DOCK Family Research Articles

Pathogenesis of DOCK family protein gene variations in immunodeficiency diseases

Pathogenesis of DOCK family protein gene variations in immunodeficiency diseases

Actin Up: An Overview of the Rac GEF Dock1/Dock180 and Its Role in Cytoskeleton Rearrangement.

Dock1, originally Dock180, was the first identified member of the Dock family of GTPase Exchange Factors. Early biochemical and genetic studies of Dock180 elucidated the functions and regulation of Dock180 and informed our understanding of all Dock family members. Dock180 activates Rac to stimulate actin polymerization in response to signals initiated by a variety of receptors. Dock180 dependent Rac activation is essential for processes such as apoptotic cell engulfment, myoblast fusion, and cell migration during development and homeostasis. Inappropriate Dock180 activity has been implicated in cancer invasion and metastasis and in the uptake of bacterial pathogens. Here, we give an overview of the history and current understanding of the activity, regulation, and impacts of Dock180.

Sponge/DOCK-dependent regulation of F-actin networks directing cortical cap behaviors and syncytial furrow ingression

In the early syncytial Drosophila embryo, rapid changes in filamentous actin networks and membrane trafficking pathways drive the formation and remodeling of cortical and furrow morphologies. Interestingly, genomic integrity and the completion of mitoses during cell cycles 10–13 depends on the formation of transient membrane furrows that serve to separate and anchor individual spindles during division. While substantial work has led to a better understanding of the core network components that are responsible for the formation of these furrows, less is known about the regulation that controls cytoskeletal and trafficking function. The DOCK protein Sponge was one of the first proteins identified as being required for syncytial furrow formation, and disruption of Sponge deeply compromises F-actin populations in the early embryo, but how this occurs is less clear. Here, we perform quantitative analysis of the effects of Sponge disruption on cortical cap growth, furrow formation, membrane trafficking, and cytoskeletal network regulation through live-imaging of the syncytial embryo. We find that membrane trafficking is relatively unaffected by the defects in branched actin networks that occur after Sponge disruption, but that Sponge acts as a master regulator of a diverse cohort of Arp2/3 regulatory proteins. As DOCK family proteins have been implicated in regulating GTP exchange on small GTPases, we also suggest that Rac GTPase activity bridges Sponge regulation to the regulators of Arp2/3 function. Finally, we demonstrate the phasic requirements for branched F-actin and linear F-actin networks in potentiating furrow ingression. In total, these results provide quantitative insights into how a large DOCK scaffolding protein coordinates the activity of a variety of different actin regulatory proteins to direct the remodeling of the apical cortex into cytokinetic-like furrows.

Dock8 deficiency attenuates microglia colonization in early zebrafish larvae

Microglia are tissue-resident macrophages that carry out immune functions in the brain. The deficiency or dysfunction of microglia has been implicated in many neurodegenerative disorders. DOCK8, a member of the DOCK family, functions as a guanine nucleotide exchange factor and plays key roles in immune regulation and neurological diseases. The functions of DOCK8 in microglia development are not fully understood. Here, we generated zebrafish dock8 mutants by CRISPR/Cas9 genome editing and showed that dock8 mutations attenuate microglia colonization in the zebrafish midbrain at early larvae stages. In vivo time-lapse imaging revealed that the motility of macrophages was reduced in the dock8 mutant. We further found that cdc42/cdc42l, which encode the small GTPase activated by Dock8, also regulate microglia colonization in zebrafish. Collectively, our study suggests that the Dock8-Cdc42 pathway is required for microglia colonization in zebrafish larvae.

DOCK4 as a Potential Biomarker Associated with Immune Infiltration in Stomach Adenocarcinoma: A Database Analysis.

