Search

Conventionally, Rho guanine nucleotide exchange factors (GEFs) are known activators of Rho guanosine triphosphatases (GTPases) that promote tumorigenesis. However, the role of Rho GEFs in non-small cell lung cancer (NSCLC) remains largely unknown. Through the screening of 81 Rho GEFs for their expression profiles and correlations with survival, four of them were identified with strong significance for predicting the prognosis of NSCLC patients. The four Rho GEFs, namely ABR, PREX1, DOCK2 and DOCK4, were downregulated in NSCLC tissues compared to normal tissues. The downregulation of ABR, PREX1, DOCK2 and DOCK4, which can be attributfed to promoter methylation, is correlated with poor prognosis. The underexpression of the four key Rho GEFs might be related to the upregulation of MYC signaling and DNA repair pathways, leading to carcinogenesis and poor prognosis. Moreover, overexpression of ABR was shown to have a tumor-suppressive effect in PC9 and H1703 cells. In conclusion, the data reveal the unprecedented role of ABR as tumor suppressor in NSCLC. The previously unnoticed functions of Rho GEFs in NSCLC will inspire researchers to investigate the distinct roles of Rho GEFs in cancers, in order to provide critical strategies in clinical practice.

Despite that androgen-deprivation therapy results in long-lasting responses, the disease inevitably progresses to metastatic castration-resistant prostate cancer. In this study, we identified miR-33b-3p as a tumor suppressor in prostate cancer. miR-33b-3p was significantly reduced in prostate cancer tissues, and the low expression of miR-33b-3p was correlated with poor overall survival of prostate cancer patients. Overexpression of miR-33b-3p inhibited both migration and invasion of highly metastatic prostate cancer cells whereas inhibition of miR-33b-3p promoted those processes in lowly metastatic cells. The in vivo results demonstrate that miR-33b-3p suppresses metastasis of tail vein inoculated prostate cancer cells to lung and lymph nodes in mice. DOCK4 was validated as the direct target of miR-33b-3p. miR-33b-3p decreased the expression of DOCK4 and restoration of DOCK4 could rescue miR-33b-3p inhibition on cell migration and invasion. Moreover, downregulation of miR-33b-3p was induced by bortezomib, the clinically used proteasome inhibitor, and overexpression of miR-33b-3p enhanced the insufficient inhibition of bortezomib on migration and invasion as well as metastasis of prostate cancer cells. In summary, our findings demonstrate that miR-33b-3p suppresses metastasis by targeting DOCK4 in prostate cancer. Our results suggest that enhancing miR-33b-3p expression may provide a promising therapeutic strategy for overcoming that proteasome inhibitor’s poor efficacy against metastatic prostate cancer.

Nasopharyngeal carcinoma (NPC) is a malignancy that is endemic to China and Southeast Asia. Radiotherapy is the usual treatment, however, radioresistance remains a major reason for failure. This study aimed to find key radioresistance regulation models and marker genes of NPC and clarify the mechanism of NPC radioresistance by RNA sequencing and bioinformatics analysis of the differences in gene expression profiles between radioresistant and radiosensitive NPC tissues. A total of 21 NPC biopsy specimens with different radiosensitivity were analyzed by RNA sequencing. Differentially expressed genes in RNA sequencing data were identified using R software. The differentially expressed gene data derived from RNA sequencing as well as prior knowledge in the form of pathway databases were integrated to find sub‐networks of related genes. The data of RNA sequencing with the GSE48501 data from the GEO database were combined to further search for more reliable genes associated with radioresistance of NPC. Survival analyses using the Kaplan–Meier method based on the expression of the genes were conducted to facilitate the understanding of the clinical significance of the differentially expressed genes. RT‐qPCR was performed to validate the expression levels of the differentially expressed genes. We identified 1182 differentially expressed genes between radioresistant and radiosensitive NPC tissue samples. Compared to the radiosensitive group, 22 genes were significantly upregulated and 1160 genes were downregulated in the radioresistant group. In addition, 10 major NPC radiation resistance network models were identified through integration analysis with known NPC radiation resistance‐associated genes and mechanisms. Furthermore, we identified three core genes, DOCK4, MCM9, and POPDC3 among 12 common downregulated genes in the two datasets, which were validated by RT‐qPCR. The findings of this study provide new clues for clarifying the mechanism of NPC radioresistance, and further experimental studies of these core genes are warranted., In this work, we identified several key radioresistance regulation models and marker genes of nasopharyngeal carcinoma (NPC) and partly demonstrated the mechanism of NPC radioresistance by RNA sequencing and bioinformatics analysis of the differences in gene expression profiles between radioresistant and radiosensitive NPC tissues.

Lung adenocarcinoma (LUAD) is a common malignancy; however, the majority of its underlying molecular mechanisms remain unknown. In the present study, weighted gene co-expression network analysis was applied to construct gene co-expression networks for the GSE19804 dataset, in order to screen hub genes associated with the pathogenesis of LUAD. In addition, with the aid of the Database for Annotation, Visualization and Integrated Discovery, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes, pathway enrichment analyses were performed on the genes in the selected module. Using the GSE40791 dataset and The Cancer Genome Atlas database, the hub genes were identified. It was discovered that the turquoise module was the most significant module associated with the tumor stage of LUAD. After performing functional enrichment analyses, it was indicated that the turquoise module was mainly enriched in signal transduction. Additionally, at the transcriptional and translational level, nine hub genes were identified and validated: Carbonic anhydrase 4 (CA4), platelet and endothelial cell adhesion molecule 1 (PECAM1), DnaJ member B4 (DNAJB4), advanced glycosylation end-product specific receptor (AGER), GTPase, IMAP family member 6 (GIMAP6), chromosome 10 open reading frame 54 (C10orf54), dedicator of cytokinesis 4 (DOCK4), Golgi membrane protein 1 (GOLM1) and platelet activating factor acetylhydrolase 1b catalytic subunit 3 (PAFAH1B3). CA4, PECAM1, DNAJB4, AGER, GIMAP6, C10orf54 and DOCK4 were expressed at lower levels in the tumor samples, whereas GOLM1 and PAFAH1B3 were highly expressed in tumor samples. In addition, all hub genes were associated with prognosis. In conclusion, one module and nine genes were recognized to be associated with the tumor stage of LUAD. These findings may enhance the understanding of the progression and prognosis of LUAD.

Genetic studies of autism spectrum disorder (ASD) have revealed multigene variations that converge on synaptic dysfunction. DOCK4, a gene at 7q31.1 that encodes the Rac1 guanine nucleotide exchange factor Dock4, has been identified as a risk gene for ASD and other neuropsychiatric disorders. However, whether and how Dock4 disruption leads to ASD features through a synaptic mechanism remain unexplored. We generated and characterized a line of Dock4 knockout (KO) mice, which intriguingly displayed a series of ASD-like behaviors, including impaired social novelty preference, abnormal isolation-induced pup vocalizations, elevated anxiety, and perturbed object and spatial learning. Mice with conditional deletion of Dock4 in hippocampal CA1 recapitulated social preference deficit in KO mice. Examination in CA1 pyramidal neurons revealed that excitatory synaptic transmission was drastically attenuated in KO mice, accompanied by decreased spine density and synaptic content of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)- and NMDA (N-methyl-D-aspartate)-type glutamate receptors. Moreover, Dock4 deficiency markedly reduced Rac1 activity in the hippocampus, which resulted in downregulation of global protein synthesis and diminished expression of AMPA and NMDA receptor subunits. Notably, Rac1 replenishment in the hippocampal CA1 of Dock4 KO mice restored excitatory synaptic transmission and corrected impaired social deficits in these mice, and pharmacological activation of NMDA receptors also restored social novelty preference in Dock4 KO mice. Together, our findings uncover a previously unrecognized Dock4-Rac1-dependent mechanism involved in regulating hippocampal excitatory synaptic transmission and social behavior.

