FAK/PYK2 promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3β
Aberrant activation of the canonical Wnt ( Wnt/β-catenin ) pathway is crucial for many cancer types , especially those of the gastrointestinal tract: genetic alterations in Wnt signaling occur in 93% of human CRCs , among which inactivation of tumor suppressor adenomatous polyposis coli ( APC ) ( 85% ) and activating mutations of proto-oncogene β-catenin ( 5% ) ( Cancer Genome Atlas Network , 2012 ) are mutually exclusive ( Sparks et al . , 1998 ) . The loss of functional APC results in less efficient GSK3 ( two isoforms in mammals , GSK3α and GSK3β ) -mediated phosphorylation of β-catenin , leading to reduced degradation of β-catenin , thus mimicking Wnt stimulation . Although APC mutation is believed to activate Wnt signaling constitutively , histopathological studies show that only about 50% colon carcinoma cells display homogeneous nuclear β-catenin staining ( Chung et al . , 2001 ) , a surrogate for Wnt signaling activity , indicating that APC mutation alone is not sufficient to cause persistent or full activation of the Wnt pathway in CRC cells , therefore Wnt signaling is regulatable in APC-mutated CRCs . But how Wnt signaling is regulated in intestinal tumorigenesis remains largely undefined . A key feature of the Wnt/β-catenin pathway is the tight control of the abundance of transcription co-activator β-catenin by the β-catenin destruction complex , consisting of GSK3 , casein kinase 1 ( CK1 ) , APC , scaffold protein Axin and β-catenin . In Wnt-off state , the cytosolic level of β-catenin is kept low by its continuous degradation: β-catenin is phosphorylated by GSK3 on serine 33 and 37 and threonine 41 , phosphorylation then triggers β-catenin recruitment of ubiquitin E3 β-TrCP ( β-transducin repeats-containing proteins ) , causing its ubiquitination and proteasomal degradation ( Wu and Pan , 2010 ) . In Wnt-on state , Wnt ligand forms a complex with the cell-surface receptor Frizzled and low-density lipoprotein receptor-related protein ( LRP ) 5/6 ( Wu and Pan , 2010 ) , and initiates a series of molecular events ultimately causing stabilization of β-catenin by suppressing phosphorylation of β-catenin ( Hernandez et al . , 2012; Kim et al . , 2013 ) as well as β-TrCP-mediated ubiquitination and proteasomal degradation of β-catenin ( Li et al . , 2012 ) . Newly synthesized β-catenin then accumulates and enters the nucleus to interact with transcription factors TCF ( T-cell factor ) /LEF ( lymphoid enhancing factor ) to activate transcription of the Wnt target genes ( Li et al . , 2012 ) . How GSK3 is modulated and fine-tuned by Wnt to achieve β-catenin stabilization remains a fundamental question in the field of Wnt signaling , with many puzzles and contradictions . We previously found that Wnt simulation induces monoubiquitination of GSK3β by β-TrCP , which is required for Wnt-induced inhibition of β-catenin recruitment of β-TrCP and subsequent stabilization of β-catenin ( Gao et al . , 2014 ) . This study addressed important questions left unanswered: What are the molecular determinants of GSK3β recruitment of β-TrCP ? β-TrCP is the substrate recruitment module of the SCFβ-TrCP ubiquitin ligase supercomplex , recruitment of β-TrCP to its substrate , for example β-catenin , generally requires substrate phosphorylation ( Cardozo and Pagano , 2004 ) . If this is also true in GSK3β's case , what is the involved phosphorylation site ( s ) ? What are the kinases responsible for catalyzing the phosphorylation ? Is this phosphorylation involved in intestinal tumorigenesis ? Our results indicate that phosphorylation of GSK3βY216 by FAK and PYK2 is a molecular determinant of GSK3β recruitment of β-TrCP , and is required for the activation of the Wnt/β-catenin pathway and crucial for Wnt-driven intestinal tumorigenesis .
β-TrCP binds to its substrates through phosphorylated residues in conserved degradation motifs ( DpSGxxpS ) ( Cardozo and Pagano , 2004 ) , but tyrosine phosphorylation of substrate is also a recruiter for β-TrCP ( Sahasrabuddhe et al . , 2015 ) . GSK3β is known to be phosphorylated at serine 9 , threonine 43 , tyrosine 216 and threonine 390 ( Gao et al . , 2014 ) . To determine which of these sites is involved in GSK3β recruitment of β-TrCP , we generated single point mutations at these residues and compared the interactions between the mutants and β-TrCP . We found that mutation of S9 to alanine ( A ) moderately inhibited GSK3β/β-TrCP interaction , whereas mutation of T43 and T390 to alanine had no impact on GSK3β affinity for β-TrCP ( Figure 1A ) . In contrast , mutation of Y216 to phenylalanine ( F ) almost completely abrogated GSK3β binding to β-TrCP . Mimicking GSK3βY216 phosphorylation by mutation Y216 to glutamic acid ( E ) restored the interaction , suggesting that it was phosphorylation that determines the recruitment of β-TrCP to GSK3β . 10 . 7554/eLife . 10072 . 