Mesoderm proliferation defects in E8.5 FAK-/- mouse embryos
knockout results in an early embryonic lethal (E.8.5) phenotype (Ilic et al., 1995
) and size comparisons between normal FAK+/+, FAK+/- and FAK-/- littermates at E8.0-8.5 show that both FAK+/- and FAK-/- embryos are smaller than FAK+/+ embryos (). By E11.5, FAK+/- embryos become indistinguishable from FAK+/+ embryos whereas FAK-/- embryos do not survive beyond E8.5-9.0 (Ilic et al., 1995
). To evaluate relative levels of cell proliferation during development, head-fold regions of FAK+/+ and FAK-/- littermate embryos were immuno-stained with antibodies to phosphorylated serine-10 histone H3 (pHis3) (). Equal numbers of pHis3-stained head-fold ectoderm cells were detected in FAK+/+ and FAK-/- embryos (). In contrast, few pHis3-stained mesoderm cells were detected in FAK-/- compared to FAK+/+ embryos () supporting the notion that a proliferation defect induced by fak
loss may occur in vivo.
Figure 1 FAK-/- embryo mesoderm cell proliferation block is p53-dependent. (A) Hoechst DNA staining of wild-type (FAK+/+), FAK+/-, and FAK embryos at E8.5. (B) Mesoderm region of FAK-/- embryos lack mitotic cells. Saggital head-fold sections of E8.0-8.5 FAK+/+ (more ...)
p53 accumulation prevents FAK-/- embryo proliferation ex vivo
To elucidate the potential role of p53 in FAK-/- embryo growth defects, we evaluated p53 protein levels in E8.5 embryo lysates (). During development, p53 protein is rapidly turned over and not normally detected before E10.5-11.0 (Gottlieb et al., 1997
). High levels of p53 and p53-responsive targets such as Mdm2 were detected in FAK-/- but not FAK+/+ embryos suggestive of p53 activation in the absence of FAK (). To evaluate the contribution of p53 in FAK-/- embryo growth, a p53-/- mutation was introduced onto the FAK-/- background by mating FAK+/- and p53-/- mice. p53 inactivation did not rescue the E8.5 embryonic lethality of FAK-/- embryos (data not shown). However, loss of p53 enabled primary FAK-/- p53-/- embryo cell growth ex vivo whereas FAK-/-p53+/+ cells failed to proliferate (). To quantify differences in cell growth, embryos were dissociated at day 6 ex vivo and cells cultured in the presence of bromodeoxyuridine (BrdU). Anti-BrdU staining revealed that FAK+/+p53+/+ and FAK+/+p53-/- cells showed a similar high rate of proliferation (50-60%) whereas <2% of cells in FAK-/-p53+/+ embryos were BrdU-positive (). Importantly, p53 inactivation released this proliferation block resulting in ~70% BrdU-positive cells from FAK-/-p53-/- embryos. This defect in FAK-/-p53+/+ embryo cell growth was not linked to the E8.0-8.5 time of extraction as cell proliferation defects and DNA aberrations were observed in cultured E7.0-7.5 FAK-/-p53+/+ but not FAK+/+p53+/+ embryos (). Together, these results support the conclusion that p53 is active in FAK-/- embryos and that p53-associated signals block FAK-/- cell proliferation.
p53-associated proliferation block in FAK-/- cells is p21-dependent
To determine whether the absence of FAK results in changes in embryo protein expression, immunoblotting was performed on FAK+/+, FAK+/-, and FAK-/- E8.0-8.5 embryo lysates (). The up-regulation of the FAK-related Pyk2 kinase was detected upon inactivation of one or both fak alleles. Comparisons of FAK+/+ and FAK-/- embryo lysates showed decreases in cyclin B and cyclin E, and increases in cyclin-dependent kinase inhibitors p57(Kip2), p27(Kip1), and p21(Cip1) in the absence of FAK (). These changes may reflect reduced cell proliferation within FAK-/- embryos, however, other cell cycle regulatory proteins such as p16(Ink4a) remained unchanged.
Figure 2 p53-mediated proliferation block of FAK-/- cells is p21-dependent. (A) Increased expression of p53, Pyk2, or cyclin-dependent kinase inhibitors and decreased expression of cyclins in FAK-/- compared to FAK+/+ littermates as determined by immunoblotting (more ...)