Despite that androgen-deprivation therapy results in long-lasting responses, the disease inevitably progresses to metastatic castration-resistant prostate cancer. In this study, we identified miR-33b-3p as a tumor suppressor in prostate cancer. miR-33b-3p was significantly reduced in prostate cancer tissues, and the low expression of miR-33b-3p was correlated with poor overall survival of prostate cancer patients. Overexpression of miR-33b-3p inhibited both migration and invasion of highly metastatic prostate cancer cells whereas inhibition of miR-33b-3p promoted those processes in lowly metastatic cells. The in vivo results demonstrate that miR-33b-3p suppresses metastasis of tail vein inoculated prostate cancer cells to lung and lymph nodes in mice. DOCK4 was validated as the direct target of miR-33b-3p. miR-33b-3p decreased the expression of DOCK4 and restoration of DOCK4 could rescue miR-33b-3p inhibition on cell migration and invasion. Moreover, downregulation of miR-33b-3p was induced by bortezomib, the clinically used proteasome inhibitor, and overexpression of miR-33b-3p enhanced the insufficient inhibition of bortezomib on migration and invasion as well as metastasis of prostate cancer cells. In summary, our findings demonstrate that miR-33b-3p suppresses metastasis by targeting DOCK4 in prostate cancer. Our results suggest that enhancing miR-33b-3p expression may provide a promising therapeutic strategy for overcoming that proteasome inhibitor’s poor efficacy against metastatic prostate cancer.

Diabetic kidney disease (DKD) has become the leading cause of end-stage renal disease but the efficacy of current treatment remains unsatisfactory. The pathogenesis of DKD needs a more in-depth research. Ubiquitin specific proteases 36 (USP36), a member of deubiquitinating enzymes family, has aroused wide concerns for its role in deubiquitinating and stabilizing target proteins. Nevertheless, the role of USP36 in diabetes has never been reported yet. Herein, we identified an increased expression of USP36 both in vitro and in vivo in diabetic renal tubular epithelial cells (TECs), and its overexpression is related to the enhanced epithelial-to-mesenchymal transition (EMT). Further investigation into the mechanisms proved that USP36 could directly bind to and mediate the deubiquitination of dedicator of cytokinesis 4 (DOCK4), a guanine nucleotide exchange factor (GEF) that could activate Wnt/β-catenin signaling pathway and induce EMT. Our study revealed a new mechanism that USP36 participates in the pathogenesis of DKD, and provided potential intervening targets accordingly.

Significance The syndrome of cytotrophoblast invasion beyond the normal boundary (in the superficial myometrium) is collectively termed placenta accreta spectrum. The incidence of this condition is rising. However, little is known about the underlying molecular changes. Global transcriptomic profiling of cytotrophoblasts isolated from these cases, as compared to gestational age-matched controls, revealed numerous changes in gene expression involving diverse pathways, including cell signaling, migration, and immune functions. DOCK4 was the most highly up-regulated mRNA in the cases. Mutations in this gene are mechanistically linked to cancer progression. Overexpression of DOCK4 in primary cytotrophoblasts increased their invasiveness. This study provides molecular insights into the pathways driving placenta accreta spectrum and suggests numerous future directions., In humans, a subset of placental cytotrophoblasts (CTBs) invades the uterus and its vasculature, anchoring the pregnancy and ensuring adequate blood flow to the fetus. Appropriate depth is critical. Shallow invasion increases the risk of pregnancy complications, e.g., severe preeclampsia. Overly deep invasion, the hallmark of placenta accreta spectrum (PAS), increases the risk of preterm delivery, hemorrhage, and death. Previously a rare condition, the incidence of PAS has increased to 1:731 pregnancies, likely due to the rise in uterine surgeries (e.g., Cesarean sections). CTBs track along scars deep into the myometrium and beyond. Here we compared the global gene expression patterns of CTBs from PAS cases to gestational age-matched control cells that invaded to the normal depth from preterm birth (PTB) deliveries. The messenger RNA (mRNA) encoding the guanine nucleotide exchange factor, DOCK4, mutations of which promote cancer cell invasion and angiogenesis, was the most highly up-regulated molecule in PAS samples. Overexpression of DOCK4 increased CTB invasiveness, consistent with the PAS phenotype. Also, this analysis identified other genes with significantly altered expression in this disorder, potential biomarkers. These data suggest that CTBs from PAS cases up-regulate a cancer-like proinvasion mechanism, suggesting molecular as well as phenotypic similarities in the two pathologies.

Proliferation and migration of vascular smooth muscle cells (VSMCs) are implicated in blood vessel development, maintenance of vascular homeostasis, and pathogenesis of vascular disorders. MicroRNAs (miRNAs) mediate the regulation of VSMC functions in response to microenvironmental signals. Because a previous study reported that miR-101, a tumor-suppressive miRNA, is a critical regulator of cell proliferation in vascular disease, we hypothesized that miR-101 controls important cellular processes in VSMCs. The present study aimed to elucidate the effects of miR-101 on VSMC function and its molecular mechanisms. We revealed that miR-101 regulates VSMC proliferation and migration. We showed that miR-101 expression is induced by bone morphogenetic protein (BMP) signaling, and we identified dedicator of cytokinesis 4 (DOCK4) as a novel target of miR-101. Our results suggest that the BMP&ndash, miR-101&ndash, DOCK4 axis mediates the regulation of VSMC function. Our findings help further the understanding of vascular physiology and pathology.

Skeletal metastasis occurs in around 75% of advanced breast cancers, with the disease incurable once cancer cells disseminate to bone, but there remains an unmet need for biomarkers to identify patients at high risk of bone recurrence. This study aimed to identify such a biomarker and to assess its utility in predicting response to adjuvant zoledronic acid (zoledronate). We used quantitative proteomics (stable isotope labelling by amino acids in cell culture‐mass spectrometry; SILAC‐MS) to compare protein expression in a bone‐homing variant (BM1) of the human breast cancer cell line MDA‐MB‐231 with parental non‐bone‐homing cells to identify novel biomarkers for risk of subsequent bone metastasis in early breast cancer. SILAC‐MS showed that dedicator of cytokinesis protein 4 (DOCK4) was upregulated in bone‐homing BM1 cells, confirmed by western blotting. BM1 cells also had enhanced invasive ability compared with parental cells, which could be reduced by DOCK4‐shRNA. In a training tissue microarray (TMA) comprising 345 patients with early breast cancer, immunohistochemistry followed by Cox regression revealed that high DOCK4 expression correlated with histological grade (p = 0.004) but not oestrogen receptor status (p = 0.19) or lymph node involvement (p = 0.15). A clinical validation TMA used tissue samples and the clinical database from the large AZURE adjuvant study (n = 689). Adjusted Cox regression analyses showed that high DOCK4 expression in the control arm (no zoledronate) was significantly prognostic for first recurrence in bone (HR 2.13, 95%CI 1.06–4.30, p = 0.034). No corresponding association was found in patients who received zoledronate (HR 0.812, 95%CI 0.176–3.76, p = 0.790), suggesting that treatment with zoledronate may counteract the higher risk for bone relapse from high DOCK4‐expressing tumours. High DOCK4 expression was not associated with metastasis to non‐skeletal sites when these were assessed collectively. In conclusion, high DOCK4 in early breast cancer is significantly associated with aggressive disease and with future bone metastasis and is a potentially useful biomarker for subsequent bone metastasis risk. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Rho GTPases, including Rho, Cdc42, Rac and ROP subfamilies, are key signaling molecules in RNA polymerase II (Pol II) transcriptional control. Our prior work has shown that plant ROP and yeast Cdc42 GTPases similarly modulate Ser2 and Ser5 phosphorylation status of the C-terminal domain (CTD) of the Pol II largest subunit by regulating CTD phosphatase degradation. Here, we present genetic and pharmacological evidence showing that Cdc42 and Rac1 GTPase signaling modulates a similar CTD Ser2 and Ser5 phosphorylation code in cultured human cancer cells. While siRNA knockdown of Cdc42 and Rac1, respectively, in HeLa cells increased the level of CTD Ser phosphatases RPAP2 and FCP1, they both decreased the level of CTD kinases CDK7 and CDK13. In addition, the protein degradation inhibitor MG132 reversed the effect of THZ1, a CDK7 inhibitor which could decrease the cell number and amount of CDK7 and CDK13, accompanied by a reduction in the level of CTD Ser2 and Ser5 phosphorylation and DOCK4 and DOCK9 (the activators for Rac1 and Cdc42, respectively). Conversely, treatments of Torin1 or serum deprivation, both of which promote protein degradation, could enhance the effect of THZ1, indicating the involvement of protein degradation in controlling CDK7 and CDK13. Our results support an evolutionarily conserved signaling shortcut model linking Rho GTPases to Pol II transcription across three kingdoms, Fungi, Plantae and Animalia, and could lead to the development of a potential synthetic-lethal strategy in controlling cancer cell proliferation or death.