003Figure 1 . β-TrCP-mediated GSK3β ubiquitination requires GSK3βY216 phosphorylation . ( A ) The whole cells lysates from HEK293T cells transfected with empty vector or constructs expressing HA-tagged GSK3β or its phosphorylation mutants were used for immunoprecipitation ( IP ) and immunoblotting ( IB ) analysis with indicated antibodies . ( B ) Ubiquitination of HA-GSK3β was evaluated by IP with anti-FLAG using whole cell lysates derived from HEK293T cells transfected with indicated constructs followed by IB analysis with anti-HA . ( C ) In vitro synthesized wild type ( WT ) and mutant HA-GSK3β proteins were immunoprecipitated with anti-HA affinity gel . Left panel: in vitro ubiquitination assay was performed by incubating immunoprecipitated HA-GSK3β with E1 , E2 , Ub and SCFβ-TrCP1 in the presence of ATP at 37°C for 2 hr . The reaction mixtures were analyzed by IB . Right panel: the immunoprecipitated HA-GSK3β was incubated with ATP at 37°C for 2 hr before subjecting to IB using antibody recognizing phosphorylated GSK3βY216 . ( D ) HEK293T cells were treated with Wnt3a-conditioned medium ( CM ) for the indicated time before harvesting for IB analysis . ( E ) HEK293T cells were transfected with indicated plasmids and treated with control-CM or Wnt3a-CM for 6 hr . Ubiquitination of HA-GSK3β WT and Y216F mutant protein were evaluated by IP with anti-FLAG followed by IB with anti-HA . ( F ) HEK293T cells were transfected with constructs expressing HA-GSK3β or its mutant . 24 hr post-transfection , the cells were harvested for IP with anti-HA followed by IB with anti-Axin1 . ( G ) Upper panel: HA-tagged GSK3β or its mutants were ectopically expressed in HEK293T cells and immunoprecipitated with anti-HA . The precipitated proteins were incubated with purified CK1 and β-catenin in the presence of ATP at 37°C for 1 hr . Kinase activity was evaluated by IB with antibody recognizing phosphorylation of β-cateninS33/S37/T41 catalyzed by GSK3β . Lower panel: the whole cell lysates from WT MEFs , GSK3β knockout ( KO ) and GSK3α knockdown ( KD ) MEFs reconstituted with empty vector or indicated HA-GSK3β mutants were used for IB . Antibody recognizing both GSK3α and GSK3β was used for detecting endogenous GSK3 and HA-tagged GSK3β . This antibody recognized HA-GSK3βY216F but not HA-GSK3βY216E mutant protein . DOI: http://dx . doi . org/10 . 7554/eLife . 10072 . 003 Consistent with β-TrCP's role in GSK3β monoubiquitination ( Gao et al . , 2014 ) , mutation of GSK3β Y216 to phenylalanine substantially reduced GSK3β ubiquitination in HEK293T cells ( Figure 1B ) and in an in vitro reconstituted system ( Figure 1C , left panel ) . In the presence of SCFβ-TrCP1 ubiquitin E3 ligase complex , WT GSK3β and its phosphorylation mimicking mutant Y216E , but not Y216F mutant , were ubiquitinated . Of note , in vitro synthesized GSK3β through Rabbit Reticulocyte Lysate Translation Systems were weakly phosphorylated at Y216 and incubation with ATP further enhances GSK3βY216 phosphorylation through GSK3β autophosphorylation ( Cole et al . , 2004 ) ( Figure 1C , right panel ) , which explains why in vitro ubiquitination of WT GSK3β was possible if GSK3β monoubiquitination requires Y216 phosphorylation . Although Wnt3a treatment did not alter p-GSK3βY216 level in HEK293T cell ( Figure 1D ) ( GSK3αY279 corresponds to GSK3βY216 , the antibody recognizing p-GSK3βY216 also recognizes p-GSK3αY279 ) , the loss of GSK3βY216 phosphorylation greatly impaired both basal and Wnt3a-induced GSK3β monoubiquitination ( Figure 1E ) . Prior studies indicate that phosphorylation of GSK3βY216 may or may not affect GSK3β kinase activity and GSK3β/Axin interaction ( Hughes et al . , 1993; Zhang et al . , 2001; Dajani et al . , 2003; Buescher and Phiel , 2010 ) . The current study showed that GSK3βY216F mutant protein interacted with Axin1 as strongly as WT GSK3β in HEK293T cells ( Figure 1F ) . Same as GSK3β ubiquitination mutant GSK3βKKKK15/27/32/36RRRR ( lysines 15/27/32/36 to arginines mutant , labeled as S4 in the figure ) , GSK3βY216F and GSK3βY216E phosphorylated β-catenin as efficiently as WT GSK3β in vitro ( Figure 1G , upper panel ) and in cells ( Figure 1G , lower panel ) . Together , these results imply that phosphorylation status on Y216 neither affects GSK3β binding affinity for Axin1 nor changes GSK3β kinase activity towards β-catenin phosphorylation . We next validated the role of GSK3βY216 in Wnt/β-catenin signaling in GSK3β knockout ( KO ) MEFs ( mouse embryonic fibroblasts ) . GSK3α and GSK3β are functionally redundant in Wnt signaling ( Doble et al . , 2007 ) , to avoid compensation from GSK3α , we generated GSK3β KO/GSK3α KD MEF cell line . Loss of both GSK3α/β increased the basal level of β-catenin ( Figure 2A , comparing lanes 1 and 5 ) , presumably due to the lack of GSK3-mediated phosphorylation of β-cateninS33/S37/T41 ( Doble et al . , 2007 ) . Reconstitution of the GSK3β KO/GSK3α KD MEFs with WT HA-GSK3β reduced the basal level of β-catenin to the level comparable to WT MEFs ( Figure 2A , comparing lanes 1 , 5 and 7 ) , likely due to the restoration of GSK3-mediated phosphorylation of β-catenin . Reconstitution these MEFs with WT HA-GSK3β and its phosphorylation mimetic mutant ( HA-GSK3βY216E ) , but not GSK3βY216F mutant , also restored Wnt-induced accumulation of β-catenin , thus validating the positive role of GSK3βY216 phosphorylation in Wnt signaling . The fact that GSK3βKKKK15/27/32/36RRRR/Y216E mutant failed to restore Wnt-induced β-catenin stabilization ( Figure 2A , lanes 13 and 14 ) functionally confirmed that Y216 phosphorylation occurs prior to ubiquitination . We have found that Wnt-induced long-term β-catenin stabilization is mediated by GSK3β monoubiquitination through suppressing β-catenin recruitment of β-TrCP ( Gao et al . , 2014 ) . In line with this , the co-IP results showed that upon Wnt3a stimulation , β-TrCP recruitment to β-catenin and ubiquitination of the phosphorylated β-cateninS33/S37/T41 ( Figure 2B , left panel ) were substantially increased in GSK3β KO/GSK3α KD MEFs reconstituted with HA-GSK3βY216F , whereas reconstitution with Y216 phosphorylation mimetic mutant ( HA-GSK3βY216E ) exerted the opposite effect . The results from the reciprocal IP assay confirmed more β-TrCP-bound p-β-cateninS33/S37/T41 in HA-GSK3βY216F cells ( Figure 2B , right panel ) . Together , these data suggested that phosphorylation of GSK3βY216 is required for the activation of the Wnt/β-catenin signaling by suppressing phosphorylated β-cateninS33/S37/T41 recruitment of β-TrCP . 