PurposeThe involvement of dedicator for cytokinesis 4 (DOCK4), a guanine nucleotide exchange factor for Rac1, in immune infiltration in stomach adenocarcinoma (STAD) remains unclear.MethodsThe UALCAN database was used to analyze the expression of the DOCK family. The Kaplan–Meier method and Gene Expression Profiling Interactive Analysis (GEPIA) databases were used to assess the prognostic value of the DOCK family in STAD. Furthermore, the correlation between expression of DOCK4 as well as other immune-related marker genes and tumor immune infiltration in STAD was explored using the TIMER and GEPIA websites. Subsequently, the relationship between DOCK4 expression and clinical characteristics was verified using the UALCAN database. Finally, DOCK4 mutation was analyzed via the TIMER2.0 and cBioPortal databases and the DOCK4 protein-protein interaction networks were constructed using the GeneMANIA and STRING websites.ResultsDOCK4 was found to be a new prognostic biomarker in STAD. DOCK4 expression in tumors was thoroughly evaluated relative to paracancerous tissues; overexpression of DOCK4 had a negative impact on the prognosis of patients with STAD. DOCK4 was found to be significantly associated with tumor immune infiltration in STAD.ConclusionIn summary, DOCK4 is a potential regulator of the recruitment and regulation of immune-infiltrating cells, thus serving as a valuable prognostic biomarker in STAD.

Abstract 139: Dock7 regulates the AKT/mTOR pathway to promote survival and sustain the transformative properties of cancer cells

Abstract Akt is a well-known target of mitogenic signaling, which is activated by phosphoinositide 3-kinase, PDK1 and mTORC2, and is commonly regarded as a master regulator of cell survival. However, there is still a good deal to learn regarding how Akt can promote survival when cancer cells are faced by the multitude of challenges they need to overcome during the process of malignant transformation. While investigating the role of Dock7, a Cdc42 and Rac guanine nucleotide exchange factor of the Dock180 family, in tumorigenesis, we discovered an unexpected connection between Dock7, Akt and cell survival. Specifically, we found that Dock7 is necessary for cancer cells to survive under the stressful conditions that arise due to the loss of a substratum or serum deprivation, through its ability to stabilize a low, basal level of Akt kinase activity. Dock7 binds to Akt, as well as associates with the known Akt substrate, TSC2, promoting its phosphorylation. Since the phosphorylation of TSC2 eliminates its ability to work with its partner TSC1 to deactivate the Rheb GTPase, a direct activator of mTORC1, we probed whether mTORC1 function is also necessary for Dock7-dependent cancer cell survival. Indeed, we determined that Dock7, as well as its signaling partner Cdc42, is required to maintain a functional level of S6 kinase activity, a downstream target of mTORC1, which was prevented by the mTORC1 inhibitor rapamycin. We further show an association between mTOR, Rheb and Dock7, but not Raptor, the defining component of mTORC1. Additionally, Raptor is dispensable for the Dock7-dependent S6 kinase activity, despite its rapamycin sensitivity. Together, these findings elucidate a novel Dock7-Akt-mTOR signaling node which promotes cancer cell survival in the absence of classical mTORC1 function to allow cells to overcome stresses faced during the development of cancer. Citation Format: Oriana Y. Teran, Miao-chong J. Lin, Matthew R. Zanotelli, Kristin F. Wilson, Richard A. Cerione. Dock7 regulates the AKT/mTOR pathway to promote survival and sustain the transformative properties of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 139.

Antimicrobial Properties of a Peptide Derived from the Male Fertility Factor kl2 Protein of Drosophila melanogaster.

Antimicrobial peptides (AMPs) are important components of innate immunity. Here, we report the antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin-p4 peptide. The antimicrobial activity of multifunctional chemerin is mainly associated with a domain localized in the middle of the chemerin sequence, Val66-Pro85 peptide (chemerin-p4). Using bioinformatic tools, we found homologs of the chemerin-p4 peptide in the proteome of D. melanogaster. One of them is MFF-p1, which is a part of the MFF kl2 protein, encoded by the gene male fertility factor kl2 (kl-2) located on the long arm of the Y chromosome. The second detected peptide (Z-p1) is a part of the Zizimin protein belonging to DOCK family, which is involved in cellular signaling processes. After testing the antimicrobial properties of both peptides, we found that only MFF-p1 possesses these properties. Here, we demonstrate its antimicrobial potential both in vitro and in vivo after infecting D. melanogaster with bacteria. MFF-p1 strongly inhibits the viable counts of E. coli and B. subtilis after 2 h of treatment and disrupts bacterial cells. The expression of kl-2 is regulated by exposure to bacteria and by the circadian clock.

DOCK4 stimulates MUC2 production through its effect on goblet cell differentiation.