Autism spectrum disorder (ASD) and dyslexia are both neurodevelopmental disorders with high prevalence in children. Both disorders have strong genetic basis, and share similar social communication deficits co-occurring with impairments of reading or language. However, whether these two disorders share common genetic risks remain elusive. DOCK4 (dedicator for cytokinesis 4), a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1, is one of few genes that are associated with both ASD and dyslexia. Dock4 is important for neuronal development and social behaviors. Two DOCK4 variations, Exon27-52 deletion (protein product: Dock4-945VS) and a missense mutation at rs2074130 (protein product: Dock4-R853H), are associated with dyslexia and/or ASD with reading difficulties. The present study explores the molecular and cellular functions of these two DOCK4 variants on neuronal development, by comparing them with the wild-type Dock4 protein. Notably, it is revealed that both mutants of Dock4 showed decreased ability to activate not only Rac1, but also another small GTPase Rap1. Consistently, both mutants were dysfunctional for regulation of cell morphology and cytoskeleton. Using Neuro-2a cells and hippocampus neurons as models, we found that both mutants had compromised function in promoting neurite outgrowth and dendritic spine formation. Electrophysiological recordings further showed that R853H partially lost the ability to promote excitatory synaptic transmission, whereas 945VS totally lost the ability. Together, we identified R853 as a previously uncharacterized site for the regulation of the integrity of Dock4 function, and provides insights in understanding the common molecular pathophysiology of ASD and dyslexia.

Cancer complexity relies on the intracellular pleiotropy of oncogenes/tumor suppressors and in the strong interplay between tumors and micro- and macro-environments. Here we followed a reductionist approach, by analyzing the transcriptional adaptations induced by three oncogenes (RAS, MYC, and HDAC4) in an isogenic transformation process. Common pathways, in place of common genes became dysregulated. From our analysis it emerges that, during the process of transformation, tumor cells cultured in vitro prime some signaling pathways suitable for coping with the blood supply restriction, metabolic adaptations, infiltration of immune cells, and for acquiring the morphological plasticity needed during the metastatic phase. Finally, we identified two signatures of genes commonly regulated by the three oncogenes that successfully predict the outcome of patients affected by different cancer types. These results emphasize that, in spite of the heterogeneous mutational burden among different cancers and even within the same tumor, some common hubs do exist. Their location, at the intersection of the various signaling pathways, makes a therapeutic approach exploitable.

Breast cancer (BC) is the most prevalent malignant cancer in the world, is the leading cause of cancer-related death female. Recently, there is accumulating evidence that long noncoding RNAs (lncRNAs) might as an important role in the progression of BC. (epithelial-mesenchymal transition (EMT) is considered to play a vital role in tumor cells migration and invasion. Nevertheless, the entire biological mechanisms and functions of lncRNAs in tumor migration, invasion, and EMT remain uncertain. In the present research, we observed that the expression of lncRNA AC073284.4 was downregulated in BC paclitaxel-resistant (PR) cells (MCF-7/PR) and tissues. Bioinformatics analysis predicted that miR-18b-5p was a direct target of AC073284.4, which has been validated by dual-luciferase reporter gene assay. We further proved that AC073284.4 could directly bind to miR-18b-5p and relieve the suppression for dedicator of cytokinesis protein 4 (DOCK4). Furthermore, the underlying functional experiments demonstrated that AC073284.4 might sponge miR-18b-5p to attenuate the invasion, metastasis, and EMT of BC cell through upregulating DOCK4 expression. In summary, AC073284.4 might serve as a competing endogenous RNA (ceRNA) in BC progression via modulating miR-18b-5p/DOCK4 axis, which weakens EMT and migration of BC. These results suggesting that AC073284.4 might function as a potential novel diagnostic biomarker in the progression of BC.

Metastases are the primary cause of cancer-related deaths. The underlying molecular and biological mechanisms remain, however, elusive, thus preventing the design of specific therapies. In melanomas, the metastatic process is influenced by the acquisition of metastasis-associated mutational and epigenetic traits and the activation of metastatic-specific signaling pathways in the primary melanoma. In the current study, we investigated the role of an adaptor protein of the Shc family (ShcD) in the acquisition of metastatic properties by melanoma cells, exploiting our cohort of patient-derived xenografts (PDXs). We provide evidence that the depletion of ShcD expression increases a spread cell shape and the capability of melanoma cells to attach to the extracellular matrix while its overexpression switches their morphology from elongated to rounded on 3D matrices, enhances cells&rsquo, invasive phenotype, as observed on collagen gel, and favors metastasis formation in vivo. ShcD overexpression sustains amoeboid movement in melanoma cells, by suppressing the Rac1 signaling pathway through the confinement of DOCK4 in the cytoplasm. Inactivation of the ShcD signaling pathway makes melanoma cells more sensitive to therapeutic treatments. Consistently, ShcD expression predicts poor outcome in a cohort of 183 primary melanoma patients.

Background: Epidermal growth factor receptor (EGFR) is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumour types have different mutation spectra. It is possible that functional differences underlie this tumour-type specific mutation spectrum. Methods: We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. Results: Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximity ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the expression of a specific set of genes, and that each mutation is associated with specific phosphorylation patterns. Finally, we demonstrate using stably expressing cell lines that EGFRvIII and EGFL858R display reduced growth and migration compared to EGFR wildtype expressing cells. Conclusion: Our results indicate that there are distinct functional differences between different EGFR mutations. The functional differences between different mutations argue for the development of mutation specific targeted therapies. (C) 2015 Elsevier Ltd. All rights reserved.

International audience; Glioblastomas (GBM) are lethal primitive brain tumours characterized by a strong intra-tumour heterogeneity. We observed in GBM tissues the coexistence of functionally divergent micro-territories either enriched in more differentiated and non-mitotic cells or in mitotic undifferentiated OLIG2 positive cells while sharing similar genomic abnormalities. Understanding the formation of such functionally divergent micro-territories in glioblastomas (GBM) is essential to comprehend GBM biogenesis, plasticity and to develop therapies. Here we report an unexpected anti-proliferative role of beta-catenin in non-mitotic differentiated GBM cells. By cell type specific stimulation of miR-302, which directly represses cyclin D1 and stemness features, beta-catenin is capable to change its known proliferative function. Nuclear beta-catenin accumulation in non-mitotic cells is due to a feed forward mechanism between DOCK4 and beta-catenin, allowed by increased GSK3-beta activity. DOCK4 over expression suppresses selfrenewal and tumorigenicity of GBM stem-like cells. Accordingly in the frame of GBM median of survival, increased level of DOCK4 predicts improved patient survival.

