysates for total Akt levels (lower panel). Similar results were obtained in at least two other independent experiments. C, total cell extracts in SDS lysis buffer were analyzed by blotting with an antibody against Ser-473-phosphorylated, active Akt (p-Akt, upper panel), or total Akt (lower panel). D, lysates were immunoprecipitated with antibodies against Akt-1, -2, and -3 proteins, or rabbit non-immune IgG (RIgG), and the kinase activity of the Akt isoforms was measured in the presence of a GSK3 fusion protein and [␥-32 P]ATP (upper panel). Each graph (lower panel) shows the quantification of the corresponding Akt isoform activity, as determined by densitometric analysis of phosphorylated substrates, normalized for the corresponding Akt protein levels determined by immunoblotting (data not shown). Values are expressed as -fold induction relative (Rel.) to the unitary level of kinase activity of each Akt isoform in proliferating cells (Ϫ). Similar results were observed in another independent experiment. ized by immunoblotting with phospho-GSK3 (Ser21/9) antibodies. Isoform-specific Akt kinase activity was assessed in presence of [␥-32 P]ATP after immunoprecipitation with antibodies to Akt 1, -2, and -3 from 200 g of protein cell extracts, as described previously (11).
Immunofluorescence-Primary keratinocytes were processed for immunostaining as described previously (19). Frozen sections from mouse skin (6-m thick) were fixed with 4% paraformaldehyde and permeabilized in TBS, 0.3% Triton X-100. Slides were blocked in 3% bovine serum albumin, 5% goat serum in TBS-Triton for 30 min; primary antibodies were incubated in TBS-Triton, 3% bovine serum albumin, followed by incubation with fluorescein isothiocyanate-or Texasred-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc.). Slides were analyzed with a Leica TCS NT4D confocal microscope (Leica, Heidelberg, Germany). The lack of bleed-trough between channels in the double labeling experiments was verified experimentally.

RESULTS
The PI3K Signaling Pathway Is Activated at the Onset of Keratinocyte Differentiation Both in Vitro and in Vivo-When exposed to high extracellular calcium concentrations, mouse primary keratinocytes withdraw from the cell cycle and undergo morphological and biochemical changes closely recapitulating the epidermal differentiation process in vivo (22). To establish whether the activity of the PI3K pathway is modulated during keratinocyte differentiation, we compared the PI3K and Akt enzymatic activities in proliferating cells and cells induced to differentiate by an increase of the extracellular calcium. The p85␣-associated PI3K activity was strongly induced within minutes and further increased up to 1 h of calcium exposure, whereas by 24 h, it diminished to levels similar to those of undifferentiated cells (see Fig. 1A, left panel, and Fig. 2A). Because active Class IA and IIA PI3Ks are often in complex with tyrosine-phosphorylated proteins (7), we also measured the PI3K activity immunoprecipitated by anti-phosphotyrosine antibodies and found a kinetic profile nearly identical to that detected in direct p85␣ immunoprecipitates (Fig. 1A, right panel). Because both phosphoinositide (PI) 4-monophosphate and PI 4,5-bisphosphate substrates were found increasingly phosphorylated to phosphoinositide 3,4-bisphosphate and phosphoinositide 3,4,5-trisphosphate, respectively, it is likely that the phosphotyrosine-associated PI3K activity induced during differentiation is mostly due to Class IA enzymes, considering that Class IIA isoforms cannot utilize the PI(4,5)P2 substrate (8). As expected, both the kinase activity and Ser 473 -phosphorylation of Akt followed closely the profile of PI3K activity (Fig. 1, B and C, and Fig. 2B); among Akt isoforms, Akt-2 and -3 were found preferentially activated during differentiation, whereas Akt1 was only marginally affected (Fig. 1D). Consistently, Akt protein targets such as GSK3␣ and -␤, the FoxO subgroup of forkhead transcription factors, and the MDM2 ubiquitin ligase (13) were all found increasingly phosphorylated at their Akt substrate sites during differentiation, as revealed by immunoblotting with the appropriate phosphorylation-specific antibodies (Fig. 2C).
