Family Of Small GTP-binding Proteins Research Articles

Identification, characterization and functional analysis of Rap1 in response to bacterial infection in tilapia

Rap1 (Ras-related protein 1), as a member of the Ras-like small GTP binding protein family, involves in the regulation of cell life activities and mediates a variety of biological events including cell junctions, cell polarity, cell proliferation and differentiation by binding to GDP/GTP. In addition, Rap1 also plays an important role in innate immune response by regulating the adhesion, diffusion and migration of white blood cells. However, it is still unclear how Rap1 affects the innate immunity of teleost fish against bacterial infections. In this study, we amplified the Rap1 from tilapia and conducted the sequence analysis of Rap1 nucleotide and amino acid sequences, then characterized its tissue distribution and expression pattern in response to pathogen infection in tilapia for the first time, and explored the bacteriostatic and bacterial binding effects of Rap1. The results showed that the Rap1 possessed a whole open reading frame (ORF) with 558 bp, encoding 185 amino acids. The Rap1 protein was highly homologous with Rap1 from other species. Conserved domain analysis revealed that Rap1 contained five G box domains that mediated its binding to ornithine. Rap1 mRNA was widely expressed in tilapia tissues, and the highest level was found in spleen. Under Streptococcus agalactiae (S. agalactiae) infection, the expression of Rap1 was significantly up-regulated in head kidney and brain. In addition, the recombinant protein Rap1 hold the ability binding to S. agalactiae and inhibit the proliferation of S. agalactiae. In conclusion, this study not only provided a reference for exploring the function of Rap1 in teleost against pathogen infection, but also enriched the basic theoretical research of Rap1 function and antibacterial immune response in teleost.

Functional Characterization of Tomato ShROP7 in Regulating Resistance against Oidium neolycopersici.

ROPs (Rho-like GTPases from plants) are a unique family of small GTP-binding proteins in plants and play vital roles in numerous cellular processes, including growth and development, abiotic stress signaling, and plant defense. In the case of the latter, the role of ROPs as response regulators to obligate parasitism remains largely enigmatic. Herein, we isolated and identified ShROP7 and show that it plays a critical role in plant immune response to pathogen infection. Real-time quantitative PCR analysis revealed that the expression of ShROP7 was significantly increased during incompatible interactions. To establish its requirement for resistance, we demonstrate that virus-induced gene silencing (VIGS) of ShROP7 resulted in increased susceptibility of tomato to Oidium neolycopersici (On) Lanzhou strain (On-Lz). Downstream resistance signaling through H2O2 and the induction of the hypersensitive response (HR) in ShROP7-silenced plants were significantly reduced after inoculating with On-Lz. Taken together, with the identification of ShROP7-interacting candidates, including ShSOBIR1, we demonstrate that ShROP7 plays a positive regulatory role in tomato powdery mildew resistance.

Abstract 2681: Novel and selective inhibitors of KRASG12D

Abstract RAS proteins are family of small GTP-binding proteins that play a major role in regulating cellular responses such as proliferation, differentiation, migration, and cell growth. RAS proteins function as molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states, which relay cellular signals in response to extracellular stimuli. Aberrant RAS signaling plays a role in up to 30% of all human cancers, with the highest incidence occurring in carcinomas of the pancreas (63–90%), colon (36–50%), and lung (19–30%). Targeting the inactive, GDP bound KRASG12C has been a promising approach for generating novel anti RAS therapies, which is validated by the approval of AMG 510 (Lumakras) by the FDA. It is also known that KRASG12D also retains GTPase activity. Consequently, KRASG12D is one of the most important chemotherapeutic drug targets. KRASG12D is commonly observed in pancreatic cancer. Here we describe preclinical profile of a potent, selective, in-vivo tool compound, VRTX144. VRTX144 was potent in HPAC cell line, representing pancreatic adenocarcinoma, and a panel of KRASG12D mutated cell lines. It was found to be several folds less active in non-KRASG12D mutant cell lines. In vitro combination strategies with other targeted therapies such as EGFR, SHP2 and CDK4/6 inhibitor is also being investigated. VRTX144 exhibited low propensity for metabolism in microsomes, and has high protein bound. VRTX144 was found to have no adverse effect in hERG upto 10 uM. Citation Format: Prashant Kashinath Bhavar, Uday Kumar Surampudi, Partha Pratim Sarma, Mahesh Mohan Shidore, Anuj Ramesh Kshirsagar. Novel and selective inhibitors of KRASG12D [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 2681.

ADP-Ribosylation Factor Family of Small GTP-Binding Proteins: Their Membrane Recruitment, Activation, Crosstalk and Functions.

Members of the ADP-ribosylation factor (ARF) family of guanine-nucleotide binding proteins play critical roles in various cellular processes, especially in regulating the secretory, and endocytic pathways. The fidelity of intracellular vesicular trafficking depends on proper activations and precise subcellular distributions of ARF family proteins regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Here we review recent progress in understanding the membrane recruitment, activation, crosstalk, and functions of ARF family proteins.