The intestinal mucosa is in continuous contact with milliard of microorganisms, thus intestinal epithelial barrier is a critical component in the arsenal of defense mechanisms required to prevent infection and inflammation. Mucin 2 (MUC2), which is produced by the goblet cells, forms the skeleton of the intestinal mucus and protects the intestinal tract from self-digestion and numerous microorganisms. Dedicator of cytokinesis 4 (DOCK4) is a member of the DOCK-B subfamily of the DOCK family of guanine nucleotide exchange factors. It is reported that DOCK4 plays a critical role in the repair of the barrier function of the intestinal epithelium after chemical damage. In this study, the role of DOCK4 in the goblet cell differentiation and MUC2 production is explored. Disordered intestinal epithelium and shortage of goblet cells were observed in DOCK4 gene knockout mice. Furthermore, DOCK4 deletion contributed to the low expression of MUC2 and the goblet cell differentiation/maturation factors including growth factor independent 1 (Gfi1) and SAM pointed domain epithelial-specific transcription factor (Spdef) in mouse ileums and colons. Overexpression of DOCK4 caused a marked increase in Gfi1, Spdef, and MUC2, while siRNA knockdown of endogenous DOCK4 significantly decreased Gfi1, Spdef, and MUC2 in HT-29 cells. In addition, MUC2, DOCK4, and the goblet cell differentiation/maturation factors mRNA levels were decreased in colorectal cancer samples compared with normal colons. A significant positive correlation was found between MUC2 and DOCK4. In conclusion, DOCK4 may serve as a critical regulator of goblet cell differentiation and MUC2 production in the intestine.

SPIKE1 Activates the GTPase ROP6 to Guide the Polarized Growth of Infection Threads in Lotus japonicus

In legumes, rhizobia attach to root hair tips and secrete nodulation factor to activate rhizobial infection and nodule organogenesis. Endosymbiotic rhizobia enter nodule primordia via a specialized transcellular compartment known as the infection thread (IT). The IT elongates by polar tip growth, following the path of the migrating nucleus along and within the root hair cell. Rho-family ROP GTPases are known to regulate the polarized growth of cells, but their role in regulating polarized IT growth is poorly understood. Here, we show that LjSPK1, a DOCK family guanine nucleotide exchange factor (GEF), interacts with three type I ROP GTPases. Genetic analyses showed that these three ROP GTPases are involved in root hair development, but only LjROP6 is required for IT formation after rhizobia inoculation. Misdirected ITs formed in the root hairs of Ljspk1 and Ljrop6 mutants. We show that LjSPK1 functions as a GEF that activates LjROP6. LjROP6 enhanced the plasma membrane localization LjSPK1 in Nicotiana benthamiana leaf cells and Lotus japonicus root hairs, and LjSPK1 and LjROP6 interact at the plasma membrane. Taken together, these results shed light on how the LjROP6-LjSPK1 module mediates the polarized growth of ITs in L. japonicus.

Abstract 1447: Elucidating the mechanism of Dock7-mediated cellular transformation

Abstract Akt is a serine/threonine protein kinase that receives signals from a plethora of external and internal stimuli in order to promote survival and regulate cellular growth and metabolism. Akt is part of the highly conserved PI3K/Akt signaling axis and is classically regulated by its interaction with the phosphoinositide, PIP3, and its subsequent phosphorylation at S473 and S308 by the mTOR complex 2 (mTORC2) and 3-Phosphoinositide-dependent protein kinase 1 (PDK1), respectively. Altered Akt activity has been linked to numerous diseases, such as cancer, diabetes and neurodegenerative diseases. Specifically, PI3K/Akt deregulation occurs in approximately one third of human cancers and ~40% of breast cancers. My research focuses on Dock7, a Guanine Nucleotide Exchange Factor (GEF) for the small GTPases Cdc42 and Rac1, and a novel player in the regulation of Akt activity and stability. Dock7 belongs to the Dock family of atypical GEFs and contains two evolutionarily conserved domains, DHR1 (a putative lipid binding domain) and DHR2 (the catalytic GEF domain). The expression level of Dock7 correlates with an unfavorable prognosis in liver cancer patients, and it is highly expressed in triple-negative breast cancers. Studies in our laboratory showed Dock7 is essential for the transformed properties of several cancer cell lines, and that it interacts with Akt in co-immunoprecipitation assays. In order to decipher the mechanism by which Dock7 mediates transformation, I created a Crispr-Cas9 Dock7 Knock-out HeLa cell line. I proceeded to confirm that these cells are not able to grow in an anchorage independent manner nor in serum free conditions, two hallmarks of transformation. Furthermore, in comparison to the phosphorylation status of Akt in parental HeLa cells, I found that in Dock7 KO HeLa cells, Akt is not phosphorylated at the S473 site in the absence of growth factors, indicative of lower basal activity. Accordingly, phosphorylation of downstream effectors of Akt is also downregulated. Besides promoting the phosphorylation state of Akt, Dock7 plays a role in the stability of the Akt protein during certain cellular stresses, highlighting a versatile and important role for Dock7 in Akt regulation and a potential target for therapeutic intervention in tumors where Akt is deregulated. Citation Format: Oriana Y. Teran Pumar, Kristin F. Wilson, Richard A. Cerione. Elucidating the mechanism of Dock7-mediated cellular transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1447.