In cell culture, p21 expression is enhanced by disruption of ECM-integrin linkages (Bao et al., 2002
) and inhibited by FAK signaling (Bryant et al., 2006
). During embryogenesis, elevated p21 levels in the absence of FAK were dependent upon p53, as p21 expression was not detected in FAK+/+, FAK+/-, or FAK-/- embryos on a p53-/- background (). To test whether elevated p21 levels contribute to the p53-dependent block in FAK-/- embryo cell proliferation, a p21-/- mutation was introduced into a FAK-/-p53+/+ background by mating FAK+/- and p21-/- mice. FAK-/-p21-/- mice exhibited embryonic lethality at ~E9.5 with increased mesoderm DNA fragmentation compared to FAK+/+p21-/- littermates (). However, cells from E8.0-8.5 FAK-/-p21-/- embryos proliferated ex vivo at ~75% the level of FAK+/+p21-/- cells (). This release of the p53-dependent cell proliferation block was verified by BrdU incorporation into FAK-/-p21-/-p53+/+ cells (~41%) but not FAK-/-p21+/+p53+/+ cells (<2%) (). These studies support the notion that fak
inactivation triggers a p53-p21-dependent cell proliferation block, yet release of this checkpoint is not sufficient to overcome an embryonic lethal FAK-null phenotype.
FAK promotes p53 turnover via FAK FERM domain-enhanced p53 ubiquitination
The inability of FAK-/-p53+/+ fibroblasts to grow in culture severely limits experimental analyses. To study the signaling linkage between FAK and p53, we generated FAK-/-p21-/- and FAK+/+p21-/- fibroblasts from E8.5 embryo littermates. In early passage FAK-/-p21-/-cells, steady state p53 levels were elevated and transient FAK re-expression resulted in decreased p53 levels (Supplemental Fig. S1A
). This effect of FAK on p53 levels was associated with enhanced p53 turnover as determined by metabolic labeling of cells (Supplemental Fig. S1B-F
To address which domains of FAK were required to promote p53 turnover, wild type (WT) FAK or various FAK mutants () were re-expressed via adenovirus (Ad) in low passage FAK-/-p21-/- fibroblasts and steady-state p53 levels were determined after 48 h by anti-p53 blotting (). Surprisingly, WT FAK and various FAK signaling mutants functioned equally to reduce p53 levels by 60 to 90% compared to Mock-transduced cells (). Over-expression of the FAK C-terminal domain (termed FAK-related non-kinase, FRNK) slightly elevated p53 levels whereas FAK residues 1-402 encompassing the FERM domain reduced p53 levels >80% (). As N-terminal truncated and activated FAK (Δ1-100) did not affect p53 levels and kinase-dead FAK decreased p53 levels (), our results support the notion that the FAK FERM domain plays a unique role in promoting p53 turnover in a kinase-independent manner.
Figure 3 FAK FERM inhibits p53 activity through enhanced Mdm2-dependent ubiquitination and proteasomal degradation. (A) Epitope-tagged FAK construct schematic. Indicated is the band 4.1, ezrin, radixin, moesin (FERM) and central kinase domains. Filled regions (more ...)
p53 levels in cells are regulated by a balance between protein expression and degradation. Addition of MG132 proteasome inhibitor for 3 h prior to cell lysis blocked FAK and FAK FERM effects on p53 () supporting the conclusion that FAK FERM facilitates p53 turnover. To determine whether changes in p53 expression affect p53 transcriptional activity, a 2.4 kb region of the p21 promoter coupled to luciferase was used as a p53 activity reporter in FAK-/-p21-/- cells (). High p53 activity was detected in growing cells and this was inhibited >3-fold by WT FAK and FAK FERM but not FRNK expression. MG132 addition prevented FAK and FAK FERM inhibition of p21 promoter activity (data not shown) and accordingly, full-length FAK, FAK FERM but not FRNK increased p53 ubiquitination (). To elucidate the ubiquitin E3 ligase for FAK FERM-mediated p53 modification, flag-p53 was transfected in Mdm2-/-p53-/- and Mdm2+/+p53-/- fibroblasts. Elevated levels of ubiquitinated p53 and p53 band shifts were detected upon Ad-FAK FERM over-expression only in Mdm2+/+ cells (). These results show that FAK FERM promotes p53 turnover through enhanced Mdm2-associated p53 ubiquitination and degradation.