To assess whether the changes of PI3K/Akt activity detected in vitro reflect events occurring in the epidermis in vivo, we analyzed both the membrane localization of the p85␣ subunit, a hallmark of type IA PI3K activation, and the distribution of active Akt in 3-day-old mouse skin. The p85␣ subunit was preferentially localized at cell-cell contacts of suprabasal differentiating keratinocytes, as indicated by the concomitant expression of the differentiation marker K1 (Fig. 3A). Active Akt was mostly localized in post-mitotic suprabasal cells and in a few basal cells loosely connected to the basement membrane that had initiated movement toward the spinous epidermal layer (Fig. 3B). Double immunofluorescence staining for active Akt coupled to either K14, a prolifer-ative keratinocyte marker (Fig. 3C), or K1, a marker of early keratinocyte differentiation (Fig. 3D), showed that the increase of active Akt coincides with the initial K1 expression and extends up to the granular epidermal layer. In contrast, the majority of K14-positive basal cells were negative for phospho-Akt staining. An increase of active Akt in the differentiated layers of the epidermis was also observed in the adult mouse skin, irrespective of the age of the animal or the stage of the hair cycle (not shown).
The PI3K/Akt Pathway Is a Key Determinant of Keratinocyte Differentiation Versus Death-The above results suggest that the PI3K/Akt signaling participates in the control of keratinocyte differentiation and/or suppresses apoptosis that it would otherwise be associated with. Withdrawal from the cell cycle and growth arrest is an intrinsic aspect of the keratinocyte differentiation program. To assess whether the expression of constitutively active Akt is sufficient to affect keratinocyte growth and/or differentiation, we employed an adenoviral vector approach, expressing Akt in a membrane-targeted form (AdAkt-Myr) (23). Infection of keratinocytes with this adenovirus caused a dose-dependent decrease of BrdUrd incorporation in both proliferating and differentiating cells compared with cells infected with a GFP-expressing adenovirus (AdGFP) (Fig. 4A), in which BrdUrd uptake was nearly identical to that of uninfected cells (data not shown). To assess whether Akt-induced growth arrest is coupled to increased keratinocyte differentiation, we analyzed the expression of epidermal differentiation markers in uninfected cells and cells infected with either the AdGFP or AdAkt-Myr virus. Cells expressing active Akt displayed higher levels of K1, loricrin, and filaggrin under both proliferating and differentiating conditions; in contrast, levels of K5, a marker of basal keratinocytes constitutively expressed by these cells in culture, remained unaffected (Fig. 4B).
To determine whether inhibition of the endogenous PI3K signaling affects keratinocyte differentiation, cells were kept under proliferating and differentiating conditions in the presence of the PI3K pharmacological inhibitors, wortmannin and Ly294002, and analyzed for the expression of differentiation markers. PI3K inhibition caused a significant decrease in the amounts of the late markers, loricrin and filaggrin, and to a lesser extent, those of the early marker K1, whereas K5 expression was not affected (Fig. 5). The effectiveness of PI3K inhibition was confirmed by the decreased levels of active Akt in differentiating cells exposed to the inhibitors (Fig. 5, lower panels).
If the PI3K/Akt pathway counteracts pro-cell death activities induced during epidermal cell differentiation (4), PI3K inhibition may result in the selective death of differentiating keratinocytes. In fact, 13 and 10% of keratinocytes induced to differentiate for 2.5 h in the presence of wortmannin and Ly294002, respectively, showed morphological features of apoptosis and high levels of active caspase-3, events that were not induced by the inhibitors under proliferating conditions (Fig. 6, A and  B). We then asked whether the more pronounced suppression of loricrin and filaggrin expression caused by PI3K inhibition (see Fig. 5) could be related to an increased susceptibility to apoptosis of keratinocytes expressing late differentiation markers. Indeed, in the presence of PI3K inhibitors, more than 75% of differentiating keratinocytes positive for active caspase-3 were also loricrin-positive, whereas only 10% of these cells showed a detectable K1 expression (Fig. 6, C and D).