Dock4 is a member of the Dock180 family of proteins that mediates cancer cell migration through activation of Rac. However, the regulatory mechanism of Dock4 remains unclear. In this study, we show that the C-terminal proline-rich region of Dock4 is essential for the Dock4 mediated promotion of cell migration in MDA-MB-231 breast cancer cells. We found that a phosphoinositide-binding protein SH3YL1 interacted with the C-terminal proline-rich region of Dock4. Interaction of SH3YL1 with Dock4 promoted Dock4-mediated Rac1 activation and cell migration. Mutations in the phosphoinositide-binding domain disrupted the ability of SH3YL1 to promote Dock4-mediated cell migration. In addition, depletion of SH3YL1 in MDA-MB-231 cells suppressed cell migration. Taken together, these results provide evidence for a novel and functionally important interaction between Dock4 and SH3YL1 to promote cancer cell migration by regulating Rac1 activity.

Objective: A study in a Caucasian population has identified two single-nucleotide polymorphisms (SNPs) in ZNF533, one in DOCK4, and two in IMMP2L, which were all significantly associated with autism. They are located in AUTS1 and AUTS5, which have been identified as autism susceptibility loci in several genome-wide screens. The present study aimed to investigate whether ZNF533, DOCK4, and IMMP2L genes are also associated with autism in a northeastern Chinese Han population. Methods: We performed a similar association study using families with three individuals (one autistic child and two unaffected parents). A family-based transmission disequilibrium test (TDT) was used to analyze the results. Results: There were significant associations between autism and the two SNPs of ZNF533 gene (rs11885327: χ 2=4.5200, P=0.0335; rs1964081: χ 2=4.2610, P=0.0390) and the SNP of DOCK4 gene (rs2217262: χ 2=5.3430, P=0.0208). Conclusions: Our data suggest that ZNF533 and DOCK4 genes are linked to a predisposition to autism in the northeastern Chinese Han population.

Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway.

This review summarises some of the knowledge we have about Crk and Grb2 family adaptor protein signalling in health and disease and outlines the current status and the challenges still remaining in the development of efficient and selective inhibitors of their protein - protein interactions. It also highlights briefly some recent successes and problems of inhibitors for proteins that functionally interact with Crk and Grb2 family adaptors, as well as opportunities, which may arise from combination therapies. Grb2 and Crk family adaptors regulate signalling pathways linked to human diseases. They are mainly composed of Src homology 2 (SH2) and Src homology 3 (SH3) domains, which serve as docking sites for signalling proteins, including various receptors, cytoplasmic kinases and GTPase regulators. Considerable insight into the biological functions and mechanisms of action of small SH2/SH3 domain adaptors has been gained in the last years from experimental approaches as diverse as targeted gene disruption and structural studies at the atomic level. This has already indicated several strategies to utilise SH2 and SH3 domain interaction inhibitors in human disease therapy. Additional molecular targets for Crk and Grb2 domain interaction blockers are expected to surface as further protein-protein interactions are discovered. Examples include newly found DOCK family proteins (DOCK3, DOCK4, and DOCK5) which are known or suspected effectors of Crk proteins and the interaction of Grb2 with the cell cycle regulator p27Kip1.

Dedicator of cytokinesis 4 (DOCK4) is a large, multi-domain signaling protein involved in modulating signaling networks in mammalian cells. Recent studies have highlighted its role across multiple tissue types in maintaining homeostasis. Deletion and/or mutations in the DOCK4 gene lead to malignant conditions and has been attributed to poor long-term prognosis. Within the hematopoietic compartment reduced expression of DOCK4 leads to dysplasia in the erythroid lineage (Sundaravel et al, PNAS, 2015). However, its functions during early stages of hematopoietic cell development have not been defined. Identification of signaling networks regulated by DOCK4 within the hematopoietic compartment will provide insights into developing strategies in overcoming functional deficits that arise due to reduced expression of DOCK4. In this study, using primary human hematopoietic stem/early progenitors (HSPCs), we identified the major downstream signaling networks regulated by DOCK4 and functional consequences of reduced expression of DOCK4. Global phospho-proteomic analysis was performed on primary human CD34+/CD90+ hematopoietic cells expressing normal levels or reduced levels of DOCK4. A similar set of experiments were also performed on cells after they have committed to the erythroid lineage. These initial studies, using 1D and 2D phospho immunoblot analysis, we showed that cells expressing reduced levels of DOCK4 exhibit increased global tyrosine phosphorylation compared to cells expressing normal levels of DOCK4 regardless of whether cells exposed to hematopoietic cytokines. Further analysis of CD34+/CD90+ cells using liquid chromatography coupled mass spectrometry (LC/MS/MS) enabled us to precisely identify multiple proteins that were differentially phosphorylated. Among the proteins that were hyper-phosphorylated, phosphatases, PTPN6 (SHP1), INPP5D (SHIP1) and LYN kinase exhibited the greatest increase in their levels of tyrosine phosphorylation. These results were further validated by immunoblot analysis using site-specific phospho-antibodies against each of the three proteins. Co-immunoprecipitation experiments using HSPCs showed that DOCK4 interacts directly with INPP5D but not with PTPN6 or LYN suggesting that INPP5D is part of the DOCK4 protein complex but not PTPN6 or LYN. We then abrogated DOCK4-INPP5D interaction by knockdown of DOCK4, which led to increased localization of phospho-INPP5D (Y1021) from the cytoplasmic compartment to the cell membrane in CD34+/CD90+ HSPCs. To determine the functional pathways impacted by reduced expression of DOCK4, we performed in-silico DAVID/GO pathway analysis using our phospho proteomic data and found that cell migration function as one of the highly enriched pathways affected, when DOCK4 levels were reduced in HSPCs. We confirmed these findings by examining the migratory abilities of HSPCs expressing low levels of DOCK4 by an in vitro transwell assay, which showed approximately 2-fold increase in HSPC migration in the cell population where DOCK4 was expressed at fifty percent level. In addition, these cells also exhibited increased cell spreading and filopodia formation. Finally, pharmacologic inhibition of enzyme activities/phosphorylation of INPP5D, LYN or PTPN6 decreased the migration of HSPCs. Based on these results; we conclude that DOCK4 is a critical signaling intermediate, which acts within the hematopoietic stem/early progenitor compartment to regulate primary human HSPCs migration by modulating downstream kinases and phosphatases. Therefore using pharmacological inhibitors of INPP5D, LYN or PTPN6 may provide an avenue to restore proper migratory patterns of HSPCs within the hematopoietic niche and potentially reverse differentiation block as observed in dysplasia. Disclosures No relevant conflicts of interest to declare.

Ephexin4 is a RhoG-specific guanine nucleotide exchange factor that interacts with the EphA2 receptor in breast cancer cells., EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.

Dendrite development is required for establishing proper neuronal connectivity. Rho-family small GTPases have been reported to play important roles in the regulation of dendritic growth and morphology. However, the molecular mechanisms that control the activities of Rho GTPases in developing dendrites are not well understood. In the present study we found Dock4, an activator of the small GTPase Rac, to have a role in regulating dendritic growth and branching in rat hippocampal neurons. Dock4 is highly expressed in the developing rat brain, predominantly in hippocampal neurons. In dissociated cultured hippocampal neurons, the expression of Dock4 protein is up-regulated after between 3 and 8 days in culture, when dendrites begin to grow. Knockdown of endogenous Dock4 results in reduced dendritic growth and branching. Conversely, overexpression of Dock4 with its binding partner ELMO2 enhances the numbers of dendrites and dendritic branches. These morphological effects elicited by Dock4 and ELMO2 require Rac activation and the C-terminal Crk-binding region of Dock4. Indeed, Dock4 forms a complex with ELMO2 and CrkII in hippocampal neurons. These findings demonstrate a new function of the Rac activator Dock4 in dendritic morphogenesis in hippocampal neurons. © 2008 Wiley-Liss, Inc.