DOCK3 is a dosage-sensitive regulator of skeletal muscle and Duchenne muscular dystrophy-associated pathologies.

DOCK3 is a member of the DOCK family of guanine nucleotide exchange factors that regulate cell migration, fusion and viability. Previously, we identified a dysregulated miR-486/DOCK3 signaling cascade in dystrophin-deficient muscle, which resulted in the overexpression of DOCK3; however, little is known about the role of DOCK3 in muscle. Here, we characterize the functional role of DOCK3 in normal and dystrophic skeletal muscle. Utilizing Dock3 global knockout (Dock3 KO) mice, we found that the haploinsufficiency of Dock3 in Duchenne muscular dystrophy mice improved dystrophic muscle pathologies; however, complete loss of Dock3 worsened muscle function. Adult Dock3 KO mice have impaired muscle function and Dock3 KO myoblasts are defective for myogenic differentiation. Transcriptomic analyses of Dock3 KO muscles reveal a decrease in myogenic factors and pathways involved in muscle differentiation. These studies identify DOCK3 as a novel modulator of muscle health and may yield therapeutic targets for treating dystrophic muscle symptoms.

The supression of DOCK family members by their specific inhibitors induces the cell fusion of human trophoblastic cells

PurposeAmong the members of the DOCK family, DOCK1–5 function as guanine-nucleotide exchange factors for small GTPase Rac1, which regulates the actin cytoskeleton. It has been reported that in model organisms the Dock-Rac axis is required for myoblast fusion. We examined the role of DOCK1–5 in trophoblast fusion herein. MethodsWe used a quantitative polymerase chain reaction (qPCR) to examine the mRNA expressions of DOCK1–5 and differentiation-related genes, i.e., fusogenic genes, in human trophoblastic cell lines, BeWo and JEG-3. We treated BeWo cells with TBOPP and C21 to inhibit DOCK1 and DOCK5. Cell dynamics and cell fusion were assessed by live imaging and immunostaining. The signaling pathways induced by DOCK1/5 inhibition were examined by western blotting. ResultsDOCK1 and DOCK5 were expressed in BeWo cells. The inhibition of DOCK1 or DOCK5 did not prevent the cell fusion induced by forskolin (a common reagent for cell fusion); it induced cell fusion. DOCK1 inhibition induced cell death, as did forskolin. DOCK1 and DOCK5 inhibition for 24 and 48 h increased the expression of the genes ASCT2 and SYNCYTIN2, which code responsive proteins of trophoblast cell fusion, respectively. ConclusionDOCK1 and DOCK5 inhibition participates in BeWo cell fusion, probably via pathways independent from forskolin-mediated pathways.

TBOPP enhances the anticancer effect of cisplatin by inhibiting DOCK1 in renal cell carcinoma

