Jason Ear

A Gαi-GIV molecular complex binds epidermal growth factor receptor and determines whether cells migrate or proliferate

Migrating cells do not proliferate and vice versa, but the mechanism involved remains unknown. Ghosh et al. reveal how this cellular decision is made by showing that a Gαi–GIV molecular complex interacts with EGF receptor and programs growth factor signaling, triggering migration when assembled and favoring mitosis when assembly is prevented.

Zebrafish High‐Throughput Screening to Study the Impact of Dissolvable Metal Oxide Nanoparticles on the Hatching Enzyme, ZHE1

The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high-throughput screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degradation of the chorionic membrane. The data indicate that 4 of 24 metal oxide nanoparticles (CuO, ZnO, Cr2 O3 , and NiO) could interfere with embryo hatching by a chelator-sensitive mechanism that involves ligation of critical histidines in the ZHE1 center by the shed metal ions. A recombinant ZHE1 enzymatic assay is established to demonstrate that the dialysates from the same materials responsible for hatching interference also inhibit ZHE1 activity in a dose-dependent fashion. A peptide-based BLAST search identifies several additional aquatic species that express enzymes with homologous histidine-based catalytic centers, suggesting that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of oxide nanoparticles that pose a hazard to aquatic species.

A GDI (AGS3) and a GEF (GIV) regulate autophagy by balancing G protein activity and growth factor signals.

This work introduces a nonreceptor GEF for Gαi subunits as a regulator of autophagy. The authors reveal how growth factors reversibly regulate autophagy by a unique mechanism that involves reversible regulation of Gαi3 activity by AGS3, a GDI, and GIV, a GEF, during initiation and reversal of autophagy, respectively.

Expression of GIV/Girdin, a metastasis-related protein, predicts patient survival in colon cancer

Metastasis accounts for the majority of cancer‐related deaths. Accurate prediction of meta‐static potential of tumors has been elusive, and the search for clinically useful markers continues. We previously reported that GIV/Girdin triggers tumor cell migration by virtue of a C‐terminal guanine‐nucle‐otide exchange factor motif that activates Gai. Here we identify GIV as a metastasis‐related protein whose full‐length transcript (GIV‐fl) is expressed exclusively in highly invasive colon, breast, and pancreatic carcinoma cells and not in their poorly invasive counterparts. A prospective, exploratory biomarker study conducted on a cohort of 56 patients with stage II colorectal cancer revealed a significant correlation between GIV‐fl expression in tumor epithelium and shortened metastasis‐free survival. Survival rate for patients with GIV‐fl‐positive tumors is significantly reduced compared with the patients with GIV‐fl‐negative tumors [P<0.0001;hazard ratio=0.076;CI=0.052–0.30 (95%)]. At the 5‐yr mark, survival is 100% in the GIV‐fl‐negative group and 62 ± 9% (mean±se; P=6×10‐5) in the GIV‐fl‐positive group. Furthermore, GIV‐fl expression predicts a risk of mortality independent of the microsatellite stability status, a well‐established prognosticator of colo‐rectal cancers. We conclude that GIV‐fl is a novel metastasis‐related protein and an independent adverse prognos‐ticator that may serve as a useful adjunct to traditional staging strategies in colorectal carcinoma.—Garcia‐Marcos, M., Jung, B. H., Ear, J., Cabrera, B., Carethers, J. M., Ghosh, P. Expression of GIV/Girdin, a metastasis‐related protein, predicts patient survival in colon cancer. FASEB J. 25, 590–599 (2011). www.fasebj.org

A Structural Determinant That Renders Gαi Sensitive to Activation by GIV/Girdin Is Required to Promote Cell Migration

Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in Gαi3 necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates Gαi subunits. Using G protein activity and in vitro pulldown assays we demonstrate that Gαi3 is a better substrate for GIV than the highly homologous Gαo. We identified Trp-258 in the Gαi subunit as a novel structural determinant for GIV binding by comparing GIV binding to Gαi3/Gαo chimeras. Mutation of Trp-258 to the corresponding Phe in Gαo decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other Gα-binding partners, i.e. Gβγ, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/RGS19 (a GTPase-activating protein), and LPAR1 (a G protein-coupled receptor). Activation of Gαi3 by GIV was also dramatically reduced when Trp-258 was replaced with Tyr, Leu, Ser, His, Asp, or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant Gαi3 W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or G protein-coupled receptor stimulation. Thus activation of Gαi3 by GIV is essential for biological functions associated with Gαi3 activation. In conclusion, we have discovered a novel structural determinant on Gαi that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on Gαi and that represents an attractive target site for designing small molecules to disrupt the Gαi-GIV interface for therapeutic purposes.

