K-ras/PI3K-Akt Signaling Is Essential for Zebrafish Hematopoiesis and Angiogenesis

The RAS small GTPases orchestrate multiple cellular processes. Studies on knock-out mice showed the essential and sufficient role of K-RAS, but not N-RAS and H-RAS in embryonic development. However, many physiological functions of K-RAS in vivo remain unclear. Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and ße3-hemoglobin, reduced blood circulation and disorganized blood vessels. Expression of either K-rasC40 that links to phosphoinositide 3-kinase (PI3K) activation, or Akt2 that acts downstream of PI3K, could rescue both hematopoietic and angiogenic defects in the K-ras knockdown. Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor. Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases

A Cdo–Bnip-2–Cdc42 signaling pathway regulates p38α/β MAPK activity and myogenic differentiation

The p38α/β mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38α/β pathway scaffold protein JLP and, via JLP, p38α/β itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo–Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38α/β activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38α/β activation during cell differentiation

Crosstalk of Ras and Rho: activation of RhoA abates Kras-induced liver tumorigenesis in transgenic zebrafish models

RAS and Rho small GTPases are key molecular switches that control cell dynamics, cell growth and tissue development through their distinct signaling pathways. While much has been learnt about their individual functions in both cell and animal models, the physiological and pathophysiological consequences of their signaling crosstalk in multi-cellular context in vivo remain largely unknown, especially in liver development and liver tumorigenesis. Furthermore, the roles of RhoA in RAS-mediated transformation and their crosstalk in vitro remain highly controversial. When challenged with carcinogens, zebrafish developed liver cancer that resembles the human liver cancer both molecularly and histopathologically. Capitalizing on the growing importance and relevance of zebrafish (Danio rerio) as an alternate cancer model, we have generated liver-specific, Tet-on inducible transgenic lines expressing oncogenic Kras[superscript G12V], RhoA, constitutively-active RhoA[superscript G14V] or dominant-negative RhoA[superscript T19N]. Double transgenic lines expressing Kras[superscript G12V] with one of the three RhoA genes were also generated. Based on quantitative bioimaging and molecular markers for genetic and signaling aberrations, we showed that the induced expression of oncogenic Kras during early development led to liver enlargement and hepatocyte proliferation, associated with elevated Erk phosphorylation, Akt2-p21Cip expression and activation. Such an increase in liver size and Akt2 expression was augmented by dominant-negative RhoA[superscript T19N], but was abrogated by the constitutive-active RhoA[superscript G14V]. Consequently, induced expression of the oncogenic Kras in adult transgenic fish led to the development of hepatocellular carcinomas. Survival studies further revealed that the co-expression of dominant-negative RhoA[superscript T19N] with oncogenic Kras increased the mortality rate compared to the other single or double transgenic lines. This study represents the first in vivo investigation of the previously unappreciated signaling crosstalk between Kras and RhoA in regulating liver overgrowth and liver tumorigenesis. Our results also implicate that activating Rho could be beneficial to suppress the Kras-induced liver malignancies.Keywords: RhoA, Zebrafish, Signaling crosstalk, Akt, Ras, Hepatocellular carcinom

Nerve Growth Factor Stimulates Interaction of Cayman Ataxia Protein BNIP-H/Caytaxin with Peptidyl-Prolyl Isomerase Pin1 in Differentiating Neurons

Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation. (213 words

Cross-Species Analyses Identify the BNIP-2 and Cdc42GAP Homology (BCH) Domain as a Distinct Functional Subclass of the CRAL_TRIO/Sec14 Superfamily

The CRAL_TRIO protein domain, which is unique to the Sec14 protein superfamily, binds to a diverse set of small lipophilic ligands. Similar domains are found in a range of different proteins including neurofibromatosis type-1, a Ras GTPase-activating Protein (RasGAP) and Rho guanine nucleotide exchange factors (RhoGEFs). Proteins containing this structural protein domain exhibit a low sequence similarity and ligand specificity while maintaining an overall characteristic three-dimensional structure. We have previously demonstrated that the BNIP-2 and Cdc42GAP Homology (BCH) protein domain, which shares a low sequence homology with the CRAL_TRIO domain, can serve as a regulatory scaffold that binds to Rho, RhoGEFs and RhoGAPs to control various cell signalling processes. In this work, we investigate 175 BCH domain-containing proteins from a wide range of different organisms. A phylogenetic analysis with ∼100 CRAL_TRIO and similar domains from eight representative species indicates a clear distinction of BCH-containing proteins as a novel subclass within the CRAL_TRIO/Sec14 superfamily. BCH-containing proteins contain a hallmark sequence motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs (‘h’ is large and hydrophobic residue and ‘s’ is small and weekly polar residue) and can be further subdivided into three unique subtypes associated with BNIP-2-N, macro- and RhoGAP-type protein domains. A previously unknown group of genes encoding ‘BCH-only’ domains is also identified in plants and arthropod species. Based on an analysis of their gene-structure and their protein domain context we hypothesize that BCH domain-containing genes evolved through gene duplication, intron insertions and domain swapping events. Furthermore, we explore the point of divergence between BCH and CRAL-TRIO proteins in relation to their ability to bind small GTPases, GAPs and GEFs and lipid ligands. Our study suggests a need for a more extensive analysis of previously uncharacterized BCH, ‘BCH-like’ and CRAL_TRIO-containing proteins and their significance in regulating signaling events involving small GTPases

Classification of BNIP2-Cdc42GAP homology domain family

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A Cooperative jack model of random coil-to-elongation transition of the FH1 domain by profilin binding explains formin motor behavior in actin polymerization

Filopodia are essential for the development of neuronal growth cones, cell polarity and cell migration. Their protrusions are powered by the polymerization of actin filaments linked to the plasma membrane, catalyzed by formin proteins. The acceleration of polymerization depends on the number of profilin-actins binding with the formin-FH1 domain. Biophysical characterization of the disordered formin-FH1 domain remains a challenge. We analyzed the conformational distribution of the diaphanous-related formin mDia1-FH1 bound with one to six profilins. We found a coil-to-elongation transition in the FH1 domain. We propose a cooperative "jack" model for the Formin-Homology-1 (FH1) domain of formins stacked by profilin-actins.6 page(s

Enhanced tumor cell killing by ultrasound after microtubule depolymerization

10.1002/btm2.10233Bioengineering and Translational Medicine63e1023

Common and Unique Transcription Signatures of YAP and TAZ in Gastric Cancer Cells

YAP and its paralog TAZ are the nuclear effectors of the Hippo tumour-suppressor pathway, and function as transcriptional co-activators to control gene expression in response to mechanical cues. To identify both common and unique transcriptional targets of YAP and TAZ in gastric cancer cells, we carried out RNA-sequencing analysis of overexpressed YAP or TAZ in the corresponding paralogous gene-knockouts (KOs), TAZ KO or YAP KO, respectively. Gene Ontology (GO) analysis of the YAP/TAZ-transcriptional targets revealed activation of genes involved in platelet biology and lipoprotein particle formation as targets that are common for both YAP and TAZ. However, the GO terms for cell-substrate junction were a unique function of YAP. Further, we found that YAP was indispensable for the gastric cancer cells to re-establish cell-substrate junctions on a rigid surface following prolonged culture on a soft substrate. Collectively, our study not only identifies common and unique transcriptional signatures of YAP and TAZ in gastric cancer cells but also reveals a dominant role for YAP over TAZ in the control of cell-substrate adhesion