10 . 7554/eLife . 10072 . 004Figure 2 . Activation of Wnt/β-Catenin signaling requires GSK3βY216 phosphorylation . ( A ) IB analysis using whole cell lysates from indicated MEFs treated with control-CM or Wnt3a-CM for 6 hr . Anti-GSK3 was used to detect endogenous GSK3 and HA-GSK3β . This antibody recognized HA-GSK3βY216F but not HA-GSK3βY216E or GSK3βY216E/S4 mutant proteins . ( B ) The GSK3β KO/GSK3α KD MEFs were transfected with vectors expressing indicated proteins , treated with Wnt3a-CM and proteasome inhibitor MG132 ( 10 µM ) for 4 hr as indicated , and then harvested for IP and IB analyses using indicated antibodies . ( C ) Left panel , SW480 cells and GSK3α/β KD SW480 cell lines expressing vector control , shRNA-resistant WT HA-GSK3β or its mutants were used for soft agar assays . Pictures were taken 16 days after plating at 40× . Right panel , quantification of soft agar assays . Data are presented as mean ±SD . Expressions of GSK3α/β ( endogenous or mutants ) and β-catenin were evaluated by IB at the time of plating . ( D ) Upper left panel , SW480 cell lines used in ( C ) were injected into both flanks of nude mice ( 5 mice in each cell group ) . Tumor sizes were measured at indicated time points . Tumor volumes were calculated and plotted . * denotes a statistically significant difference ( p < 0 . 01 ) comparing with shRNA control group . Bottom left panel , tumor weighs from different groups at the end of the experiment . Values are means ±SD ( n = 10 ) . Right panel , representative tumors from different groups . ( E ) IB analysis using whole cell lysates from indicated MEFs treated with control-CM or Wnt3a-CM for 6 hr . Anti-GSK3 was used to detect endogenous GSK3 and Flag-GSK3α . This antibody recognized Flag-GSK3αY279F but not Flag-GSK3αY279E mutant protein . ( F ) Left panel: IB characterization of the SW480 cell lines . Middle panel: indicated SW480 cell lines were injected into both flanks of nude mice ( 5 mice in each cell group ) . Tumors from different groups were weighed at the end of the experiment . Values are means ±SD ( n = 10 ) . * denotes a statistically significant difference ( p < 0 . 01 ) comparing with shRNA control group . Right panel , representative tumors from different groups . DOI: http://dx . doi . org/10 . 7554/eLife . 10072 . 004 CRC cell lines including SW480 ( APC-deficient , WT β-catenin ) are Wnt autocrine cell lines ( Bafico et al . , 2004 ) . In SW480 cells , KD of both GSK3α and GSK3β greatly inhibited cell growth in soft agar ( Figure 2C ) and in nude mice ( Figure 2D ) . Reconstitution of the cells with WT HA-GSK3β or HA-GSK3βY216E but not HA-GSK3βY216F restored cell growth in vitro and in vivo , implying that phosphorylation of GSK3βY216 is functionally important in CRC cells . GSK3αY279 corresponds to GSK3βY216 . Given the redundant role of GSK3α and GSK3β in Wnt signaling ( Doble et al . , 2007 ) , we next examined whether phosphorylated GSK3αY279 and GSK3βY216 also function redundantly in Wnt signaling and in CRC cells . The results showed that reconstitution of the GSK3β KO/GSK3α KD MEFs with WT Flag-GSK3α or Flag-GSK3αY279E , but not GSK3αY279F mutant restored Wnt-induced accumulation of β-catenin ( Figure 2E ) . In GSK3α/β KD SW480 cells , reconstitution with WT Flag-GSK3α or Flag-GSK3αY279E but not Flag-GSK3αY279F restored in vivo tumor growth in a cell line xenograft assay ( Figure 2F ) . Together , these data indicated that GSK3αY279 and GSK3βY216 function redundantly in the Wnt/β-catenin pathway and in CRC cells . To identify the kinases responsible for GSK3βY216 phosphorylation , we performed a pilot screening experiment using various kinase inhibitors . In line with a prior report that GSK3βY216 can be phosphorylated by PYK2 ( Sayas et al . , 2006 ) , we found that treatment of HEK293T cells with PF-562271 , a dual inhibitor of FAK/PYK2 , inhibited GSK3βY216 and GSK3αY279 phosphorylation in the presence and absence of Wnt3a ( Figure 3A ) . FAK and PYK2 are closely related non-receptor tyrosine kinases , participating in diverse signaling pathways and control a number of cellular processes ( Mitra et al . , 2005; Schaller , 2010 ) . Unlike ubiquitously expressed FAK , PYK2 has a more restricted tissue expression and is highly enriched in the brain and hematopoietic cells ( Avraham et al . , 2000 ) . Our IB results ( Figure 3B ) showed that while HEK293T and HT-29 cells predominantly expressed FAK , SW480 cells primarily expressed PYK2 . Both FAK and PYK2 were expressed in DLD-1 cells . In HEK293T cells , KD of FAK abrogated GSK3αY279 and GSK3βY216 phosphorylation and Wnt-induced β-catenin accumulation ( Figure 3C ) . In CRC cells