A Daple-Akt feed-forward loop enhances noncanonical Wnt signals by compartmentalizing β-catenin.

Cellular proliferation is antagonistically regulated by canonical and noncanonical Wnt signals; their dysbalance triggers cancers. We previously showed that a multimodular signal transducer, Daple, enhances PI3-K→Akt signals within the noncanonical Wnt signaling pathway and antagonistically inhibits canonical Wnt responses. Here we demonstrate that the PI3-K→Akt pathway serves as a positive feedback loop that further enhances noncanonical Wnt signals by compartmentalizing β-catenin. By phosphorylating the phosphoinositide- (PI) binding domain of Daple, Akt abolishes Daple's ability to bind PI3-P-enriched endosomes that engage dynein motor complex for long-distance trafficking of β-catenin/E-cadherin complexes to pericentriolar recycling endosomes (PCREs). Phosphorylation compartmentalizes Daple/β-catenin/E-cadherin complexes to cell-cell contact sites, enhances noncanonical Wnt signals, and thereby suppresses colony growth. Dephosphorylation compartmentalizes β-catenin on PCREs, a specialized compartment for prolonged unopposed canonical Wnt signaling, and enhances colony growth. Cancer-associated Daple mutants that are insensitive to Akt mimic a constitutively dephosphorylated state. This work not only identifies Daple as a platform for cross-talk between Akt and the noncanonical Wnt pathway but also reveals the impact of such cross-talk on tumor cell phenotypes that are critical for cancer initiation and progression

Tyrosine-Based Signals Regulate the Assembly of Daple⋅PARD3 Complex at Cell-Cell Junctions.

Polarized distribution of organelles and molecules inside a cell is vital for a range of cellular processes and its loss is frequently encountered in disease. Polarization during planar cell migration is a special condition in which cellular orientation is triggered by cell-cell contact. We demonstrate that the protein Daple (CCDC88C) is a component of cell junctions in epithelial cells which serves like a cellular "compass" for establishing and maintaining contact-triggered planar polarity. Furthermore, these processes may be mediated through interaction with the polarity regulator PARD3. This interaction, mediated by Daple's PDZ-binding motif (PBM) and the third PDZ domain of PARD3, is fine-tuned by tyrosine phosphorylation on Daple's PBM by receptor and non-receptor tyrosine kinases, such as Src. Hypophosphorylation strengthens the interaction, whereas hyperphosphorylation disrupts it, thereby revealing an unexpected role of Daple as a platform for signal integration and gradient sensing for tyrosine-based signals within the planar cell polarity pathway

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein-protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs

Prognostic relevance of CCDC88C (Daple) transcripts in the peripheral blood of patients with cutaneous melanoma

A loss of balance between G protein activation and deactivation has been implicated in the initiation of melanomas, and non-canonical Wnt signaling via the Wnt5A/Frizzled (FZD) pathway has been shown to be critical for the switch to an invasive phenotype. Daple [CCDC88C], a cytosolic guanine nucleotide exchange modulator (GEM) which enhances non-canonical Wnt5A/FZD signaling via activation of trimeric G protein, Gαi, has been shown to serve opposing roles-as an inducer of EMT and invasiveness and a potent tumor suppressor-via two isoforms, V1 (full-length) and V2 (short spliced isoform), respectively. Here we report that the relative abundance of these isoforms in the peripheral circulation, presumably largely from circulating tumor cells (CTCs), is a prognostic marker of cutaneous melanomas. Expression of V1 is increased in both the early and late clinical stages (p \u3c 0.001, p = 0.002, respectively); V2 is decreased exclusively in the late clinical stage (p = 0.003). The two isoforms have opposing prognostic effects: high expression of V2 increases relapse-free survival (RFS; p = 0.014), whereas high expression of V1 tends to decrease RFS (p = 0.051). Furthermore, these effects are additive, in that melanoma patients with a low V2-high V1 signature carry the highest risk of metastatic disease. We conclude that detection of Daple transcripts in the peripheral blood (i.e., liquid biopsies) of patients with melanoma may serve as a prognostic marker and an effective strategy for non-invasive long-term follow-up of patients with melanoma

Zebrafish Models of Ribosome-Associated Disorders for Identifying Novel Therapeutics