The treatment of renal cell carcinoma (RCC) with chemotherapy remains a challenge; therefore, improving the knowledge of the molecular mechanisms underlying RCC chemoresistance and developing novel therapeutic strategies is important. Dedicator of cytokinesis 1 (DOCK1), the first member of the DOCK family to be discovered, displays various roles during tumorigenesis; however, its role during RCC progression is not completely understood. Therefore, the present study aimed to clarify the function of DOCK1 and 1-[2-(3′-(trifluoromethyl)-(1,1′-biphenyl)-4-yl)-2-oxoethyl]-5-pyrrolidinylsulfonyl-2 (1H)-pyridone (TBOPP), a DOCK1-sensitive inhibitor, during RCC development and chemoresistance. The results of CCK-8 and EdU assay indicated that TBOPP decreased RCC cell viability and proliferation compared with the control group, and sensitized RCC cells to cisplatin. Moreover, RCC cells with high DOCK1 expression levels displayed increased resistance to cisplatin, whereas DOCK1 knockdown enhanced the lethal effects of cisplatin on RCC cells. Furthermore, the results determined by western blotting, CCK-8 and cell apoptosis assay indicated that TBOPP effectively reduced DOCK1 expression levels compared with the control group, and the TBOPP-mediated cisplatin sensitizing effect was mediated by DOCK1 inhibition. The present study suggests that DOCK1 plays a vital role in RCC cell chemoresistance to cisplatin; therefore, TBOPP may serve as a novel therapeutic agent for RCC chemoresistance.

DOCK2 Expression Affects Leukemogenesis and Disease Progression in a Murine Model of FLT3/ITD Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and an internal tandem duplication (ITD) mutation portends a poor prognosis. FLT3/ITD activates Rac1 GTPase, and both proteins play key roles in DNA damage response (DDR). Disease progression in FLT3/ITD AML is often heralded by copy-neutral loss of heterozygosity (CN-LOH) at the FLT3 locus, which is typically caused by duplication of the mutant allele through homologous recombination with loss of the wild-type (WT) allele. We previously identified dedicator of cytokinesis 2 (DOCK2) as a FLT3/ITD-interacting protein. The DOCK family of proteins acts as guanine nucleotide exchange factors for the Rho family of GTPases, including Rac1. Expression of DOCK2 is limited to hematopoietic cells, and it is expressed in leukemic blasts in FLT3/ITD AML. Knockdown of DOCK2 in FLT3/ITD cell lines results in reduced Rac1 activity, decreased expression and activity of FLT3 and DDR factors, and increased sensitivity to cytarabine. In this study, we used mouse models to further investigate the relationship between DOCK2 and FLT3/ITD. Mice with a Dock2 mutation (Dock2/Hsd) that results in a marked reduction in Dock2 expression were bred with mice that carry a Flt3/ITD mutation (Flt3+/ITD). Quantitative PCR of mouse bone marrow (BM) revealed that homozygous expression of Dock2/Hsd markedly decreased the expression of Flt3 and key DDR factors in both WT and Flt3+/ITD mice. Flow cytometric analysis of the BM demonstrated that Dock2/Hsd resulted in partial reversal of the myeloproliferative neoplasm that develops in Flt3+/ITD mice, with a significant reduction in multipotent progenitors (MPP) in Flt3+/ITD;Dock2Hsd/Hsd (0.026%) vs. Flt3+/ITD mice (0.075%; P=0.048) at 3 months. To assess the role of DOCK2 in leukemogenesis and disease progression, the Dock2/Hsd mutation was introduced into a murine model of FLT3/ITD acute leukemia that carries both Flt3/ITD and the Nup98-HoxD13 (NHD13) fusion gene. It was previously shown that Flt3+/ITD;NHD13 mice develop acute leukemia with 100% penetrance and short latency. We found that the median survival of Flt3+/ITD;NHD13 mice (117 days) was not significantly affected by heterozygous expression of Dock2/Hsd (123 days); however, the median survival of Flt3+/ITD; NHD13; Dock2Hsd/Hsd mice was extended to 198 days (P=0.001). In contrast, Dock2/Hsd provided no survival benefit to mice that carried either Flt3+/ITD or NHD13 alone. The prolonged survival of Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice was accompanied by delayed onset of LOH at the Flt3/ITD locus. At 3 months, approximately half of Flt3+/ITD;NHD13;Dock2+/+ (47%; n=19) and Flt3+/ITD;NHD13;Dock2+/Hsd (57%; n=14) mice exhibited LOH in the peripheral blood, whereas only 11% of Flt3+/ITD; NHD13;Dock2Hsd/Hsd mice showed LOH (n=18; P=0.008 vs. Dock2+/Hsd and P=0.029 vs. Dock2+/+). Most of the moribund Flt3+/ITD;NHD13 mice (n=13) developed AML with minimal maturation (70%), and the remainder developed T-lymphoblastic leukemia with an early T-precursor phenotype (ETP-ALL). In contrast, moribund Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice (n=14) exhibited a more diverse disease spectrum: 30% mixed phenotype acute leukemia, 21% AML with minimal maturation, 21% ETP-ALL, 21% AML with maturation, and 7% acute erythroid leukemia (AEL). All moribund Flt3+/ITD;NHD13 mice showed complete LOH in BM; however, only 68% of moribund Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice exhibited complete LOH, with the rest showing partial or no LOH. Notably, all mice that exhibited partial or no LOH developed AEL or AML with maturation, diseases that were present in NHD13 mice but were not observed in Flt3+/ITD;NHD13 mice. This suggests that suppression of Dock2 mitigates the effects of Flt3/ITD, allowing the NHD13 mutation to drive the development of disease. These findings demonstrate that decreased DOCK2/Rac1 activity delays LOH at the FLT3 locus and disease progression in a murine model of FLT3/ITD leukemia. Thus, pharmacologic inhibition of the Rac1 signaling pathways via DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. Disclosures Small: Pharos I, B & T: Consultancy, Research Funding; InSilico Medicine: Membership on an entity's Board of Directors or advisory committees. Duffield:Boston Biomedical/Sumitomo Dainippon Pharma Co., Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; MedImmune: Consultancy.