The Activation of the PI3K/Akt Pathway Depends on Both Tyrosine Kinase Activity and E-cadherin Engagement during Keratinocyte Differentiation-E-cadherin plays a pivotal role in keratinocyte cell-cell adhesion and differentiation (27) and may mediate survival signals in epithelia (20,21). To test whether E-cadherin-dependent cell adhesion is a trigger for PI3K/Akt activation in differentiating keratinocytes, we compared the levels of active Akt in cells that were either treated with an E-cadherin function-blocking antibody, DECMA-1, or a control antibody prior to differentiation induction. The effectiveness of cadherin inhibition was confirmed by the displacement from cell contacts of p120 ctn , a protein recruited at the cell membrane via its direct binding to cadherins during cell junction assembly (28) (Fig. 7A). Keratinocytes treated with DECMA-1 showed a nearly complete suppression of Akt activation in response to calcium relative to control-treated cells (Fig.  7B); in contrast, these cells retained a normal Akt activation in response to epidermal growth factor stimulation. Notably, functional inhibition of E-cadherin not only blocked the PI3K/Akt pathway but also caused a decreased expression of the keratinocyte differentiation markers, loricrin and filaggrin (Fig. 7C), and increased levels of active caspase-3 and morphological hallmarks of apoptosis ( Fig. 7D and data not shown).
Because higher levels of active PI3K were associated with phosphotyrosine-containing proteins during differentiation (see Fig. 1A), it was important to verify whether this increased activity depends on tyrosine phosphorylation events induced during this process. With this aim, cells were exposed to an array of protein kinase inhibitors under both proliferating and differentiating conditions, and the level of Ser 473 -phosphorylated Akt was analyzed as read out of the PI3K pathway activation state. Differentiation-induced Akt phosphorylation was abolished by treatment with the broad specific tyrosine kinase inhibitor, genistein, and was substantially reduced by both the Src family kinase inhibitor, PP1, and the EGFR-specific inhibitor, tyrphostin AG1478 (Fig. 7E). Importantly, the differentiation-induced increase in active Akt was specifically sensitive to tyrosine kinase inhibition, as it was not affected by the treatment of cells with inhibitors of the protein kinase C family of protein kinases, the functions of which are also critical for keratinocyte differentiation (29,30).
Keratinocyte Differentiation Triggers the Tyrosine Kinase-dependent Association of PI3K with E-cadherin-Catenin Protein Complexes-The above results suggest that the activation of the PI3K pathway may depend on cooperative signals from the EGFR and Src-like tyrosine kinases and the E-cadherin adhesion molecule. Therefore, we tested whether PI3K associates with E-cadherin-catenin complexes during differentiation and whether this association is tyrosine phosphorylationdependent. Co-immunoprecipitation experiments revealed that both the p85␣ and the p110␣ PI3K subunits associate with E-cadherin-catenin protein complexes already in growing cells (Fig. 8A, left panel). Upon induction of differentiation, p85␣ was increasingly associated with E-cadherin and ␤and ␥-catenins, whereas p110␣ displayed higher association with ␥-catenin. p120 ctn , which also associates with E-cadherin during differentiation (19), was found increasingly associated with both p85␣ and p110␣; however, the level of p120 ctn in complex with p85␣ was much higher (Fig. 8A, right panel). Small amounts of cadherin-catenin proteins were found associated with p110␤, which is also expressed in keratinocytes, but these associations were not regulated during differentiation (data not shown).
