Enhancement of β-catenin activity by BIG1 plus BIG2 via Arf activation and cAMP signals

Abstract

Multifunctional β-catenin, with critical roles in both cell-cell adhesion and Wnt-signaling pathways, was among HeLa cell proteins coimmunoprecipitated by antibodies against brefeldin A-inhibited guanine nucleotide-exchange factors 1 and 2 (BIG1 or BIG2) that activate ADP-ribosylation factors (Arfs) by accelerating the replacement of bound GDP with GTP. BIG proteins also contain A-kinase anchoring protein (AKAP) sequences that can act as scaffolds for multimolecular assemblies that facilitate and limit cAMP signaling temporally and spatially. Direct interaction of BIG1 N-terminal sequence with β-catenin was confirmed using yeast two-hybrid assays and in vitro synthesized proteins. Depletion of BIG1 and/or BIG2 or overexpression of guanine nucleotide-exchange factor inactive mutant, but not wild-type, proteins interfered with β-catenin trafficking, leading to accumulation at perinuclear Golgi structures. Both phospholipase D activity and vesicular trafficking were required for effects of BIG1 and BIG2 on β-catenin activation. Levels of PKAphosphorylated β-catenin S675 and β-catenin association with PKA, BIG1, and BIG2 were also diminished after BIG1/BIG2 depletion. Inferring a requirement for BIG1 and/or BIG2 AKAP sequence in PKA modification of β-catenin and its effect on transcription activation, we confirmed dependence of S675 phosphorylation and transcription coactivator function on BIG2 AKAP-C sequence.ADP-ribosylation factor | cell migration | AKAP | phospholipase D M ultifunctional β-catenin is an essential component of intercellular adherens junctions (AJs) that link cell surface cadherin and actin cytoskeleton via interactions with both. β-catenin is also a transcriptional coactivator in several pathways, including Wnt-initiated signaling (1, 2), which is critical in embryonic development and tissue homeostasis or pathology (2, 3). In the absence of "canonical" catenin-dependent Wnt signaling, cytoplasmic β-catenin is maintained at low levels via phosphorylations of S45 and S33, S37 and T41 by, respectively, casein kinase-1 and glycogen synthase kinase-3 (GSK-3) (1, 2). Subsequent degradation of β-catenin via an ubiquitin/proteasome pathway involves axin and adenomatous polyposis coli (APC) as scaffold proteins, plus β-transducin repeat-containing protein (β-TrCP), an E3 ubiquitin ligase. In one well-known model, binding of Wnt to Frizzled (Fz) and its coreceptor low-density lipoprotein receptor-like protein 5 or 6 (LRP5/6) resulted in GSK-3 sequestration in multivesicular bodies, thereby reducing its cytosolic level (4, 5). With lower GSK-3 activity, "stabilized" β-catenin (1, 2) entered the nucleus and, along with members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family, activated transcription via Wnt-target gene promoters, including c-myc and cyclin D1 (6-8). In β-catenin knockout mice, embryogenesis was arrested at gastrulation (9) and aberrant Wnt signaling led to tumor development (2, 3).Although relationships between labile and stabilized β-catenin pools remain poorly understood, ...

Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis

“…In addition, activation of PI3K/ Akt also induces VEGF expression in ECs (53,54). Consistent with previous findings in HeLa cells (28) and thyroid cancer (52), knockdown of BIG1 and BIG2 in HUVECs significantly reduces phosphorylation of Akt. Although it is still not clear how BIG1 and BIG2 interfere with phosphorylation of Akt, we show here that incubation with SC79, which specifically enhances Akt phosphorylation, can rescue the suppression of VEGF expression after BIG1 or BIG2 knockdown in HUVECs.…”

“…In previous reports, depletion of BIG1 or BIG2 in HeLa cells decreased phosphorylation of Akt at S473 (28), and knockdown of BIG1 in papillary thyroid cancer cells also inhibited Akt signaling to attenuate epithelialmesenchymal transition and reduce cell migration and invasion (52). Because PI3K-Akt signaling is known to mediate VEGF expression in ECs (53,54), we next examined the effects of BIG1 or BIG2 knockdown on activation of Akt in HUVECs.…”

“…Immunofluorescence staining of HUVECs on Millicell EZ SLIDE 4-well glass slides (MilliporeSigma) was performed following a previous report (28). To stain F-actin, cells were incubated with Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific) for 1 h and counterstained with 1 mg/ml of DAPI (MilliporeSigma).…”

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors

“…In addition, Li et al found that BIG1 physically interacted with β‐catenin, and that knockdown of BIG1 or BIG2 tethered β‐catenin at perinuclear Golgi structures without changing β‐catenin expression. These results imply that the accumulation of β‐catenin at Golgi structures after inhibition of BIG1 or BIG2 might contribute to the suppression of Wnt signaling by delaying β‐catenin transit to the nuclei (Li, Le, Kato, Moss, & Vaughan, ). We found that BFA decreased β‐catenin transcripts (Figure d); therefore, these Golgi disruptors might not only suppress the transcription of β‐catenin, but also alter its distribution in cells.…”

Podocyte-specific Rac1 deficiency ameliorates podocyte damage and proteinuria in STZ-induced diabetic nephropathy in mice

“…β-catenin is a key component of Wnt signaling pathway and active β-catenin is found in diverse kidney diseases including focal segmental glomerulosclerosis (FSGS) and DN 19 , 30 . Excess cytoplasmic β-catenin is often targeted by a N-terminal phosphorylation/ubiquitylation-mediated degradation system 31 – 35 . N-terminus dephosphorylation of β-catenin is sufficient to induce stabilized β-catenin migrating into the nucleus 10 , 28 .…”