DOCK4, a member of DOCK180 family proteins, was originally identified as a product of a gene deleted during tumor progression. Although its tumor suppression properties have been reported, the regulation mechanism of this protein has not been fully elucidated. DOCK4 shares two conserved domains called as DHR-1 and DHR-2 domain as other members including DOCK180. Although DHR-1 in DOCK180 is reported to bind to PIP(3), whether that of DOCK4 exhibits similar function has yet not been examined. In a search for novel PIP(3) binding proteins by the PIP(3) analog beads binding assay, we found that DOCK4 and its novel splicing variant, whose exon1 and exon52 are different from the known one, bind to PIP(3). Binding assay using deletion mutants of DOCK4 revealed that the binding region falls into the DHR-1 domain. These results raise the possibility that DOCK4 may be regulated by PIP(3) to exert its function.

Recognition of cognate Rho GTPases by guanine-nucleotide exchange factors (GEF) is fundamental to Rho GTPase signaling specificity. Two main GEF families use either the Dbl homology (DH) or the DOCK homology region 2 (DHR-2) catalytic domain. How DHR-2-containing GEFs distinguish between the GTPases Rac and Cdc42 is not known. To determine how these GEFs specifically recognize the two Rho GTPases, we studied the amino acid sequences in Rac2 and Cdc42 that are crucial for activation by DOCK2, a Rac-specific GEF, and DOCK9, a distantly related Cdc42-specific GEF. Two elements in the N-terminal regions of Rac2 and Cdc42 were found to be essential for specific interactions with DOCK2 and DOCK9. One element consists of divergent amino acid residues in the switch 1 regions of the GTPases. Significantly, these residues were also found to be important for GTPase recognition by Rac-specific DOCK180, DOCK3, and DOCK4 GEFs. These findings were unexpected because the same residues were shown previously to interact with GTPase effectors rather than GEFs. The other element comprises divergent residues in the beta3 strand that are known to mediate specific recognition by DH domain containing GEFs. Remarkably, Rac2-to-Cdc42 substitutions of four of these residues were sufficient for Rac2 to be specifically activated by DOCK9. Thus, DOCK2 and DOCK9 specifically recognize Rac2 and Cdc42 through their switch 1 as well as beta2-beta3 regions and the mode of recognition via switch 1 appears to be conserved among diverse Rac-specific DHR-2 GEFs.

Introduction: Invasion and growth of brain tumours require cooperation of the tumour cell and endothelial cell compartments. Limited efficacy of anti-angiogenic therapy in glioblastoma is associated with activation of VEGF independent mechanisms, and the development of hypoxia. Those can be overcome by targeting intracellular signaling pathways, and designing strategies for blood vessel normalization. Here we investigate the role of the Rac1 exchange factor Dock4, a regulator of the small GTPase Rac1, in cancer cell invasion and the microenvironment of glioblastoma. Materials and Methods: We assessed Dock4 expression in glioblastoma patient samples. The invasive potential of cancer cells with Dock4 knockdown was tested in spheroid assays in collagen matrix. To investigate the role of Dock4 in the microenvironment, the murine glioma cell line CT2A was injected intracranially into Dock4 heterozygous mice and wild type mice. Mice of each genotype were randomly allocated into two groups: control or treatment with radiation therapy, 2Gy daily x 5 days or 5 Gy daily x 3 days, delivered using an image guided small animal irradiation platform (SARRP). Results: We demonstrate that Dock4 expression in patient samples was associated with high nestin positivity. Knocking down Dock4 suppressed invasion of patient derived neurospheres in collagen. Heterozygous Dock4 genetic deletion in the microenvironment reduced blood vessel size, and reversed morphological abnormalities including the lining of blood vessels by cancer cells in syngeneic tumours. In this pilot study, tumours growing in Dock4 heterozygous mice showed improved response to radiation compared to tumours growing in wild type mice. Conclusions: These findings ­suggest that targeting the Dock4 pathway may provide a means of improving radiotherapy by normalizing the blood vessels, which may provide a therapeutic opportunity in parallel with inhibiting glioblastoma invasion.

DOCK180 is one of the two principal proteins bound to the SH3 domain of the adaptor protein CrkII. Here, we have studied the involvement of DOCK180 in integrin signaling. DOCK180 was neither phosphorylated nor bound to CrkII in quiescent NIH 3T3 cells and 3Y1 cells. We found that DOCK180 was phosphorylated and bound to CrkII in NIH 3T3 cells stimulated with integrin and also in 3Y1 cells transformed by v-src or v-crk. The binding of DOCK180 to CrkII correlated with the binding of CrkII to p130(Cas), which is a major CrkII SH2 domain-binding protein at focal adhesions. In a reconstitution experiment, expression of DOCK180 induced hyperphosphorylation of p130(Cas) and a concomitant increase in the amount of CrkII bound to p130(Cas). Similarly, binding of DOCK180 to CrkII was also enhanced by the coexpression of p130(Cas). Finally, we found that coexpression of p130(Cas) and CrkII with DOCK180 induced local membrane spreading and accumulation of DOCK180-CrkII-p130(Cas) complexes at focal adhesions. These findings suggest that DOCK180 positively regulates signaling from integrins to CrkII-p130(Cas) complexes at focal adhesions.

Acute myeloid leukemia with a normal karyotype (NK-AML) has been assigned to an intermediate prognostic risk group. However, there is a marked variability in outcome within this group of AML, suggesting a significant biological and molecular heterogeneity. Chromosomal abnormalities may be cryptic by metaphase cytogenetics, but still have an impact on the process of leukemogenesis and/or the clinical outcome of NK-AML. Therefore, we analyzed the genomic complement of NK-AML cases to search for the presence of submicroscopic genomic imbalances.We applied high-resolution oligo-based aCGH analysis to a cohort of AML patients with a normal karyotype, and FISH to validate the aCGH findings. Relative gene expression levels were examined by qPCR.High-resolution aCGH analysis of 21 NK-AML patients revealed one patient with a rare submicroscopic deletion at 7q31.1. This female patient exhibited a rapid disease progression and a dismal outcome. The deletion was mono-allelic, approximately 189,1 kb in size, and encompassed the major 3' part of the DOCK4 gene. The expression level of the DOCK4 gene in her leukemic cells was decreased when compared to CD34+ cells from healthy controls. When compared to AML patients with -7/del(7q) as the sole chromosomal anomaly, the DOCK4 expression level was found to be similarly low.This is the first report of an acquired partial deletion of the DOCK4 gene in a patient with de novo NK-AML. DOCK4 is a strong tumor suppressor candidate, required for GTPase activation in signal transduction pathways controlling cellular proliferation, adhesion, migration and phagocytosis. Our findings may be relevant for understanding of the process of leukemogenesis and the response to therapy in a subset of NK-AML patients.

Precise regulation of neurite growth and differentiation determines accurate formation of synaptic connections, whose disruptions are frequently associated with neurological disorders. Dedicator of cytokinesis 4 (Dock4), an atypical guanine nucleotide exchange factor for Rac1, is found to be associated with neuropsychiatric diseases, including autism and schizophrenia. Nonetheless, the neuronal function of Dock4 is only beginning to be understood. Using mouse neuroblastoma (Neuro-2a) cells as a model, this study identifies that Dock4 is critical for neurite differentiation and extension. This regulation is through activation of Rac1 and modulation of the dynamics of actin-enriched protrusions on the neurites. In cultured hippocampal neurons, Dock4 regulates the establishment of the axon-dendrite polarity and the arborization of dendrites, two critical processes during neural differentiation. Importantly, a microdeletion Dock4 mutant linked to autism and dyslexia that lacks the GEF domain leads to defective neurite outgrowth and neuronal polarization. Further analysis reveals that the SH3 domain-mediated interaction of Dock4 is required for its activity toward neurite differentiation, whereas its proline-rich C terminus is not essential for this regulation. Together, our findings reveal an important role of Dock4 for neurite differentiation during early neuronal development.