Ribosomes are large complex molecular machineries required for the synthesis of proteins via a process called protein translation. The importance of this molecular machine is conserved and is found within all living cells. Two subunits (designated the small and large subunits) form a functional ribosome. Each subunit is a complex of ribosomal RNA and a variety of proteins. Within the ribosome, amino acids are linked together depending on the order specified by the mRNA molecule. Due to the importance of ribosomes in maintaining cellular function, one would expect defects in ribosome function, or biogenesis, to have a systemic effect on the organism. However, what is typically seen with disorders involving aberrant ribosome functions, a.k.a. ribosomopathy, is a tissue-specific defect. Specifically, patients normally present themselves with bone marrow failure, which ultimately leads to severe anemia. Other common features in patients include a shorter stature and a higher predisposition to certain cancers. Diamond Blackfan anemia (DBA) is the first described ribosome-associated disorder with ribosomal protein S19, RPS19, being the most commonly mutated gene found in patients. Since then, the number of ribosomal proteins found mutated in DBA patients has expanded and includes genes such as RPL11, RPL5, and RPS24. Another ribosomopathy is 5q-syndrome. In this disease, a large chromosomal deletion is found in patients. Within the commonly deleted region (CDR), it is believe that ribosomal protein S14, RPS14, is the candidate gene associated with the macrocytic anemia seen in the disease.The effects of p53 signaling during ribosome stress have been well documented in both patient bone marrow and in models of ribosomopathies. Aberrant ribosome function has been shown to increase p53 signaling through stabilization of p53 through a RP-MDM2-p53 checkpoint mechanism in the cell. Upon increases in p53 activity, p53-mediated cell cycle arrest and apoptosis occurs. Reversal of this increase in p53 signaling has been shown to relive the phenotypic and anemic defects in cellular and animal models. However, the use of p53 inhibition towards patient care has been cautioned due to risk of cancer progression. p53 is a major tumor suppressor gene in the cell and abolishing its activity has been shown to elevate cancer risk. Patients with malfunctioning ribosome proteins are at a higher risk of tumor development; therefore, targeting p53 may further enhance tumor risk. This dilemma raises the need for novel therapeutics in tackling the treatment of ribosomopathies and to further understand disease pathology.Animal models are indispensible for understanding the molecular mechanism and pathology of a disease. Zebrafish is an attractive vertebrate model to use in biological studies due to its rapid development, small size, transparent nature, and ease of manipulating gene function. Furthermore, there are many conserved genes between mammals and zebrafish, thereby making the model useful for understanding gene function. The advent of genome-editing technologies applied to the zebrafish model (i.e. TALEN, CRISPR/Cas9) has expanded the use of the zebrafish to model human diseases. In this work, we model two ribosome-associated disorders in the zebrafish, DBA and 5q-sydrome, via targeted RPL11 and RPS14, respectively. Using these models, we show that a delay in late-stage erythropoiesis occurs and is followed by cell death upon ribosome stress. The delay in late-stage erythropoiesis is independent of p53 signaling and may, in part, be due to overexpression of Lft1. We demonstrated the ability of RAP-011, an activin receptor type IIA ligand trap, to increase erythropoiesis in the zebrafish and restore erythroid levels in our zebrafish models. Furthermore, the effects of RAP-011 are independent of p53 signaling, and thus, offer a new strategy in the treatment of ribosomopathies

A Daple-Akt feed-forward loop enhances noncanonical Wnt signals by compartmentalizing β-catenin.

Cellular proliferation is antagonistically regulated by canonical and noncanonical Wnt signals; their dysbalance triggers cancers. We previously showed that a multimodular signal transducer, Daple, enhances PI3-K→Akt signals within the noncanonical Wnt signaling pathway and antagonistically inhibits canonical Wnt responses. Here we demonstrate that the PI3-K→Akt pathway serves as a positive feedback loop that further enhances noncanonical Wnt signals by compartmentalizing β-catenin. By phosphorylating the phosphoinositide- (PI) binding domain of Daple, Akt abolishes Daple's ability to bind PI3-P-enriched endosomes that engage dynein motor complex for long-distance trafficking of β-catenin/E-cadherin complexes to pericentriolar recycling endosomes (PCREs). Phosphorylation compartmentalizes Daple/β-catenin/E-cadherin complexes to cell-cell contact sites, enhances noncanonical Wnt signals, and thereby suppresses colony growth. Dephosphorylation compartmentalizes β-catenin on PCREs, a specialized compartment for prolonged unopposed canonical Wnt signaling, and enhances colony growth. Cancer-associated Daple mutants that are insensitive to Akt mimic a constitutively dephosphorylated state. This work not only identifies Daple as a platform for cross-talk between Akt and the noncanonical Wnt pathway but also reveals the impact of such cross-talk on tumor cell phenotypes that are critical for cancer initiation and progression