DOCK family proteins: key players in immune surveillance mechanisms

Dedicator of cytokinesis (DOCK) proteins constitute a family of evolutionarily conserved guanine nucleotide exchange factors (GEFs) for the Rho family of GTPases. Although DOCK family proteins do not contain the Dbl homology domain typically found in other GEFs, they mediate the GTP-GDP exchange reaction through the DOCK homology region-2 (DHR-2) domain. In mammals, this family consists of 11 members, each of which has unique functions depending on the expression pattern and the substrate specificity. For example, DOCK2 is a Rac activator critical for migration and activation of leukocytes, whereas DOCK8 is a Cdc42-specific GEF that regulates interstitial migration of dendritic cells. Identification of DOCK2 and DOCK8 as causative genes for severe combined immunodeficiency syndromes in humans has highlighted their roles in immune surveillance. In addition, the recent discovery of a naturally occurring DOCK2-inhibitory metabolite has uncovered an unexpected mechanism of tissue-specific immune evasion. On the other hand, GEF-independent functions have been shown for DOCK8 in antigen-induced IL-31 production in helper T cells. This review summarizes multifaced functions of DOCK family proteins in the immune system.

T-cell mitochondrial dysfunction and lymphopenia in DOCK2-deficient patients

This study delineates the mitochondrial defects in DOCK2-deficient T cells contributing to the T cell lymphopenia characteristic of this primary immunodeficiency.

Dissociation mechanism of GDP from Cdc42 via DOCK9 revealed by molecular dynamics simulations.

Cell division control protein 42 homolog (Cdc42) influences a variety of cellular responses such as cell migration and polarity. Deregulation of Cdc42 has been associated with several human diseases and developmental disorders. Over-activation of Cdc42 through guanine nucleotide exchange factor (GEF) is a critical event for Cdc42 involved cancer metastasis. Members of DOCK family of GEF are important activators of Cdc42. However, this activation mechanism is still unknown. Molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) calculations were employed to investigate the central step of the activation of Cdc42: the dissociation mechanism of GDP from Cdc42 via DOCK9. Simulation results show that Mg2+ ion has a remarkable influence on the conformational change of switch I of Cdc42 through residue Pro34 which functions as a "clasp" to control the flexibility of switch I. In the GDP dissociation process, the Mg2+ ion leave first to result in a suitable conformation of Cdc42 for following DOCK9 binding to. When DOCK9 binds to Cdc42, it changes the orientations of residues Lys16, Thr17, Cys18 and Phe28 of Cdc42 to weaken the interactions between Cdc42 and GDP to release GDP. This study first elucidates the dissociation mechanism of GDP from Cdc42 via DOCK9 and identifies the essential residues of Cdc42 in this process. These simulation results are consistent with the recent findings of biochemical and amino acid mutational studies, and the observations are beneficial to understand the activation mechanism of Cdc42 and to provide insights for designing compounds targeting on Cdc42 related cancer metastasis.

High expression of DOCK2 indicates good prognosis in acute myeloid leukemia.