To assess whether the increased association of p85␣ with E-cadherincatenin complexes depends on tyrosine phosphorylation in differentiating cells, we analyzed the levels of E-cadherin and ␥-catenin in complex with p85␣ in differentiating keratinocytes exposed to genistein, PP1, and AG1478, which inhibited the differentiation-induced Akt engagement (see Fig. 7E). Consistently, treatment with these tyrosine kinase inhibitors reduced significantly the association of E-cadherin and ␥-catenin with p85␣ (Fig. 8B).
Increased Tyrosine Phosphorylation of YXXM PI3K Binding Motifs on Catenin and EGFR Proteins during Keratinocyte Differentiation-Because E-cadherin-associated ␤and ␥-catenins and p120 ctn become increasingly tyrosine-phosphorylated during keratinocyte differentiation (19,24), we reasoned that these phosphorylation events might generate docking sites for the SH2 domains of the p85 subunit and thus couple cadherins and tyrosine kinases in the activation of PI3K. Amino acid sequence analysis of catenins with the Scansite computer software (31) revealed one putative YXXM PI3K binding site on both ␥-catenin and p120 ctn , conserved among rodents and humans but not on the closely related ␤-catenin (Fig. 9A, inset). Moreover, Western analysis of keratinocyte extracts with an antibody detecting phosphorylated YXXM motifs (32) identified four bands induced early on in differentiation, three of which had molecular masses compatible with those of ␥-catenin (85 kDa) and the two main splicing isoforms of p120 ctn (100 and 120 kDa) (Fig. 9A). Therefore, we tried to establish whether these proteins could be detected by the PI3K docking motif antibody after immunoprecipitation from proliferating and differentiating cells, in parallel with the analysis of their overall tyrosine phosphorylation levels. As described previously (19), the tyrosine phosphorylation of ␤and ␥-catenin in complex with E-cadherin was found increased early on during differentiation (Fig. 9B, left panels). Consistent with the presence of YXXM motifs, ␥-catenin, but not ␤-catenin, was also increasingly detected by the PI3K docking motif antibody, either in complex with Infected cells were kept for 24 h in low calcium medium and exposed to low or high calcium for an additional 24 h. Cells were pulse-labeled with BrdUrd for the last 3 h prior to the termination of the experiment, and the percentage of BrdUrd-positive nuclei was determined in a minimum of six representative fields (Ͼ100 cells/field). Similar results were obtained in two other independent experiments. B, mouse keratinocytes were transduced with either AdAkt-Myr or AdGFP control viruses for 48 h (multiplicity of infection ϭ 20) and kept under growing (Ϫ) or differentiating conditions (Ca ϩϩ ) for the indicated times. Cell lysates were blotted with antibodies to filaggrin, loricrin, K1 , K5, and total Akt. Filaggrin is synthesized as a high molecular weight precursor, profilaggrin, which is subsequently processed, and the diffuse bands correspond to the multiple products of this process. K5 was used as a loading control.   Keratinocytes treated with DECMA-1 or CH-19 control antibodies were analyzed for the amounts of active (p-Akt) or total Akt proteins under proliferating conditions (Ϫ) and after differentiation induction (Ca ϩϩ ) or exposure to epidermal growth factor (25 nM) for the indicated times. C, analysis of keratinocyte differentiation as a function of E-cadherin inhibition. Cells treated with either DECMA-1 or CH-19 antibodies were kept under growing condition (Ϫ) or induced to differentiate for 6 h. Total cell extracts were analyzed by immunoblotting with antibodies to filaggrin, loricrin, K1, and K5, active (p-Akt), and total Akt, as indicated. D, keratinocytes exposed to increased extracellular calcium for 6 h in the presence of CH-19 or DECMA-1 antibodies (as indicated) were double stained for p120 ctn (green) and active caspase-3 (red). Note that low amounts of active caspase-3 can be visualized in the nuclei of a few control cells, and a pronounced increase of activated caspase is detected in keratinocytes treated with the DECMA-1 antibody. Arrowheads