predominantly expressing either FAK ( HT-29 cells ) or PYK2 ( SW480 cells ) , KD of FAK or PYK2 alone was sufficient to reduce the levels of phosphorylated GSK3αY279 and GSK3βY216 and total β-catenin ( Figure 3D , E ) . However , in DLD-1 cells expressing FAK and PYK2 , simultaneous KD of both kinases were required to inhibit GSK3Y279/Y216 phosphorylation and to reduce the level of β-catenin ( Figure 3F ) , strongly suggesting that FAK and PYK2 function redundantly to phosphorylate GSK3Y279/Y216 to promote Wnt/β-catenin signaling . To further confirm that GSK3 is a phosphorylation substrate of FAK/PYK2 , we examined whether FAK/PYK2 binds to GSK3β and FAK/PYK2's ability to phosphorylate GSK3βY216 in vitro . In line with prior findings that GSK3β interacts with FAK and PYK2 in vivo ( Huang et al . , 2002; Sayas et al . , 2006 ) , our co-IP results showed that GSK3β interacted with PYK2 in SW480 cells and with FAK in HT-29 cells ( Figure 3G ) . However , whether they bind directly or indirectly is not known . The result of in vitro kinase assay confirmed that FAK and PYK2 were able to directly phosphorylate GSK3βY216 in vitro ( Figure 3H ) . Together , these results reveal that FAK and PYK2 mediate GSK3Y279/Y216 phosphorylation in CRC cells . 10 . 7554/eLife . 10072 . 005Figure 3 . Phosphorylation of GSK3βY216 by FAK/PYK2 . ( A ) HEK293T cells were pre-treated with or without PF-562271 ( 4 µM ) for 4 hr followed by incubation with control-CM or Wnt3a-CM containing PF-562271 ( 4 µM ) for additional 4 hr as indicated . The whole cell lysates were subjected for IB with indicated antibodies . ( B ) FAK and PYK2 expressions were evaluated by IB analysis . ( C ) Parental , FAK KD and shRNA control HEK293T cells were treated with control-CM or Wnt3a-CM for 4 hr before harvesting for IB analysis using indicated antibodies . ( D–F ) FAK KD , PYK2 KD or FAK/PYK2 KD cell lines and shRNA control cell lines were generated using lentiviral-based shRNA targeting FAK or PYK2 or control shRNA . The whole cell lysates were used to evaluate the levels of p-GSK3βY216 and β-catenin by IB with indicated antibodies . ( G ) GSK3β in SW480 and HT29 cells was immunoprecipitated with anti-GSK3β followed by IB with anti-PYK2 and anti-FAK , respectively . Normal IgG was used as IP control . ( H ) In vitro synthesized HA-GSK3β was immunoprecipitated with anti-HA . Beads-bound HA-GSK3β was then incubated with purified FAK or PYK2 recombined protein ( or kinase buffer as a negative control ) in the presence of ATP at 37°C for 2 hr . The reaction mixtures were subjected to IB analysis using indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 10072 . 005 The above results identified GSK3β as a new substrate of FAK kinase . FAK has kinase-dependent and kinase-independent scaffolding activities ( Cance et al . , 2013 ) . If FAK/PYk2 regulates Wnt/β-catenin through their kinase activity by phosphorylating GSK3βY216 , there should be differences in the time courses of FAK/PYK2 inhibitor-mediated suppression of FAK/PYK2 kinase activity , GSK3βY216 phosphorylation and β-catenin . The results confirmed that treatment with PF-562271 caused sequential inhibition of PYK2 kinase activity represented as phosphorylated PYK2Y402 ( Park et al . , 2004 ) , GSK3βY216 phosphorylation and β-catenin ( Figure 4A ) . To ultimately validate that it is the kinase activity that mediates FAK/PYK2 regulation of Wnt/β-catenin signaling , we examined GSK3βY216 phosphorylation-dependent activation of Wnt/β-catenin signaling in FAK kinase-deficient MEFs ( FAKR454/R454 knockin MEFs ) . A knock-in point mutation of lysine 454 to arginine within the catalytic domain inactivates FAK kinases activity ( Lim et al . , 2010 ) ( Figure 4B , represented by abolished phosphorylation of FAKY397 ) but leaves FAK's scaffolding activity intact ( Lim et al . , 2008 ) . Consistent with our finding that FAK and PYK2 function redundantly in phosphorylating GSK3 , despite of suppressed FAK activity in FAKR454/R454 MEFs , KD of PYK2 was required to inhibit GSK3βY216 phosphorylation . shRNA KD of PYK2 also abrogated Wnt3a-induced β-catenin accumulation; whereas overexpression of GSKβY216E restored Wnt3a-indcued β-catenin stabilization in the FAKR454/R454/PYK2 KD MEFs ( Figure 4C ) , implying that FAK/PYK2 stabilize β-catenin through phosphorylating GSKβY216 . We further found that β-TrCP recruitment to and ubiquitination of the phosphorylated β-cateninS33/S37/T41 were greatly increased in FAKR454/R454/PYK2 KD MEFs , ectopic expression of GSKβY216E exerted the opposite effect ( Figure 4D ) . Together , these results indicated that FAK/PYK2 stabilizes β-catenin through FAK/PYK2 kinase-mediated phosphorylation of GSKβY216 . In line with the observation that elevated p-GSK3βY216 was significantly higher in 5FU treatment-resistant CRC patients than in 5FU-responsive patients ( Grassilli et al . , 2013 ) , we also found that overexpression of GSKβY216E substantially prevented PF-562271 treatment-mediated inhibition of cell proliferation in SW480 cells ( Figure 4E ) . 10 . 7554/eLife . 10072 . 