FAK FERM F2 lobe facilitates FAK nuclear localization
As activated p53 is primarily nuclear-localized and exogenous FAK FERM is nuclear-enriched (Golubovskaya et al., 2005
; Stewart et al., 2002
), analyses were undertaken to determine the determinants of FAK FERM nuclear targeting. Fusions of FAK residues (1-402) to green fluorescent protein (GFP) exhibit strong nuclear localization independent of Y397 phosphorylation (Supplemental Fig. S2A
). The classic nuclear localization sequence typically contains clusters of basic amino acids. The avian FAK FERM domain is a three lobed (F1-F3) structure (Lietha et al., 2007
). Primary sequence alignments of the murine FAK FERM F2 lobe region showed that FAK and Pyk2 contained patches of basic amino acids that are not conserved in other FERM-containing proteins such as ezrin, radixin, moesin, or merlin (). Although these FAK F2 FERM domain basic residues are separated by primary sequence, they comprise surface-exposed clusters (). The largest patch consists of residues lysine (K) 190, K191, K216, K218, arginine (R) R221, and K222 whereas a second smaller basic patch is comprised of R204, R205, and K209. Residues R177 and R178 are partially exposed within the FAK FERM F2 lobe structure ().
Figure 4 Determinants of FAK nuclear localization. (A) Structure-based alignment of FAK FERM F2 lobe residues (Lietha et al., 2007) with other FERM-containing proteins. Conserved basic residues within FAK and Pyk2 are highlighted in blue, total conserved FERM (more ...)
Alanine substitutions were introduced within the FAK FERM F2 lobe and the effects on FAK FERM nuclear localization were monitored by fluorescence microscopy (data not shown) and cellular fractionation into nuclear (N) or cytosolic (C) extracts (). Mutations in the largest basic patch (K190/191A or K216/218A) blocked whereas mutations in a second smaller patch (K204/R205A) slightly reduced FERM nuclear accumulation. R177/R178A mutations also prevented FERM nuclear localization whereas mutation of surface exposed basic residues in the FAK FERM F3 lobe (R312/K313A) or the SUMOylation site (K152R) did not affect FERM nuclear targeting (). To prove the role of the FERM domain promoting FAK nuclear localization, GFP-FAK WT and GFP-FAK R177/R178A were expressed in FAK-/- fibroblasts or human umbilical endothelial cells (HUVECs) (data not shown) and GFP localization monitored by time-lapse imaging in the presence or absence of the nuclear export inhibitor leptomycin B (). In vehicle-treated cells, both FAK constructs were detected in focal contacts and the cytoplasm and leptomycin B addition promoted WT but not FAK R177/R178A nuclear accumulation within 4 h.
To analyze whether endogenous FAK is present in the nucleus, nuclear (N) or cytosolic (C) HUVEC extracts were analyzed for FAK distribution (). Approximately 5-10% of HUVEC FAK is nuclear-associated and tyrosine phosphorylated. To determine whether cellular stress may enhance FAK nuclear accumulation, HUVECs were treated with the protein kinase inhibitor staurosporine (). Within 30 min, staurosporine enhanced endogenous FAK, GFP-FAK, but not FERM F2 lobe mutated GFP-FAK R177/178A nuclear accumulation. This was accompanied by FAK dephosphorylation, loss of FAK from focal contacts, and preceded staurosporine-induced HUVEC apoptosis occurring within 4-8 h (Supplemental Fig. S2B-F
). Staurosporine caused a rapid redistribution of GFP-FAK R177/178A from focal contacts to punctate cytoplasmic clusters but not to the nucleus (Supplemental Fig. S2E
). Loss of integrin-mediated cell adhesion is another cellular stressor, and this was also found to increase FAK nuclear accumulation within 30 min (). Together, these results support the importance of the FAK FERM F2 lobe in nuclear targeting. Moreover, the distribution of FAK at focal contacts, in the cytoplasm, or the nucleus is affected by cellular stress.