Rac GEF Dock4, recently reported as a candidate genetic risk factor for autism, dyslexia, and schizophrenia, is highly concentrated in dendritic spines in hippocampal neurons and is implicated in spine formation through interaction with the actin-binding protein cortactin., In neuronal development, dendritic spine formation is important for the establishment of excitatory synaptic connectivity and functional neural circuits. Developmental deficiency in spine formation results in multiple neuropsychiatric disorders. Dock4, a guanine nucleotide exchange factor (GEF) for Rac, has been reported as a candidate genetic risk factor for autism, dyslexia, and schizophrenia. We previously showed that Dock4 is expressed in hippocampal neurons. However, the functions of Dock4 in hippocampal neurons and the underlying molecular mechanisms are poorly understood. Here we show that Dock4 is highly concentrated in dendritic spines and implicated in spine formation via interaction with the actin-binding protein cortactin. In cultured neurons, short hairpin RNA (shRNA)–mediated knockdown of Dock4 reduces dendritic spine density, which is rescued by coexpression of shRNA-resistant wild-type Dock4 but not by a GEF-deficient mutant of Dock4 or a truncated mutant lacking the cortactin-binding region. On the other hand, knockdown of cortactin suppresses Dock4-mediated spine formation. Taken together, the results show a novel and functionally important interaction between Dock4 and cortactin for regulating dendritic spine formation via activation of Rac.

The Drosophila sponge (spg)/CG31048 gene belongs to the dedicator of cytokinesis (DOCK) family genes that are conserved in a wide variety of species. DOCK family members are known as DOCK1–DOCK11 in mammals. Although DOCK1 and DOCK2 involve neurite elongation and immunocyte differentiation, respectively, the functions of other DOCK family members are not fully understood. Spg is a Drosophila homolog of mammalian DOCK3 and DOCK4. Specific knockdown of spg by the GMR-GAL4 driver in eye imaginal discs induced abnormal eye morphology in adults. To mark the photoreceptor cells in eye imaginal discs, we used a set of enhancer trap strains that express lacZ in various sets of photoreceptor cells. Immunostaining with anti-Spg antibodies and anti-lacZ antibodies revealed that Spg is localized mainly in R7 photoreceptor cells. Knockdown of spg by the GMR-GAL4 driver reduced signals of R7 photoreceptor cells, suggesting involvement of Spg in R7 cell differentiation. Furthermore, immunostaining with anti-dpERK antibodies showed the level of activated ERK signal was reduced extensively by knockdown of spg in eye discs, and both the defects in eye morphology and dpERK signals were rescued by over-expression of the Drosophila raf gene, a component of the ERK signaling pathway. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rap1 in and around the plasma membrane of the eye disc cells. Together, these results indicate Spg positively regulates the ERK pathway that is required for R7 photoreceptor cell differentiation and the regulation is mediated by interaction with Rap1 during development of the compound eye.

CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Western blotting with CRK SH3 has demonstrated that it binds to 135- to 145-, 160-, and 180-kDa proteins. The 135- to 145-kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange protein. Here, we report on the molecular cloning of the 180-kDa protein, which is designated DOCK180 (180-kDa protein downstream of CRK). The isolated cDNA contains a 5,598-bp open reading frame encoding an 1,866-amino-acid protein. The deduced amino acid sequence did not reveal any significant homology to known proteins, except that an SH3 domain was identified at its amino terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulated diffusely in the cytoplasm and did not have any effect on cell morphology, farnesylated DOCK180 was localized on the cytoplasmic membrane and changed spindle 3T3 cells to flat, polygonal cells. These results suggest that DOCK180 is a new effector molecule which transduces signals from tyrosine kinases through the CRK adaptor protein.

Cell morphogenesis, which requires rearrangement of the actin cytoskeleton, is essential to coordinate the development of tissues such as the musculature and nervous system during normal embryonic development. One class of signaling proteins that regulate actin cytoskeletal rearrangement is the evolutionarily conserved CDM (C. elegans Ced-5, human DOCK180, Drosophila Myoblast city, or Mbc) family of proteins, which function as unconventional guanine nucleotide exchange factors for the small GTPase Rac. This CDM-Rac protein complex is sufficient for Rac activation, but is enhanced upon the association of CDM proteins with the ELMO/Ced-12 family of proteins. We identified and characterized the role of Drosophila Sponge (Spg), the vertebrate DOCK3/DOCK4 counterpart as an ELMO-interacting protein. Our analysis shows Spg mRNA and protein is expressed in the visceral musculature and developing nervous system, suggesting a role for Spg in later embryogenesis. As maternal null mutants of spg die early in development, we utilized genetic interaction analysis to uncover the role of Spg in central nervous system (CNS) development. Consistent with its role in ELMO-dependent pathways, we found genetic interactions with spg and elmo mutants exhibited aberrant axonal defects. In addition, our data suggests Ncad may be responsible for recruiting Spg to the membrane, possibly in CNS development. Our findings not only characterize the role of a new DOCK family member, but help to further understand the role of signaling downstream of N-cadherin in neuronal development.

Background Autism spectrum disorders (ASDs) are characterized by social, communication, and behavioral deficits and complex genetic etiology. A recent study of 517 ASD families implicated DOCK4 by single nucleotide polymorphism (SNP) association and a microdeletion in an affected sibling pair. Methods The DOCK4 microdeletion on 7q31.1 was further characterized in this family using QuantiSNP analysis of 1M SNP array data and reverse transcription polymerase chain reaction. Extended family members were tested by polymerase chain reaction amplification of junction fragments. DOCK4 dosage was measured in additional samples using SNP arrays. Since QuantiSNP analysis identified a novel CNTNAP5 microdeletion in the same affected sibling pair, this gene was sequenced in 143 additional ASD families. Further polymerase chain reaction-restriction fragment length polymorphism analysis included 380 ASD cases and suitable control subjects. Results The maternally inherited microdeletion encompassed chr7:110,663,978-111,257,682 and led to a DOCK4-IMMP2L fusion transcript. It was also detected in five extended family members with no ASD. However, six of nine individuals with this microdeletion had poor reading ability, which prompted us to screen 606 other dyslexia cases. This led to the identification of a second DOCK4 microdeletion co-segregating with dyslexia. Assessment of genomic background in the original ASD family detected a paternal 2q14.3 microdeletion disrupting CNTNAP5 that was also transmitted to both affected siblings. Analysis of other ASD cohorts revealed four additional rare missense changes in CNTNAP5. No exonic deletions of DOCK4 or CNTNAP5 were seen in 2091 control subjects. Conclusions This study highlights two new risk factors for ASD and dyslexia and demonstrates the importance of performing a high-resolution assessment of genomic background, even after detection of a rare and likely damaging microdeletion using a targeted approach.