DOCK family proteins are evolutionarily conserved guanine nucleotide exchange factors for Rho GTPase with different cellular functions. It has been demonstrated that DOCK1 had adverse prognostic effect in acute myeloid leukemia (AML). We first analyzed data of 85 AML patients who were treated with chemotherapy and had available DOCK1 to DOCK11 expression information and found that DOCK1 and DOCK2 had prognostic significance in AML. In view of the known prognosis of DOCK1 in AML, we then explored the prognostic role of DOCK2. One hundred fifty-six AML patients with DOCK2 expression data were extracted from The Cancer Genome Atlas (TCGA) database and enrolled in this study. Patients were divided based on treatment modality into the chemotherapy group and the allogeneic hematopoietic stem cell transplant (allo-HSCT) group. Each group was divided into two groups by the median expression levels of DOCK2. In the chemotherapy group, high DOCK2 expression was associated with longer event-free survival (EFS, P=0.001) and overall survival (OS, P=0.007). In the allo-HSCT group, EFS and OS were not significantly different between high and low DOCK2 expression groups. Multivariate analysis showed that high DOCK2 expression was an independent favorable prognostic factor for both EFS and OS in all patients (all P<0.05). In conclusion, our results indicated that high DOCK2 expression, in contrast to DOCK1, conferred good prognosis in AML.

Resistance protein Pit interacts with the GEF OsSPK1 to activate OsRac1 and trigger rice immunity

Resistance (R) genes encode intracellular nucleotide-binding/leucine-rich repeat-containing (NLR) family proteins that serve as critical plant immune receptors to induce effector-triggered immunity (ETI). NLR proteins possess a tripartite domain architecture consisting of an N-terminal variable region, a central nucleotide-binding domain, and a C-terminal leucine-rich repeat. N-terminal coiled-coil (CC) or Toll-interleukin 1 receptor (TIR) domains of R proteins appear to serve as platforms to trigger immune responses, because overexpression of the CC or TIR domain of some R proteins is sufficient to induce an immune response. Because direct downstream signaling molecules of R proteins remain obscure, the molecular mechanisms by which R proteins regulate downstream signaling are largely unknown. We reported previously that a rice R protein named Pit triggers ETI through a small GTPase, OsRac1, although how Pit activates OsRac1 is unclear. Here, we identified OsSPK1, a DOCK family guanine nucleotide exchange factor, as an interactor of Pit and activator for OsRac1. OsSPK1 contributes to signaling by two disease-resistance genes, Pit and Pia, against the rice blast fungus Magnaporthe oryzae and facilitates OsRac1 activation in vitro and in vivo. The CC domain of Pit is required for its binding to OsSPK1, OsRac1 activation, and the induction of cell death. Overall, we conclude that OsSPK1 is a direct and key signaling target of Pit-mediated immunity. Our results shed light on how R proteins trigger ETI through direct downstream molecules.

Microtubule-Dependent Confinement of a Cell Signaling and Actin Polymerization Control Module Regulates Polarized Cell Growth

Cell types with wildly varying shapes use many of the same signaling and cytoskeletal proteins to dynamically pattern their geometry [1-3]. Plant cells are encased in a tough outer cell wall, and growth patterns are indirectly controlled by the cytoskeleton and its ability to locally specify the material properties of the wall [4, 5]. Broad and non-overlapping domains of actin and microtubules are predicted to create sharp cell-wall boundaries with distinct mechanical properties [6] that are often proposed to direct growth patterns and cell shape [1, 6, 7]. However, mechanisms by which the cytoskeleton is patterned at the spatial and temporal scales that dictate cell morphology are not known. Here, we used combinations of live-cell imaging probes and unique morphology mutants in Arabidopsis to discover how the microtubule and actin systems are spatially coordinated to pattern polarized growth in leaf epidermal cells. The DOCK family guanine nucleotide exchange factor (GEF) SPIKE1 [8, 9] clusters and activates conserved heteromeric WAVE/SCAR and ARP2/3 complexes at the cell apex to generate organized actin networks that define general cytoplasmic flow patterns. Cortical microtubules corral punctate SPIKE1 signaling nodules and restrict actin polymerization within a broad microtubule-depletion zone at the cell apex. Our data provide a useful model for cell-shape control, in which a GEF, actin filament nucleation complexes, microtubules, and the cell wall function as interacting systems that dynamically pattern polarized growth.