indicate cells with apoptotic features. Bar, 33 m. E, cell extracts obtained from keratinocytes pretreated with Me 2 SO (DMSO) vehicle, genistein (100 M), or the indicated kinase inhibitors (5 M) for 1 h and kept under growing (Ϫ) or differentiating conditions for another hour were blotted with antibodies to active (p-Akt) and total Akt proteins. SEPTEMBER 23, 2005 • VOLUME 280 • NUMBER 38 E-cadherin or after direct immunoprecipitation (Fig. 9B, right panels). Because the association between p85␣ and cadherin-catenins was suppressed by tyrosine kinase inhibition (Fig. 8B), we asked whether the ␥-catenin reactivity for the PI3K docking motif antibody was also blocked under the same conditions. In fact, although the reactivity of ␥-catenin for this antibody increased in differentiating control cells, this event was suppressed by a concomitant tyrosine kinase inhibition, as revealed by the analysis of E-cadherin immune complexes obtained from the same cell extracts used for the experiment shown in Fig. 8B  (Fig. 9C). Immunoprecipitation of p120 ctn followed by anti-phosphotyrosine immunoblotting revealed a robust increase in its tyrosine phosphorylation during differentiation (Fig. 9D, left panel); p120 ctn isoforms FIGURE 8. Association of PI3K and E-cadherin-catenin protein complexes during keratinocyte differentiation. A, keratinocyte extracts from proliferating cells (Ϫ) and cells stimulated to differentiate with calcium for 1 h were immunoprecipitated with antibodies against the p110␣ or the p85␣ subunits of PI3K or with non-immune IgG. Immune complexes were analyzed by immunoblotting with either a mixture of antibodies against E-cadherin, ␤and ␥-catenin (left panel) or an antibody against p120 ctn (p120, right panel). B, tyrosine kinase inhibition interferes with the association of p85-PI3K with E-cadherin (E-cad.) and ␥-catenin (␥-cat.). Keratinocyte extracts from proliferating cells (Ϫ) and cells stimulated with calcium for 1 h in the presence of Me 2 SO (DMSO) or the indicated tyrosine kinase inhibitors were immunoprecipitated (IP) with antibodies to p85␣ or non-immune IgG and analyzed by immunoblotting with antibodies to E-cadherin, ␥-catenin, and p85␣, as indicated. FIGURE 9. Increased protein phosphorylation on YXXM motifs in keratinocyte differentiation. A, total extracts from keratinocytes under growing conditions (Ϫ) or induced to differentiate for the indicated times were analyzed by immunoblotting with antibodies to the phosphorylated YXXM p85-PI3K binding motif (p-p85 PI3Kbm). The inset at the bottom shows the putative YXXM motifs on ␥-catenin, p120 ctn , and EGFR proteins, as identified by the Scansite computer software (31). B, lysates from proliferating cells (Ϫ) or cells stimulated with calcium for 1 h were immunoprecipitated (IP) with antibodies for E-cadherin (left panels) or ␥-catenin (right panels). Immune complexes were blotted with anti-phosphotyrosine (p-Tyr), p-p85-PI3Kbm, and ␤and ␥-catenin antibodies, as indicated. C, E-cadherin immune complexes from keratinocytes treated with Me 2 SO (DMSO) or the indicated tyrosine kinase inhibitors as in Fig. 8B were blotted with the p-p85-PI3Kbm and ␥-catenin (␥-cat.) antibodies. Note the loss of reactivity of ␥-catenin for the p-p85-PI3Kbm antibody in the presence of tyrosine kinase inhibition. D, the E-cadherin immunoprecipitates as in B were analyzed along with p120 ctn immunoprecipitates from the same cell extracts by immunoblotting with anti-phosphotyrosine antibodies, p-p85-PI3Kbm antibodies, and antibodies to p120 ctn (as indicated). E, lysates from proliferating (Ϫ) and differentiating cells (1h) treated with Me 2 SO or the EGFR inhibitor AG1478 were immunoprecipitated with EGFR antibodies. Immune complexes were analyzed by immunoblotting with p-p85-PI3Kbm and EGFR antibodies (upper and lower panels, respectively). F, frozen section of a 3-day-old mouse epidermis analyzed by double immunofluorescence staining with p-p85-PI3K-bm (red) and fluorescein isothiocyanate-conjugated K1 antibodies (green). Arrowheads indicate the stratum corneum; the dotted line designates the basement membrane. Bar, 10 m.