Background: Autism spectrum disorders (ASDs) are characterized by social, communication, and behavioral deficits and complex genetic etiology. A recent study of 517 ASD families implicated DOCK4 by single nucleotide polymorphism (SNP) association and a microdeletion in an affected sibling pair. Methods: The DOCK4 microdeletion on 7q31.1 was further characterized in this family using QuantiSNP analysis of 1M SNP array data and reverse transcription polymerase chain reaction. Extended family members were tested by polymerase chain reaction amplification of junction fragments. DOCK4 dosage was measured in additional samples using SNP arrays. Since QuantiSNP analysis identified a novel CNTNAP5 microdeletion in the same affected sibling pair, this gene was sequenced in 143 additional ASD families. Further polymerase chain reaction-restriction fragment length polymorphism analysis included 380 ASD cases and suitable control subjects. Results: The maternally inherited microdeletion encompassed chr7:110,663,978-111,257,682 and led to a DOCK4-IMMP2L fusion transcript. It was also detected in five extended family members with no ASD. However, six of nine individuals with this microdeletion had poor reading ability, which prompted us to screen 606 other dyslexia cases. This led to the identification of a second DOCK4 microdeletion co-segregating with dyslexia. Assessment of genomic background in the original ASD family detected a paternal 2q14.3 microdeletion disrupting CNTNAP5 that was also transmitted to both affected siblings. Analysis of other ASD cohorts revealed four additional rare missense changes in CNTNAP5. No exonic deletions of DOCK4 or CNTNAP5 were seen in 2091 control subjects. Conclusions: This study highlights two new risk factors for ASD and dyslexia and demonstrates the importance of performing a highresolution assessment of genomic background, even after detection of a rare and likely damaging microdeletion using a targeted approach.

Breast cancer is the most common cancer among women in developed countries. The prevalence of the disease is increasing in the world. Its annual incidence among Iranian women is about 7000 cases. RAP1A, a tumor suppressor gene, is located at 1p13.3 and plays an important role in the cellular adhesion pathway and is involved in the pathogenesis of breast cancer. The DOCK4 gene, which is located at 7q31.1, specifically activates RAP1A gene. In the present study, DNA samples from 64 cases of sporadic breast tumors (referred to Mehrad Hospital in Tehran) were screened using PCR-SSCP method and the number of observed variations compared with the control group (100 normal women). Mutation detection for coding exons of RAP1A gene and the 500 bp upstream of transcription initiation site as promoters of both DOCK4 and RAP1A were carried out and compared with the control group. The promoter region of DOCK4 showed a heterozygous mutation with G>A transition at nucleotide −303 in a fibroadenoma case. With regard to RAP1A we found a heterozygous mutation, G>A transition in an adenoid cystic carcinoma case, and another heterozygous mutation, G>T transversion in an intraductal papilloma case both at nucleotide +45. A homozygous variation, T>A transversion was also found at nucleotide +29 of a fibroadenoma case. The differences in the frequency of variations mentioned above were not statistically significant. However Fisher's exact showed significant difference for T>A transversion. Although, the higher frequency of these mutations and variations may be related to the disease, a larger sample size is needed for the confirmation of our findings.

The canonical Wnt/beta-catenin pathway is a highly conserved signaling cascade that is involved in development and stem cell renewal. The deregulation of this pathway is often associated with increased cell growth and neoplasia. The small GTPase Rac has been shown to influence canonical Wnt signaling by regulating beta-catenin stability through an unknown mechanism. We report that DOCK4, a guanine nucleotide exchange factor (GEF) for Rac and a member of the CDM family of unconventional GEFs, mediates Wnt-induced Rac activation in the canonical Wnt/beta-catenin pathway. DOCK4 expression regulates cellular beta-catenin levels in response to the Wnt signal, in vitro. Biochemical studies demonstrate that DOCK4 interacts with the beta-catenin degradation complex, consisting of the proteins adenomatosis polyposis coli, Axin and glycogen synthase kinase 3beta (GSK3beta). This molecular interaction enhances beta-catenin stability and Axin degradation. Furthermore, we observe that DOCK4 is phosphorylated by GSK3beta, which enhances Wnt-induced Rac activation. Using a T-cell factor reporter zebrafish we confirm that DOCK4 is required for Wnt/beta-catenin activity, in vivo. These results elucidate a novel intracellular signaling mechanism in which a Rac GEF, DOCK4 acts as a scaffold protein in the Wnt/beta-catenin pathway.

INTRODUCTION: Development of brain metastatic lesions requires activation of signaling pathways for cancer cell invasion. Metastatic disease is intrinsically linked to endothelial cell activation for brain colonization and angiogenic growth for disease progression. DOCK180 family of Rho guanine nucleotide exchange factors (GEFs) are implicated in invasion, and recent studies show they control blood vessel development. The study aims to elucidate the role of the Rac GEF DOCK4 in breast cancer brain metastases. METHOD: i) Patient samples were analysed for DOCK4 expression levels which were related to histological parameters in primary tumours, and expression levels in brain metastases, ii) the role of DOCK4 in the brain vasculature was studied in heterozygous Dock4 null mice and mouse model of breast cancer brain metastasis, iii) the DOCK4 signalling pathway was delineated using biochemical assays and proteomics approaches. RESULTS: i) Significant correlation was observed between DOCK4 expression and histological grade in primary breast tumours, independent of ER and LN status. There was a trend towards reduced survival in cases with moderate/ strong DOCK4 expression. Moderate/ strong DOCK4 expression was observed in brain metastases specimens. ii) There was significant decrease in brain blood vessel lumen size in Dock4 + /- mice and number of blood vessels with dilated lumens in EO771 intracranial tumours in Dock4 + /- mice. iii) DOCK4 interacts with the Cdc42 GEF DOCK9 and RhoG effector ELMO to control filopodia formation and blood vessel lumen morphogenesis. CONCLUSION: DOCK4 and interaction partners are potential targets and biomarkers in brain metastases through regulation of cancer cell invasion and angiogenesis.

Cell migration is essential for normal development and many pathological processes including tumor metastasis. Rho family GTPases play important roles in this event. In particular, Rac is required for lamellipodia formation at the leading edge during migration. Dock4 is a member of the Dock180 family proteins, and Dock4 mutations are present in a subset of human cancer cell lines. However, the function and the regulatory mechanism of Dock4 remain unclear. Here we show that Dock4 is regulated by the small GTPase RhoG and its effector ELMO and promotes cell migration by activating Rac1. Dock4 formed a complex with ELMO, and expression of active RhoG induced translocation of the Dock4-ELMO complex from the cytoplasm to the plasma membrane and enhanced the Dock4- and ELMO-dependent Rac1 activation and cell migration. On the other hand, RNA interference-mediated knockdown of Dock4 in NIH3T3 cells reduced cell migration. Taken together, these results suggest that Dock4 plays an important role in the regulation of cell migration through activation of Rac1, and that RhoG is a key upstream regulator for Dock4.

The Rho family of small GTPases are important regulators of multiple cellular activities and, most notably, reorganization of the actin cytoskeleton. Dbl-homology (DH)-domain-containing proteins are the classical guanine nucleotide exchange factors (GEFs) responsible for activation of Rho GTPases. However, members of a newly discovered family can also act as Rho-GEFs. These CZH proteins include: CDM (Ced-5, Dock180 and Myoblast city) proteins, which activate Rac; and zizimin proteins, which activate Cdc42. The family contains 11 mammalian proteins and has members in many other eukaryotes. The GEF activity is carried out by a novel, DH-unrelated domain named the DOCKER, CZH2 or DHR2 domain. CZH proteins have been implicated in cell migration, phagocytosis of apoptotic cells, T-cell activation and neurite outgrowth, and probably arose relatively early in eukaryotic evolution.