006Figure 4 . FAK/PYK2 promotes the Wnt/β-catenin pathway through phosphorylating GSK3βY216 . ( A ) Time courses of PF-562271 treatment-induced inhibition of phosphorylation of PYK2 , GSK3β and β-catenin in SW480 cells . The whole cell lysates were used for the IB analysis using indicated antibodies . ( B ) The whole cell lysates from indicated cells were used for IB analysis . ( C ) The WT MEFs , FAKR454/R454/PYK2 KD MEFs transfected with empty vector or vector expressing indicated GSK3β mutants were treated with Wnt-3a CM or control-CM for 6 hr before harvesting for IB analysis . ( D ) The WT MEFs and FAKR454/R454/PYK2 KD MEFs transfected with empty vector or vector expressing HA-GSK3βY216E were treated with Wnt3a-CM and proteasome inhibitor MG132 for 4 hr and then harvested for IP and IB analyses . KI/KD: FAKR454/R454/PYK2 KD MEFs expressing empty vector . KI/KD/Y216E: FAKR454/R454/PYK2 KD MEFs expressing HA-GSK3βY216E . ( E ) Parental SW480 cells and SW480 cell lines expressing vector control , HA-GSK3β or its mutants were treated with 2 µM PF-562271 for 4 days . Cell viability was determined by CCK-8 assay , using SW480 parental cell treated with DMSO as control . Data are presented as mean ±SD . * denotes a statistically significant difference ( p < 0 . 01 ) between PF-562271-treated parental SW480 cells and cells overexpressing HA-GSK3βY216E . ( F ) Upper left panel: indicated HT-29 cell lines were injected into both flanks of nude mice ( 5 mice in each cell group ) . Animals were treated with either vehicle ( 5% Gelucire ) or PF-562271 ( 33 mg/kg in vehicle ) as indicated by oral gavage twice daily for 3 weeks . Tumor sizes were measured at indicated time points . Tumor volumes were calculated and plotted . * denotes a statistically significant difference ( p < 0 . 01 ) comparing with vehicle-treated group . Upper right panel , tumor weighs from different groups at the end of the experiment . Values are means ±SD ( n = 10 ) . Bottom panel , representative tumors from different groups . DOI: http://dx . doi . org/10 . 7554/eLife . 10072 . 006 FAK can directly phosphorylate β-catenin at tyrosine 142 ( Chen et al . , 2012 ) . We next examined whether PF-562271 treatment inhibits tumor growth when Y142 in β-catenin is mutated . The results from xenograft experiments showed that although overexpression of β-cateninY142E partially jeopardized the anti-tumor effect of PF562271 , PF562271 treatment significantly inhibited in vivo growth of SW480 cells ectopically expressing Flag-β-cateninY142E ( Figure 4F ) . While this result further proved the importance of FAK/PYK2-mediated GSK3 phosphorylation , it also suggested that phosphorylation of β-cateninY142 plays a role in FAK-targeted therapy for CRC . Intestine-specific deletion of FAK almost completely suppressed intestinal adenoma formation in APC-mutant mouse model for colorectal cancer ( Ashton et al . , 2010 ) , proving that FAK is required for the oncogenicity of APC mutation in intestinal tumorigenesis . But the underlying mechanisms remain unclear . To determine whether FAK is involved in APC-driven tumorigenesis through its catalytic activity , we examined the anti-tumorigenic effects of dual FAK/PYK2 kinase inhibitor PF-562271 in APCmin/+ mice , a genetic model predisposed to the formation of intestinal adenomas due to aberrant activation of the Wnt/β-catenin signaling . We found that PF-562271 treatment significantly inhibited both polyp formation ( number ) and polyp expansion ( size ) ( Figure 5A , B ) . The results of IB confirmed that loss of functional APC upregulated intestinal expression of c-Myc ( β-catenin target gene ) and FAK ( c-Myc target ) ( Figure 5C , left panel ) . PF-562271 treatment inhibited intestinal FAK kinase activity , evidenced by reduced level of p-FAKY397 ( Avraham et al . , 2000 ) , and remarkably reduced β-catenin level , accompanied by reduced expressions of c-Myc and FAK , consistent with the notion that FAK functions as both ‘effector’ and ‘regulator’ of β-catenin . In line with the previous report ( Carothers et al . , 2006 ) , we also found that intestinal PYK2 expression was substantially increased in tumors of APCmin/+ mice ( Figure 5C , right panel ) , PF-562271 treatment abrogated the induction ( Figure 5C , left panel ) . 10 . 7554/eLife . 10072 . 007Figure 5 . Inhibition of FAK/PYK2 kinase activity suppresses adenoma formation in APCmin/+ mice . ( A ) Representative staining results of ileum ( distal ) segments from APCmin/+ mice treated with vehicle or PF-562271 were shown . ( B ) Adenoma formation on entire small intestine was quantitatively assessed in APCmin/+ mice treated with vehicle ( n = 5 ) or PF-562271 ( n = 5 ) . Data was presented as mean ±S . D . ( C ) Left panel: tissue lysates extracted from the scraped intestinal mucosa from indicated mice were used for IB analysis . Each lane represented a single mouse . Right panel: tissue lysates extracted from C57 control mouse , APCmin/+ mouse and polyps were used for IB . ( D ) Tissue lysates used in ( C , left panel ) were immunoprecipitated with anti-GSK3β . The beads-bound immunoprecipitates were resolved by SDS-PAGE and probed with indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 10072 . 