β-catenin, functioning as a major component of both Wnt signaling and cell-cell adhesion, plays a central role in cell proliferation, differention, polarity, morphogenesis, and development (40
). β-catenin deficiency arrests mouse embryo development at gastrulation (43
). β-catenin localized in different cellular compartments forms distant complexes that carry out differential cellular functions. We show here that β-catenin distribution can be dynamically regulated, and phosphorylation of β-catenin by AKT results in the relocalization of some β-catenin from cell-cell contracts and increase in its transcriptional activity.
Activating mutations of Wnt components lead to nuclear localization of β-catenin and are involved in tumor formation and development (44
). Wnt-independent signaling, nevertheless, is also involved in regulation of β-catenin transactivation and tumorigenesis (26
). β-catenin-TCF/LEF-1 signaling can be activated by growth factors, such as EGF, HGF/scatter factor (SF), insulin-like growth factor (IGF)-I, IGF-II, and insulin (15
). In response to insulin stimulation, phosphatidylinositol (PI) 3-kinase-activated AKT phosphoryates GSK-3β at Ser9, which leads to inactivation of GSK-3β and augmentation of β-catenin-TCF/LEF-1 transcriptional activity (47
). In addition to this indirect regulation, our results show that AKT phosphorylates β-catenin both in vitro
and in vivo
. The mutation of S552 of β-catenin into Ala or Asp did not change its phosphorylation level by GSK-3β or its half-life, in contrast to WT, indicating that phosphorylation of β-catenin at S552 by AKT does not alter β-catenin protein stability. Instead, phosphorylation at this site enables some β-catenin to disassociate from cell-cell contacts and accumulate in the nucleus. Mutation of the AKT phosphorylation site reduced total transcriptional activity of TCF/LEF-1 induced by AKT () about 57%. This partial effect of AKT on TCF/LEF-1 transcriptional activity is likely because AKT can still indirectly enhance upregulation of β-catenin transcriptional activity by phosphorylation and inhibition of GSK-3β. Thus, AKT can activate β-catenin-TCF/LEF-1 transcriptional activity both by indirect stabilization of β-catenin through inhibition of GSK-3β and direct phosphorylation of β-catenin, which enhances β-catenin nuclear accumulation.
In addition to sequential phosphorylation of β-catenin at its N-terminus by CK1 and GSK-3β (44
), β-catenin can be phosphorylated by CK2 at Thr393 (52
) and PKA at Ser675 (33
), which enhance β-catenin transctivation by stabilizing β-catenin protein. Paradoxically, it was reported that CK2-dependent phosphorylation of the E2 ubiquitin conjugating enzyme UBC3B induces the interaction between UBC3B and β-TrCP and enhances β-catenin degradation (53
). PKA-phosphorylated β-catenin at S675 promotes the association with its co-activator, CREB-binding protein, thus enhancing β-catenin-TCF/LEF-1 transcriptional activity, although different effects of this phosphorylation on β-catenin stability were observed (33
). S552 was shown to be a weak phosphorylation site of PKA (33
). However, we did not detect measurable reduction of phosphorylation levels of the β-catenin S552A mutant by PKA compared to WT. In contrast, chemical inhibition of AKT and mutation of S552 of β-catenin, but not PKA phosphorylation site S675, abolished phosphorylation by both AKT1 and AKT2 and detection by anti-phospho-RRXS/T antibody, although this antibody may also recognize other –3 position R-bearing phospho-S/T peptides, such as the substrate motif for PKA and PKC. Active AKT phosphorylates proteins containing the consensus sequence RXRXXS/T (31
). However, several reports indicate that AKT is also able to phosphorylate Ser or Thr when present in the consensus sequence RXXS/T (54
) or other non-consensus sequences (56
). We demonstrated that AKT phosphorylates S552 in the 489-RRTS-552 sequence of β-catenin, providing additional evidence showing that RXXS/T can be a phosphorylation sequence by AKT.
As a family of conserved regulatory proteins that mainly bind to a plethora of functionally diverse signaling molecules that are phosphorylated at the Ser/Thr residues (39
), 14-3-3 can regulate the subcellular distribution of its associated proteins. This is exemplified by the nuclear import of Chk2-phosphorylated the MDM2 homolog MDMX promoted by 14-3-3 (57
). 14-3-3ζ, in association with β-catenin, can facilitate transactivation of β-catenin by AKT (38
). 489-RRTSMG-554 of β-catenin is a binding motif for 14-3-3ζ. β-catenin S552A weakens its binding to 14-3-3ζ and has a reduced transcriptional activity, whereas β-catenin S552D enhances this binding and increases its transactivation, in contrast to WT β-catenin. Furthermore, S552D β-catenin has accumulated nuclear localization, which supports that 14-3-3ζ may mediate dynamic nucleus-cytosol transportation of its binding proteins (58
). It has been shown that AKT1 and AKT2 have distinct roles in tumor cell growth and migration, while AKT1 and AKT2 can either have positive or negative roles, depending on the cell types and experimental setting (59
). Our results show that activated forms of AKT1 and AKT2 are able to phosphorylate β-catenin in vitro
and in vivo
. Furthermore, expression of constitutively activated AKT1 or AKT2 results in the relocalization of β-catenin from cell–cell contacts and subsequent transactivation. Thus, it seems that AKT1 and AKT2, can affect the same substrate for regulation of its functions.
S552D β-catenin mutant has a higher transcriptional activity than WT or S552A β-catenin, which correlates with their differential levels of nuclear accumulation. Furthermore, A431 cells stably expressing S552D β-catenin mutant showed much enhanced invasion in contrast to the cells stably expressing either WT or S552 β-catenin. Transactivation of β-catenin increases the transcription of genes that promote tumor cell growth, such as MYC
(which encodes cyclin D
), and JUN
), and genes that promote tumor cell invasion, such as matrix metalloproteinase-7 (MMP7)
) and TWIST1
). Further investigation is needed to determine whether phosphorylation of β-catenin at S552 induces a specific set of downstream genes that promote tumor development.
EGFR overexpression or mutation has been found in many types of cancers, with activation of AKT by EGFR or other growth factor receptors, Ras, and regulation of PI 3-K and PTEN contributing to tumor development (72
). EGF-induced phosphorylation and transactivation of β-catenin are dependent on the activity of AKT. We have previously shown that EGFR activation results in Wnt-independent β-catenin transactivation by downregulation of caveolin-1, whereas overexpression of caveolin-1 suppresses the effect of EGFR on β-catenin (16
). Downregulation and internalization of caveolin-1 by EGF will release inhibited signaling molecules normally sequestered in caveolae, such as growth factor receptors, Ha-Ras, and PI 3-K (16
), which may in turn contribute to AKT activation. Activated AKT can stabilize β-catenin through inhibition of GSK-3β and/or directly phosphorylate β-catenin, resulting in its disassociation from cell-cell contacts and nuclear accumulation. The combined effects enhance β-catenin-TCF/LEF-1 transcriptional activity, which in turn contributes to tumor cell invasion and tumor development.