PI3K/Akt in Keratinocyte Differentiation Versus Death
were also detected by the PI3K docking motif antibody, although with a lower reactivity compared with ␥-catenin (Fig. 9D, middle panel). A distinct protein associated with p120 ctn and co-migrating with ␥-catenin was also recognized by the antibody.
The 180-kDa protein visualized by the PI3K docking motif antibody in total keratinocyte extracts (Fig. 9A) was likely the EGFR, the cytoplasmic domain of which contains one YXXM motif at the Tyr 920 (33). Consistent with this possibility, the EGFR was also increasingly detected by this antibody after direct immunoprecipitation from differentiating cells, reactivity that was abolished by exposure of cells to the AG1478 inhibitor (Fig. 9E). Notably, the PI3K docking motif antibody increasingly visualized cell-cell contacts of suprabasal differentiating cells in the intact mouse skin (Fig. 9F), with a pattern that was reminiscent of that described for the p85␣ subunit of PI3K (see Fig. 3A). These findings suggest that phosphorylation of proteins at YXXM motifs is likely to occur at the level of keratinocyte cell-cell adhesive structures in vivo.

DISCUSSION
In this work we have explored the question of what determines the choice of the epidermal keratinocyte between differentiation and death and found that the PI3K/Akt signaling is a key determinant. In cultured keratinocytes, the kinase activities of PI3K and Akt are induced early on after the switch between cell proliferation and differentiation and stay sustained for several hours, declining prior to the onset of the latest stages of differentiation. Based on the membrane localization of the p85␣ PI3K subunit and the distribution of active Akt, a similar activation of this pathway is likely to occur in suprabasal keratinocytes in vivo, irrespective of age or stage of the hair cycle. Our study reveals a critical role for endogenous PI3K signaling in protecting differentiating keratinocytes from premature death because inhibition of this pathway caused caspase-3 activation and apoptosis selectively in the differentiating, but not proliferating, keratinocytes. Other key cell regulatory molecules, specifically the product of the c-myc proto-oncogene, the p21 CDK inhibitor, and members of the protein kinase C and apoptosis signal-regulating kinase families of protein kinases, stimulate the differentiation process of keratinocytes while promoting apoptosis in different cell types or even epidermal cells in different contexts (4). Thus, it is tempting to speculate that PI3K signaling normally counterbalances the potential death-inducing activities of these or other pathways, allowing differentiation to proceed. The role of the PI3K pathway in this context is further indicated by the increased phosphorylation of Akt target molecules, such as FoxO-1 and -4, MDM2, and GSK3␣ and -␤, and their functions in cell survival (14).
Besides protecting differentiating keratinocytes from apoptosis, we have found that activation of this pathway functions as a trigger of the differentiation process, consistent with previous findings that Akt1-and -2 double knock-out mice have impaired epidermal differentiation (15). In particular, the keratinocyte growth arrest induced by active Akt expression was somewhat unexpected, considering the implications of the PI3K/Akt pathway in cell proliferation in other contexts (34). However, similar growth inhibitory effects of Akt were described previously in Caco-2 cells (35), human endothelial cells (36), and even human keratinocytes (37), and these events were often associated with cell differentiation. Akt-dependent inhibitory mechanisms on cell proliferation include: (i) a block of the Ras-mitogen-activated protein kinase mitogenic pathway at the level of Raf, (ii) the induction of a p53-p21 dependent growth arrest (36), and (iii) the engagement of the NF-B signaling via phosphorylation of I␣ (38), which in keratinocytes could account for both growth arrest and stimulation of cell survival (39,40).