FAK nuclear localization is important for p53 inhibition
Primary FAK-/-p21-/- fibroblasts are a unique model system to determine the role of FAK in p53 regulation. Increased expression of the FAK-related Pyk2 kinase was detected FAK-/-p21-/- fibroblasts and lentiviral-mediated Pyk2 shRNA was used to maintain low levels of Pyk2 for FAK re-expression studies (data not shown). To address whether FAK FERM nuclear localization is important for p53 regulation, stable pooled populations of GFP-FAK FERM (1-402) or GFP-FRNK-expressing FAK-/-p21-/- fibroblasts were established. WT, K152R SUMOylation mutant, and R312/K313A FAK FERM F3 lobe mutated proteins were nuclear-localized, F2 lobe FERM mutants (R177/R178A and K190/K191A) were cytoplasmic-distributed, and GFP-FRNK localized to focal contacts ( and Supplemental Table 2
). Steady state p53 levels in FAK FERM-expressing cells were evaluated by immunoblotting and compared to parental FAK-/-p21-/- and GFP-expressing controls. Cells expressing WT FERM and the SUMOylation (K152R) FERM mutant exhibited reduced p53 levels (). Importantly, mutations in the FERM F2 lobe (K190/K191A and R177/R178A) blocked FAK FERM effects on lowering p53 levels (). However, mutations in the FAK FERM F3 lobe (R312/K313A) also blocked the ability of FAK FERM to reduce p53 levels () even though R312/K313A FAK FERM was nuclear-localized (). As p53 transcriptional activity paralleled p53 expression levels in FAK FERM reconstituted FAK-/-p21-/- fibroblasts (data not shown), the combined results support the notion that FAK FERM nuclear localization is required but not sufficient for p53 regulation.
Figure 5 Separate FAK FERM lobes mediate p53 binding, nuclear localization, and Mdm2 association. (A) The indicated GFP-FAK FERM (1-402) constructs or GFP-FRNK were stably-expressed in FAK-/-p21-/- (Pyk2 shRNA) fibroblasts and intracellular distribution visualized (more ...)
To determine whether full-length FAK modulates p53 activity in a similar manner as the FAK FERM, co-transfection assays were performed in FAK-/-p53-/- fibroblasts (). The p21 promoter-luciferase reporter was responsive to p53 re-expression resulting in ~14-fold increased activity. Co-expression of WT or SUMOylation mutant K152R FAK resulted in >65% inhibition of p53 activity whereas equal expression of F2 lobe mutated R177/R178A FAK lowered p53 activity only 5-10% (). F2 lobe mutated K190/K191A or F3 lobe mutated R312/K313A FAK only weakly inhibited p53 activity. Taken together, these results support the conclusion that FAK FERM F2 lobe-mediated nuclear translocation is important for p53 inhibition. However, the fact that FERM F3 lobe mutations (R312/K313A) did not affect nuclear translocation, yet disrupted the ability of FAK to inhibit p53, indicates that FAK-mediated regulation of p53 involves multiple FERM-associated mechanisms.
FAK FERM acts as a scaffold to facilitate p53 and Mdm2 association
Co-immunoprecipitation (co-IP) analyses have shown that FAK and p53 can form a complex (Golubovskaya et al., 2005
), but the binding determinants for p53 within the FAK FERM domain remain unknown. GFP-FAK FERM or GFP-FRNK constructs were expressed in human A549 cells and evaluated for endogenous p53 association by co-IP analyses (). p53 associated with FAK FERM but not FRNK and p53 binding to FAK FERM was weakened by mutations in the F2 (R177/R178A) but not F3 (R312/K313A) lobes (). Similar results were obtained by GST-p53 pull down assays (Supplemental Fig. S3A
). Interestingly, F2 lobe mutated K190/K191A FAK FERM can form a complex with p53 (), but these FERM mutations disrupt nuclear localization () and p53 regulation (). Additionally, F3 lobe mutated R312/K313A FAK FERM localizes to the nucleus (), binds to p53 (), but does not promote p53 turnover (). Together, these results support the notion that FERM-mediated nuclear localization, p53 binding, and FERM-enhanced p53 turnover are separable events.
To determine whether individual FAK FERM lobes can associate with p53 or Mdm2 in cells, the F1, F2, or F3 regions were transiently-expressed as GST fusion proteins in 293T cells (). The FAK FERM F1 lobe strongly associated with p53 in vivo whereas weak binding was detected with FAK FERM F2 lobe, and no p53 binding was detected with the FAK FERM F3 lobe (). This result supports the notion that the FAK FERM p53 binding site likely spans the F1 and F2 FERM lobe regions. For Mdm2, FAK FERM F3 lobe bound the strongest with only weak interactions detected with F1 or F2 FAK FERM lobes (). Interestingly, F3 lobe mutated R311/K312A FERM bound Mdm2 and surprisingly, F2 lobe mutated R177/R178A FERM exhibited enhanced Mdm2 binding (Supplemental Fig. S3B
). Co-transfection studies revealed that both F2 and F3 lobe FERM mutations disrupted FAK FERM-enhanced p53 ubiquitination () and support the notion that FAK-enhanced p53 ubiquitination may be part of a nuclear-localized complex. Accordingly, although F2 lobe mutated K190/K191 FERM can bind p53, this mutant is cytoplasmic-distributed and fails to promote p53 ubiquitination. For F3 lobe mutated R312/K313A FERM, it is likely that this mutation disrupts the binding of another protein other than Mdm2 that is needed to facilitate p53 ubiquitination.