007 When assessing intestinal GSK3βY216 phosphorylation by IB , we detected multiple bands at high molecular weight that might represent phosphorylated GSK3αY279 and/or phosphorylated GSK3βY216 with other types of modifications , possibly ubiquitination . Unable to discriminate these possibilities by straight western blotting analysis , we chose to immunoprecipitate intestinal GSK3β using anti-GSK3β and then resolved the immunoprecipitates on SDS-PAGE gels , followed by IB analysis with anti-ubiquitin antibody and antibody recognizing phosphorylated GSK3βY216 . In contrast to that endogenous ubiquitinated GSK3β in non-Wnt-treated resting cells was barely detectable in cell culture ( Gao et al . , 2014 ) , to our surprise , substantial amount of the ubiquitinated intestinal GSK3β was readily detected in both C57BL/6J mice and APCmin/+ mice with much higher levels observed in APCmin/+ mice ( Figure 5D ) . This finding highlights the importance of studying the role of posttranslational modifications in relevant biological contexts . The intestinal level of p-GSK3βY216 was also much higher in APCmin/+ mice than in control mice , whereas PF-562271 treatment greatly suppressed GSK3βY216 phosphorylation and GSK3β ubiquitination . Overall , these results strongly support that FAK/PYK2 kinase contributes to APC-driven intestinal tumorigenesis through phosphorylating GSK3βY216 to promote Wnt/β-catenin signaling . Most colorectal carcinomas develop from adenomas via adenoma-carcinoma-sequence driven by the accumulation of genetic and epigenetic mutations ( Fearon and Vogelstein , 1990 ) ; Our results ( Figure 5 ) imply that FAK/PYK2/GSK3βY216/β-catenin regulation axis is an early participant and driving force for intestinal tumorigenesis in APCmin/+ mice . If this is also true in the development of human CRC , dysregulation of FAK/PYK2 expression , GSK3βY216 phosphorylation and β-catenin accumulation should occur at the adenoma stage of the adenoma–carcinoma-sequence . In a proof-of-concept experiment , we analyzed serial sections of colon specimen from the same tissue block . We found that the IHC staining intensities of FAK , PYK2 , GSK3β , p-GSK3α/βY279/Y216 and β-catenin were increased during the course of the normal-adenoma–carcinoma progression ( Figure 6A ) , demonstrating that induction of FAK/PYK2 expression , GSK3α/βY279/Y216 phosphorylation and β-catenin accumulation did occur at the early stage ( the adenoma stage ) of colon cancer development . The positive correlation between these elevated expressions also suggested that these inductions were related events . 10 . 7554/eLife . 10072 . 008Figure 6 . FAK , PYK2 , p-GSK3βY216 and β-catenin positively correlate in human colorectal cancer tissues . ( A ) Hematoxylin and eosin ( H&E ) and immunohistochemical staining of the colorectal adenoma , carcinoma and adjacent normal tissues from a single patient . ( B ) 34 tumor samples on tissue microarray were immunohistochemical stained and scored . Score distribution and average score of each antibody staining were summarized . ( C ) Scoring differences between tumor and matched normal tissues from twenty-six patients . * Denotes p-GSK3βY216 , FAK or PYK2 scores in different directions . _ highlights the numbers of patients whose TMA scores of p-GSK3βY216 , FAK or PYK2 , and β-catenin move in the same direction . ( D ) Hematoxylin and eosin ( H&E ) and immunohistochemical staining of colorectal carcinoma and adjacent normal tissues from a familial adenomatous polyposis patient . ( E ) Schematic diagram of the FKA/PYK2/p-GSK3βY216/β-catenin axis and its role in intestinal tumorigenesis . DOI: http://dx . doi . org/10 . 7554/eLife . 10072 . 008 To determine whether the positive correlations between the levels of FAK , PYK2 , p-GSK3α/βY279/Y216 and β-catenin are commonly seen in human CRC tissues , we performed IHC for these markers on CRC tissue microarray containing 34 cases of colorectal adenocarcinoma with 26 matched and 8 unmatched adjacent normal tissues . The results showed that the levels of FAK , PYK2 , GSK3β , p-GSK3α/βY279/Y216 and β-catenin were all significantly elevated in tumor tissues , with more than 1 score point difference between normal and tumor tissue on average ( Figure 6B ) . In the vast majority of samples , FAK/p-GSK3α/βY279/Y216/β-catenin ( 19 of 26 samples ) and PYK2/p-GSK3α/βY279/Y216/β-catenin ( 18 out of 26 samples ) were regulated in the same way , supporting that they are related events in the development of colonic cancer ( Figure 6C ) . To definitively link the upregulation of FAK/PYK2/p-GSK3α/βY279/Y216/β-catenin axis in human CRC to the loss of functional APC , we analyzed serial sections of colon specimens from a patient with FAP , an inherited colorectal cancer syndrome caused by germline mutation of APC . The levels of FAK/PYK2 , p-GSK3α/βY279/Y216 and β-catenin were all found substantially elevated in colon carcinomas ( Figure 6D ) , thus providing proof-of-concept evidence linking these molecular changes to the loss of functional APC in CRC patients .