Another important question relates to the mechanisms upstream of PI3K activation in differentiating keratinocytes. Both tyrosine kinases and cadherin adhesion molecules have been implicated previously in the activation of PI3K signaling (7, 11). Our findings indicate that tyrosine kinases and E-cadherin are likely to work cooperatively in the engagement of the PI3K pathway during keratinocyte differentiation, as this event was abolished either by interference with E-cadherin adhesive functions or by inhibition of the Src-and the EGFR kinases. In particular, the functions of E-cadherin are selectively required during differentiation, as keratinocytes exposed to cadherin-blocking antibodies failed to activate Akt during this process but retained a normal Akt activation in response to epidermal growth factor. This indicates that the mechanisms upstream of PI3K in differentiation are likely distinct from those elicited by mitogenic growth factors. The rapid activation (within minutes) of PI3K/Akt signaling in response to high extracellular calcium suggests that this event does not require the full assembly and/or maturation of adherens junctions, which takes place over the course of hours (19,41), but may be triggered by the earliest engagement of E-cadherin. The subsequent remodeling of adhesive structures may instead be important for maintaining sustained levels of PI3K activity during differentiation. Besides suppressing Akt activation, interference with E-cadherin functions also inhibited the expression of loricrin and filaggrin and promoted caspase-3 activation, which suggests that the PI3K signaling downstream of E-cadherin may be essential for coupling prosurvival and pro-differentiation cellular mechanisms, and might explain, at least in part, the phenotype of mice with a conditional ablation of E-cadherin in the epidermis (21). Importantly, not only PI3K was found to rely on E-cadherin functions for activation but also its physical association with cadherin-catenin complexes was increased during differentiation in a tyrosine kinase-dependent manner. Class IA PI3K activation generally occurs upon binding to phosphorylated YXXM amino acid motifs (17); we found that tyrosine phosphorylation of ␥-catenin and p120 ctn during differentiation is likely to provide such binding sites and thus a direct link between E-cadherin and tyrosine kinases and PI3K activation. Our results suggest that ␥-catenin may represent a main docking protein for PI3K within cadherin-catenin complexes; this idea is supported by the preferential co-precipitation of ␥-catenin with the p110␣ catalytic subunit and by its prominent reactivity for an antibody recognizing phosphorylated YXXM motifs. Tyrosine kinase inhibition, besides suppressing the activation of the PI3K pathway in differentiation, blocked in parallel the association of PI3K with cadherin-catenin complexes and the reactivity of ␥-catenin to the PI3K docking motif antibody. Taken together, these findings suggest that phosphorylation of the PI3K binding site on ␥-catenin and the recruitment of PI3K to cadherin-catenin complexes are likely related events in PI3K signaling activation. Additional docking sites for PI3K may be provided by p120 ctn , but the weaker reactivity of the molecule for the PI3K binding motif antibody suggests accessory roles in PI3K activation, at least in this context. We cannot formally rule out that either ␤-catenin or other unidentified proteins may also be involved in the recruitment of PI3K to keratinocyte adhesive structures via different mechanisms.
The EGFR protein, the kinase activity of which was found important for PI3K activation during keratinocyte differentiation, may also be involved in the direct recruitment of PI3K. The EGFR also bears a PI3K docking site at the Tyr 920 (33), and a protein with the EGFR molecular weight was detected by the PI3K docking motif antibody both in total keratinocytes extracts and after direct immunoprecipitation of the EGFR from differentiating cells. Because Src kinases can also phosphorylate the Tyr 920 of the EGFR (33), this suggests potential cross-talks between these families of kinases in PI3K activation at this level. The diagram in Fig. 10 summarizes the proposed mechanisms of activation of the PI3K signaling in keratinocyte differentiation and their biological roles. Thus, the PI3K/Akt signaling downstream of E-cadherin and tyrosine kinases is likely to be a critical integrator of signals promoting keratinocyte differentiation versus death.