To support the notion that FAK may act as a scaffold to enhance the formation of a p53-Mdm2 complex, FAK-/-p53-/- fibroblasts were transfected with flag-p53, HA-Mdm2 and increasing amounts of recombinant GST or GST-FAK added to cell lysates. A p53-Mdm2 association was detected in the absence of FAK and this was not affected by purified GST addition (). GST-FAK addition increased the amount of p53-bound Mdm2 through the formation of a p53-Mdm2-FAK complex in a dose-dependent manner. However, excess GST-FAK did not enhance p53-Mdm2 complex formation even though increased GST-FAK bound to p53 (). This result is consistent with a biphasic scaffolding effect of FAK in facilitating p53 and Mdm2 interactions. Together with the FERM mutational results, these studies support the conclusion that nuclear-localized FAK functions as a FERM-associated scaffold for p53 and Mdm2 binding that facilitates p53 ubiquitination leading to p53 turnover.
FAK regulation of cell proliferation and p53-dependent apoptosis in human fibroblasts
As our studies support the importance of FAK in regulating p53 activation during development and in mouse fibroblasts, lentiviral-mediated FAK shRNA knockdown was used to test whether a FAK-p53 signaling axis exists in human fibroblasts and HUVECs. Within 72 h, >80% of cells showed strong lentiviral GFP expression and neither anti-FAK or scrambled (Scr) shRNA expression promoted cell apoptosis (Supplemental Fig. S4
). However, >75% reduction in FAK expression was associated with ~2-fold increased p53-p21 protein levels and reduced cell proliferation (, and Supplemental Fig. S5
). To determine whether decreased FAK and elevated p53 levels would sensitize cells to DNA damaging agents, Scr and FAK shRNA-expressing fibroblasts were treated with cisplatin. FAK shRNA cells showed ~4-fold increased apoptosis compared to cisplatin-treated Scr shRNA cells () and the combination FAK shRNA plus cisplatin lead to elevated p53 protein levels compared to cisplatin-treated Scr shRNA fibroblasts ().
Figure 6 FAK controls human diploid fibroblast proliferation and p53-dependent apoptosis. (A) Lysates from scrambled (Scr) and FAK shRNA infected cells after 72 h were analyzed by anti-FAK, p53, p21, actin, and GFP blotting. (B) FAK shRNA inhibits human fibroblast (more ...)
To verify that increased cisplatin-stimulated apoptosis was due to decreased FAK, human fibroblasts were treated with FAK shRNA and after 48 h, cells were transduced with murine Ad-FAK WT or Ad-FAK Δ1-100 containing a deletion in the FAK FERM domain (). This truncation of FAK activates FAK kinase activity and facilitates FAK signaling (Mitra and Schlaepfer, 2006
), but FAK Δ1-100 does not function to regulate p53 levels in mouse fibroblasts (). WT FAK re-expression blocked cisplatin-stimulated apoptosis in FAK shRNA cells whereas Δ1-100 FAK had no effect (). Importantly, Δ1-100 FAK exhibited high activity levels as determined by anti-phosphotyrosine blotting of fibroblast lysates and led to the robust activation of survival-associated signaling pathways such as Akt (). However, only WT FAK reduced p53 levels and prevented p21 expression in cisplatin-treated cells (). Importantly, cisplatin-stimulated apoptosis in FAK shRNA-expressing fibroblasts was dependent upon p53 as siRNA-mediated p53 reduction blocked both increased p21 expression and cell death ().
To prove the importance of the FAK FERM domain function in preventing cisplatin-stimulated apoptosis, cell survival analyses were performed after Ad-FERM WT or Ad-FERM R177/R178A expression in FAK shRNA knockdown human fibroblasts (). FAK FERM but not R177/178A FERM blocked p53 accumulation and only WT FAK FERM functioned to prevent apoptosis. Theses results are strong support for the biological importance of the FAK FERM domain in the regulation of p53-dependent cell survival.