The current study has revealed a novel FAK/PYK2/GSK3βY216/β-catenin regulatory circuit in intestinal tumorigenesis . Our findings indicate that: loss of functional APC induces the expression of FAK and PYK2 , elevated FAK/PYK2 phosphorylates GSK3βY216 to stabilize β-catenin , which initiates and supports intestinal tumorigenesis ( Figure 6E ) . Phosphorylation of GSK3βY216 was discovered about two decades ago ( Hughes et al . , 1993 ) , but until now , its role in Wnt/β-catenin signaling remains mysterious . Our results has uncovered a previously unrealized role of GSK3βY216 phosphorylation: it triggers GSK3β recruitment of β-TrCP and subsequent GSK3β monoubiquitination by β-TrCP , an event required for Wnt-induced inhibition of β-catenin degradation , thus identifying phosphorylation of GSK3βY216 as an ‘activator’ of the Wnt/β-catenin pathway . Phosphorylation of GSK3βY216 has been shown to be required for GSK3β's full kinase activity measured by in vitro phosphorylation of c-jun ( Hughes et al . , 1993 ) ; however , mutation of Y216 did not impair GSK3β's ability to phosphorylate Tau ( Buescher and Phiel , 2010 ) . The reason for the discrepancy is not clear , but it might be explained by the fact that different substrates were used in these studies . The current study indicates that phosphorylation of GSK3βY216 per se does not affect GSK3β activity towards β-catenin , thus ruling out the possibility that GSK3βY216 regulates the Wnt/β-catenin pathway through modulating GSK3β kinase activity . GSK3 is emerging as a prominent drug target for the treatment of various disorders including cancer and currently more than 50 GSK3 kinase inhibitors have been developed ( McCubrey et al . , 2014 ) . The kinase activity per se of GSK3 plays a dual role in Wnt signaling: GSK3 phosphorylation of Wnt coreceptor LRP6 on its PPPSP motifs is required for the activation of the pathway ( positive role ) , whereas phosphorylation of β-catenin by GSK3 triggers its degradation ( negative role ) ( Wu and Pan , 2010 ) . The kinase-mediated double role feature makes targeting GSK3 catalytic activity potentially problematic since it suppresses GSK3-mediated degradation of β-catenin , thus activating Wnt signaling therefore being potentially oncogenic . Indeed , 60 days of lithium treatment resulted in overall modest but significant increase in the tumor size in APCmin/+ mice ( Gould et al . , 2003 ) , implying that GSK3 kinase inhibitor does pose an oncogenic risk , at least in the context of Wnt-driven carcinogenesis . The current study suggests a novel way of GSK3 inhibition: targeting GSK3α/βY279/Y216 phosphorylation , which opens a new avenue for the design and development of GSK3 inhibitors . FAK plays a pivotal role in angiogenesis , tumor cell invasion and metastasis ( Sulzmaier et al . , 2014 ) . FAK is also required for tumor initiation: organ-specific deletion of FAK suppresses mammary tumorigenesis ( Luo et al . , 2009; Pylayeva et al . , 2009 ) , intestinal tumorigenesis ( Ashton et al . , 2010 ) , skin tumor formation ( McLean et al . , 2004 ) and hepatocarcinogenesis ( Shang et al . , 2015 ) , suggesting that inhibition of FAK may present a promising strategy for treating these diseases . FAK has kinase-dependent and kinase-independent scaffolding functions ( Cance et al . , 2013 ) ; whether and how these two activities contribute to FAK's role in a particular type of cancer remains largely unexplored . In c-Met/β-catenin-driven hepatocarcinogenesis , FAK kinase activity was not required for β-catenin-induced Cyclin D1 expression ( Shang et al . , 2014 ) . However , pharmacologic inhibition of FAK kinase activity suppressed KRAS-driven lung adenocarcinomas ( Konstantinidou et al . , 2013 ) , suggesting an involvement of FAK kinase activity in KRAS-driven lung cancer . This study indicates that FAK contributes APC-driven intestinal tumorigenesis through its kinase activity . Currently several FAK inhibitors are undergoing clinical trials and the first phase I trial has been very promising . A clinical challenge that lies ahead is to identify tumor types and patient subgroups that are most responsive to FAK inhibitors . To this end , the current study has identified a potentially responsive tumor type—colorectal cancer—and potentially responsive patient subpopulations—CRC patients with APC mutations . FAK is known to regulate β-catenin dynamics by directly phosphorylating β-cateninY142 ( Chen et al . , 2012 ) . Phosphorylation of β-cateninY142 acts as a switch from the adhesive to the transcriptional role of β-catenin ( Piedra et al . , 2003 ) ; as a result , FAK facilitates vascular endothelial cadherin-β-catenin dissociation and endothelial cells junctional breakdown ( Chen et al . , 2012 ) . This study has revealed a new role of FAK in the regulation of β-catenin: stabilizing β-catenin by phosphorylating GSK3α/βY279/Y216 . FAK regulates mammary stem cell and intestinal tissue regeneration ( Luo et al . , 2009; Ashton et al . , 2010; Sulzmaier et al . , 2014 ) ; given the importance of Wnt/β-catenin pathway in stem cell , our study could provide a potential mechanistic explanation how FAK is involved in stem cell and tissue regeneration . PYK2 is closely related to FAK in sequence and structure with 48% amino acid identity , and they function redundantly and non-redundantly in a function and context-dependent manner ( Schaller , 2010 ) . Despite its low expression in normal human intestines ( Avraham et al . , 1995 ) , we and others find that intestinal PYK2 is elevated in adenomas in APCmin/+ mice and in CRC patient tissues ( Carothers et al . , 2006 ) . The current study clearly show that by phosphorylating GSK3α/βY279/Y216 , FAK and PYK2 function redundantly in the regulation of Wnt/β-catenin signaling in CRC cells , raising the possibility that FAK and PYK2 could also function redundantly in Wnt-driven intestinal tumorigenesis . However , if so , why was intestine-specific deletion of FAK alone sufficient to cause complete abrogation of adenoma formation in APC-mutant mice ( Ashton et al . , 2010 ) ? Why did PYK2 not compensate for the loss of FAK in intestinal tumorigenesis ? A prior study ( Carothers et al . , 2006 ) and our data ( Figure 5C , right panel ) showed that drastic PYK2 elevation occurred in tumors in APCmin/+ mice , indicating that PYK2 is upregulated during the process of intestinal tumorigenesis . FAK induction , although further enhanced in tumors , however , is triggered by APC mutation—the initiating event for tumorigenesis in the APC-mutant mice , through activated Wnt pathway target c-Myc ( Ashton et al . , 2010 ) ; therefore FAK induction by APC mutation should occur earlier than PYK2 elevation . If so , it explains why inhibition of FAK by intestinal deletion of FAK was sufficient to suppress tumor development , it also explains why PF562271 treatment inhibited PYK2 elevation in APCmin/+ mice ( Figure 5C , left panel ) . Nevertheless , regardless of the exact mechanisms mediating in vivo PYK2 regulation during intestinal tumorigenesis , elevated PYK2 in tumors further enhances Wnt-dependent tumorigenesis through phosphorylating GSK3 to promote the Wnt pathway—a positive feedback loop that reinforces the tumor development process . Tumors undergoing targeted therapy often relapse due to the utilization of autonomous parallel-redundant signaling . The current study indicates that both FAK and PYK2 are elevated in already established colon tumors in CRC patients and that effective inhibition of the Wnt/β-catenin pathway in CRC cells expressing FAK and PYK2 will only be achieved by simultaneously targeting both kinases; we believe these results provide a solid mechanistic justification for clinical use of FAK/PYK2 dual inhibitor instead of FAK-specific inhibitor for better clinical outcomes in CRC patients .
Data are presented as mean ±SD . The difference between two groups was evaluated using Student's t-test ( two tailed ) . p values less than 0 . 05 were considered statistically significant . Detailed information for DNA constructs , shRNA , antibodies and reagents , and detailed methods for cell culture , immunoprecipitation ( IP ) , immunoblotting ( IB ) , transfection , lentivirus and retrovirus production and infection , in vitro kinase assay , ubiquitination assay , soft agar assay , cell proliferation assay , and animal experiments are provided below .
Aberrant activation of Wnt/β-catenin signaling plays an unequivocal role in colorectal cancer , but identification of effective Wnt inhibitors for use in cancer remains a tremendous challenge . New insights into the regulation of this pathway could reveal new therapeutic point of intervention , therefore are greatly needed . Here we report a novel FAK/PYK2/GSK3βY216/β-catenin regulation axis: FAK and PYK2 , elevated in adenomas in APCmin/+ mice and in human colorectal cancer tissues , functioned redundantly to promote the Wnt/β-catenin pathway by phosphorylating GSK3βY216 to reinforce pathway output—β-catenin accumulation and intestinal tumorigenesis . We previously showed that Wnt-induced β-catenin accumulation requires Wnt-induced GSK3β/β-TrCP interaction; the current study revealed that phosphorylation of GSK3βY216 was a molecular determinant of GSK3β recruitment of β-TrCP . Pharmacological inhibition of FAK/PYK2 suppressed adenoma formation in APCmin/+ mice accompanied with reduced intestinal levels of phospho-GSK3βY216 and β-catenin , indicating that FAK/PYK2/GSK3βY216 axis is critical for the activation of Wnt/β-catenin signaling in APC driven intestinal tumorigenesis .
The cells in our body communicate with each other to coordinate many essential processes , including cell division and the formation of tissues and organs . The Wnt signaling pathway is crucial for cell communication across all animal species , but activating this pathway at the wrong time can cause cancer to develop . As part of the investigation into treatments for colon and other intestinal cancers , researchers have tried to identify drugs that inhibit Wnt signaling . This search would be easier if we understood more about how the Wnt signaling pathway is controlled . When the protein GSK3 is active , it can switch the Wnt signaling pathway ‘off’ , and inactivating GSK3 can turn the Wnt signaling pathway ‘on’ . Enzymes known as kinases can inactivate GKS3 by adding a phosphate group to it , in a process known as phosphorylation . It was unknown which kinases phosphorylate GSK3 , and whether this affects how cancerous tumors develop in the colon . Gao , Chen et al . analyzed cells taken from human and mice and observed that two kinases called PYK2 and FAK phosphorylate one form of GSK3 , known as GSK3β . Furthermore , both kinases are required to inactivate GSK3β completely , and so turn on the Wnt signaling pathway . During the early stages of colon cancer , cells first develop into polyps , which subsequently become cancerous . Gao , Chen et al . treated mice that had genetic mutations that made them susceptible to colon cancer with a chemical compound that inhibits both FAK and PYK2 . These mice grew fewer polyps than untreated mice , and the polyps that did grow tended to be smaller . Tissue samples taken from humans in the early stages of colon cancer—as the polyps progress towards becoming cancerous—had high levels of FAK , PYK2 and phosphorylated GSK3β . Overall , this suggests that drugs that simultaneously inhibit FAK and PYK2 may be an effective treatment for colon cancer , although further studies will be needed to confirm this .