Tyrosine Phosphorylation of the Gα-Interacting Protein GIV Promotes Activation of Phosphoinositide 3-Kinase During Cell Migration

GIV links ligand stimulation of various receptors to downstream activation of a kinase involved in cell migration. GIVing Migration a Boost Phosphoinositide 3-kinase (PI3K) can promote cell migration, and the activation of PI3K occurs downstream of ligand binding to several cell surface receptors. Lin et al. show that the guanine nucleotide exchange factor GIV (Gα-interacting vesicle-associated protein) may link ligand stimulation of these receptors to activation of PI3K. GIV was tyrosine phosphorylated by various receptor and non-receptor tyrosine kinases, and these phosphorylation events enabled GIV to bind to a regulatory subunit of PI3K, increase PI3K activity at the plasma membrane, and promote cell migration. The metastatic and invasive extent of a breast carcinoma was positively correlated to tyrosine phosphorylation of GIV and its association with the regulatory subunit of PI3K. Thus, manipulations that prevent or decrease the tyrosine phosphorylation of GIV could potentially be used to slow the progression of invasive cancers. GIV (Gα-interacting vesicle-associated protein; also known as Girdin) enhances Akt activation downstream of multiple growth factor– and G protein (heterotrimeric guanosine 5′-triphosphate–binding protein)–coupled receptors to trigger cell migration and cancer invasion. We demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at tyrosine-1764 and tyrosine-1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the amino- and carboxyl-terminal Src homology 2 domains of p85α, a regulatory subunit of PI3K; stabilized receptor association with PI3K; and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85α increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIV-PI3K interaction a potential therapeutic target within the PI3K-Akt pathway.

Defects of protein production in erythroid cells revealed in a zebrafish Diamond–Blackfan anemia model for mutation in RPS19

Diamond–Blackfan anemia (DBA) is a rare congenital red cell aplasia that classically presents during early infancy in DBA patients. Approximately, 25% of patients carry a mutation in the ribosomal protein (RP) S19 gene; mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 have been reported. How ribosome protein deficiency causes defects specifically to red blood cells in DBA has not been well elucidated. To genetically model the predominant ribosome defect in DBA, we generated an rps19 null mutant through the use of TALEN-mediated gene targeting in zebrafish. Molecular characterization of this mutant line demonstrated that rps19 deficiency reproduced the erythroid defects of DBA, including a lack of mature red blood cells and p53 activation. Notably, we found that rps19 mutants’ production of globin proteins was significantly inhibited; however, globin transcript level was either increased or unaffected in rps19 mutant embryos. This dissociation of RNA/protein levels of globin genes was confirmed in another zebrafish DBA model with defects in rpl11. Using transgenic zebrafish with specific expression of mCherry in erythroid cells, we showed that protein production in erythroid cells was decreased when either rps19 or rpl11 was mutated. L-Leucine treatment alleviated the defects of protein production in erythroid cells and partially rescued the anemic phenotype in both rps19 and rpl11 mutants. Analysis of this model suggests that the decreased protein production in erythroid cells likely contributes to the blood-specific phenotype of DBA. Furthermore, the newly generated rps19 zebrafish mutant should serve as a useful animal model to study DBA. Our in vivo findings may provide clues for the future therapy strategy for DBA.

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

GIV, a guanidine exchange factor for trimeric Gi, contains a unique domain that functions like a SH2 domain. GIVs SH2-like domain binds autophosphorylated RTKs. Binding of GIVs SH2 to RTKs enables the receptors to activate trimeric Gi. Inhibition of GIV:RTK interaction abolishes GIV-dependent Akt enhancement downstream of RTKs.

The Molecular Basis of the Caskin1 and Mint1 Interaction with CASK

Calcium/calmodulin-dependent serine protein kinase (CASK) is a conserved multi-domain scaffolding protein involved in brain development, synapse formation, and establishment of cell polarity. To accomplish these diverse functions, CASK participates in numerous protein-protein interactions. In particular, CASK forms competing CASK/Mint1/Velis and CASK/Caskin1/Velis tripartite complexes that physically associate with the cytoplasmic tail of neurexin, a transmembrane protein enriched at presynaptic sites. This study shows that a short linear EEIWVLRK peptide motif from Caskin1 is necessary and sufficient for binding CASK. We also identified the conserved binding site for the peptide on the CASK calmodulin kinase domain. A related EPIWVMRQ peptide from Mint1 was also discovered to be sufficient for binding. Searching all human proteins for the Mint1/Caskin1 consensus peptide ExIWVxR revealed that T-cell lymphoma invasion and metastasis 1 (TIAM1) contains a conserved EEVIWVRRE peptide that was also found to be sufficient for CASK binding in vitro. TIAM1 is well known for its role in tumor metastasis, but it also possesses overlapping cellular and neurological functions with CASK, suggesting a previously unknown cooperation between the two proteins. This new peptide interaction motif also explains how Caskin1 and Mint1 form competing complexes and suggests a new role for the cellular hub protein CASK.

RAP-011 improves erythropoiesis in zebrafish model of Diamond-Blackfan anemia through antagonizing lefty1

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with β-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.