The driving forces for the regulation of cell morphology are the Rho family GTPases that coordinate the assembly of the actin cytoskeleton. This dynamic feature is a result of tight coupling between the cytoskeleton and signal transduction and is facilitated by actin-binding proteins (ABPs). Mutations in the actin bundling and PDZ domain-containing protein harmonin are the causes of Usher syndrome type 1C (USH1C), a syndrome of congenital deafness and progressive blindness, as well as certain forms of non-syndromic deafness. Here, we have used the yeast two-hybrid assay to isolate molecular partners of harmonin and identified DOCK4, an unconventional guanine exchange factor for the Rho family of guanosine triphosphatases (Rho GEF GTPases), as a protein interacting with harmonin. Detailed molecular analysis revealed that a novel DOCK4 isoform (DOCK4-Ex49) is expressed in the brain, eye and inner ear tissues. We have further provided evidence that the DOCK4-Ex49 binds to nucleotide free Rac as effectively as DOCK2 and DOCK4 and it is a potent Rac activator. By immunostaining using a peptide antibody specific to DOCK4-Ex49, we showed its localization in the inner ear within the hair bundles along the stereocilia (SC). Together, our data indicate a possible Rac-DOCK4-ABP harmonin-activated signaling pathway in regulating actin cytoskeleton organization in stereocilia.

The modifier of cell adhesion protein (MOCA), or Dock3, initially identified as presenilin-binding protein (PBP), belongs to the Dock180 family of proteins and is localized specifically in neurons. Here we demonstrate that MOCA binds to Rac1 and enhances its activity, which leads to the activation of c-Jun NH2-terminal kinase (JNK) and causes changes in cell morphology. Farnesylated MOCA, which is localized in the plasma membrane, enhances the activation of Rac1 and JNK more markedly than wild-type MOCA, and cells expressing farnesylated MOCA show flattened morphology similar to those expressing a constitutive active mutant of Rac1, Rac1Q61L. On poly-d-lysine-coated dishes, endogenous MOCA is concentrated on the leading edge of broad membrane protrusions (lamellipodia) where actin filaments are co-localized. MOCA is also concentrated with actin on the growth cone in primary cultures of cortical neurons. These observations suggest that MOCA may induce cytoskeletal reorganization and changes in cell adhesion by regulating the activity of Rac1.

The small GTPase Rac has a central role in regulating the actin cytoskeleton during cell migration and axon guidance. Elmo has been identified as an upstream regulator of Rac1 that binds to and functionally cooperates with Dock180 (refs 2-4). Dock180 does not contain a conventional catalytic domain for guanine nucleotide exchange on Rac, but possesses a domain that directly binds to and specifically activates Rac1 (refs 5, 6). The small GTPase RhoG mediates several cellular morphological processes, such as neurite outgrowth in neuronal cells, through a signalling cascade that activates Rac1 (refs 7-12); however, the downstream target of RhoG and the mechanism by which RhoG regulates Rac1 activity remain unclear. Here we show that RhoG interacts directly with Elmo in a GTP-dependent manner and forms a ternary complex with Dock180 to induce activation of Rac1. The RhoG-Elmo-Dock180 pathway is required for activation of Rac1 and cell spreading mediated by integrin, as well as for neurite outgrowth induced by nerve growth factor. We conclude that RhoG activates Rac1 through Elmo and Dock180 to control cell morphology.

The Rho family GTPases Rac, Rho and Cdc42 are critical in regulating the actin-based cytoskeleton, cell migration, growth, survival and gene expression. These GTPases are activated by guanine nucleotide-exchange factors (GEFs). A biochemical search for Cdc42 activators led to the cloning of zizimin1, a new protein whose overexpression induces Cdc42 activation. Sequence comparison combined with mutational analysis identified a new domain, which we named CZH2, that mediates direct interaction with Cdc42. CZH2-containing proteins constitute a new superfamily that includes the so-called 'CDM' proteins that bind to and activate Rac. Together, the results suggest that CZH2 is a new GEF domain for the Rho family of proteins.

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac.

BACKGROUND: EGFR is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumor types have different mutation spectra. It is possible that functional differences underlie this tumor-type specific mutation spectrum. METHODS: We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. RESULTS: Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximilty ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the expression of a specific set of genes, and that each mutation is associated with specific phosphorylation patterns. Finally, we demonstrate using stably expressing cell lines that EGFRvIII and EGFL858R display reduced growth and migration compared to EGFR wildtype expressing cells. CONCLUSION: Our results indicate that there are distinct functional differences between different EGFR mutations. The functional differences between different mutations argue for the development of mutation specific targeted therapies.

The Rho family of low-molecular-weight G proteins, which consists of the Rho, Rac, and Cdc42 subfamilies, regulates the actin cytoskeleton (Hall 1998). Microinjection of constitutive active forms of these proteins into serum-starved fibroblasts demonstrated that Rho stimulates the organization of actin stress fibers, Rac stimulates the formation of lamellipodia or membrane ruffling, and Cdc42 induces the formation of filopodia (Nobes and Hall 1995). Like other G proteins, the Rho-family proteins are activated by guanine nucleotide exchange proteins (GEPs) and inactivated by GTPase-activating proteins (GAPs) (Lamarche and Hall 1994; Nobes and Hall 1994). The catalytic domains of GEPs for Rho share amino-acid sequences with the Dbl-oncogene product; thus, this domain is designated as the Dbl-homology domain (Cerione and Zheng 1996). It has been also reported that PIP2 and lipid kinases bind to and activate Rho family members (Tolias et al. 1995, 1998; Zheng et al. 1996). A third group of Rho regulator is RhoGDI, a GDP-dissociation inhibitor of Rho (Sasaki and Takai 1998). RhoGDI binds to GDP-bound Rho proteins and retains them in the cytoplasm in an inactive form (Sasaki et al. 1993). CrkII is a cellular homolog of the v-Crk oncogene product and consists almost entirely of SH2 and SH3 domains (Matsuda et al. 1992). Two major CrkII SH2-binding proteins are paxillin and p130Cas, both of which are components of focal adhesions (for review, see Kiyokawa et al. 1997). More recently, it has been shown that CrkII–p130Cas complexes regulate integrin-mediated cell movement and spreading (Vuori et al. 1996; Klemke et al. 1998). The amino-terminal SH3 domain of CrkII binds to several proteins; among the most prominent are C3G and DOCK180 (Matsuda and Kurata 1996). Homologs of DOCK180 have recently been isolated from Drosophila melanogaster and Caenorhabditis elegans and designated as mbc and ced-5, respectively (Erickson et al. 1997; Wu and Horvitz 1998). ced-5 mutants are defective in the engulfment of cell corpses and the migration of the gonadal distal tip cells. The latter phenotype was rescued by the expression of human DOCK180. mbc was originally isolated as a gene that was essential for muscle development, suggesting that mbc is also involved in cellular motility. Human DOCK180 also appears to be involved in cytoskeletal reorganization. Expression of membrane-targeted DOCK180 induced spreading of NIH-3T3 cells (Hasegawa et al. 1996). DOCK180 forms a complex with CrkII and p130Cas only after integrin stimulation of NIH-3T3 cells. Expression of DOCK180 with CrkII and p130Cas induces cell spreading and accumulation of DOCK180 at focal adhesions (Kiyokawa et al. 1998). However, the biochemical function of DOCK180 is unknown. Here, we demonstrate that DOCK180 activates and associates with the Rac1 GTPase and that DOCK180 requires Rac1 for the induction of cell spreading in NIH-3T3 cells.

The Rac and Cdc42 GTPases share several regulators and effectors, yet perform distinct biological functions. The factors determining such specificity in vivo have not been identified. In a mutational screen inDrosophila to identify Rac-specific signaling components, we isolated 11 alleles of myoblast city (mbc). mbcmutant embryos exhibit defects in dorsal closure, myogenesis, and neural development. DOCK180, the mammalian homolog of Mbc, associates with Rac, but not Cdc42, in a nucleotide-independent manner. These results suggest that Mbc is a specific upstream regulator of Rac activity that mediates several morphogenetic processes inDrosophila embryogenesis.