Multiple Omics Analysis of the Rac3 Roles in Different Types of Human Cancer

Rac Family Small GTPase 3 (Rac3) is a member of the Rho family of small GTP-binding proteins which play critical roles in the occurrence, progression, and metastasis of various tumors. Nevertheless, previous studies have focused on a single type of human cancer, so that the roles of Rac3 in different cancer types have not been sufficiently clarified. With the progress of biological detection and bioinformatics technology, the emerging cancer genomics databases make it possible to perform a pan-cancer analysis. Therefore, for the first time, we performed multiple omics analysis to investigate the roles of Rac3 in differential expression, survival prognosis, mutative status, DNA methylation, functions, immune infiltration, and immunotherapy across 33 human cancer types. We found that Rac3 expression was abnormal in 17 cancer types and significantly different in both molecular and immune subtypes of 10 cancers (p<0.05). These Rac3 expression dysregulations in cancer tissues significantly affected their corresponding survival prognosis. Our results also indicated that genetic alterations of Rac3 occurred in 27 cancer types and were significantly associated with prognosis. Moreover, methylation of Rac3 was associated with dysfunctional T-cell phenotypes and affected the prognosis of the brain, melanoma, and breast tumors, and Rac3 Copy Number Alterations (CNA) might affect the infiltration levels of different immune cells in nine cancers. Rac3 and Rac3-related genes were enriched in the axon guidance, actin cytoskeleton regulation, and neurotrophin signaling pathway, and involved the regulation of cellular localization and actin cytoskeleton organization, and small GTPase mediated signal transduction. Then, we further explored the correlations between Rac3 expression and 25 immune cells and cancer-associated fibroblasts across 33 human cancers. Furthermore, we found that Rac3 achieved higher predictive power (AUC>0.5) than three standardized biomarkers of tumor immune responses and was associated with cytotoxic T-cell levels (CTLs) and the risk of immunotherapeutic responses in three cancer types. Collectively, our study contributes to a comprehensive and integrative understanding of the oncogenic roles of Rac3 across human cancers and offers a new clue for treatment strategies.

Association Mapping and Functional Analysis of Rice Cold Tolerance QTLs at the Bud Burst Stage

Cold tolerance at the bud burst stage (CTB) is a key trait for direct-seeded rice. Although quantitative trait loci (QTL) affecting CTB in rice have been mapped using traditional linkage mapping and genome-wide association study (GWAS) methods, the underlying genes remain unknown. In this study, we evaluated the CTB phenotype of 339 cultivars in the Rice Diversity Panel II (RDP II) collection. GWAS identified four QTLs associated with CTB (qCTBs), distributed on chromosomes 1–3. Among them, qCTB-1-1 overlaps with Osa-miR319b, a known cold tolerance micro RNA gene. The other three qCTBs have not been reported. In addition, we characterised the candidate gene OsRab11C1 for qCTB-1-2 that encodes a Rab protein belonging to the small GTP-binding protein family. Overexpression of OsRab11C1 significantly reduced CTB, while gene knockout elevated CTB as well as cold tolerance at the seedling stage, suggesting that OsRab11C1 negatively regulates rice cold tolerance. Molecular analysis revealed that OsRab11C1 modulates cold tolerance by suppressing the abscisic acid signalling pathway and proline biosynthesis. Using RDP II and GWAS, we identified four qCTBs that are involved in CTB and determined the function of the candidate gene OsRab11C1 in cold tolerance. Our results demonstrate that OsRab11C1 is a negative regulator of cold tolerance and knocking out of the gene by genome-editing may provide enhanced cold tolerance in rice.

Effects of decreased Rac activity and malignant state on oral squamous cell carcinoma in vitro.

Rac proteins, members of the Rho family of small GTP-binding proteins, have been implicated in transducing a number of signals for various biological mechanisms, including cell cytoskeleton organization, transcription, proliferation, migration, and cancer cell motility. Among human cancers, Rac proteins are highly activated by either overexpression of the genes, up-regulation of the protein, or by mutations that allow the protein to elude normal regulatory signaling pathways. Rac proteins are involved in controlling cell survival and apoptosis. The effects of Rac inhibition by the Rac-specific small molecule inhibitor NSC23766 or by transfection of dominant negative Rac (Rac-DN) were examined on three human-derived oral squamous cell carcinoma cell lines that exhibit different malignancy grades, OSC-20 (grade 3), OSC-19 (grade 4C), and HOC313 (grade 4D). Upon suppression of Rac, OSC-19 and HOC313 cells showed significant decreases in Rac activity and resulted in condensation of the nuclei and up-regulation of c-Jun N-terminal kinase (JNK), leading to caspase-dependent apoptosis. In contrast, OSC-20 cells showed only a slight decrease in Rac activity, which resulted in slight activation of JNK and no change in the nuclei. Fibroblasts treated with NSC23766 also showed only a slight decrease in Rac activity with no change in the nuclei or JNK activity. Our results indicated that apoptosis elicited by the inhibition of Rac depended on the extent of decreased Rac activity and the malignant state of the squamous cell carcinoma. In addition, activation of JNK strongly correlated with apoptosis. Rac inhibition may represent a novel therapeutic approach for cancer treatment.

The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis

Cytotoxic necrotic factor (CNF) of Yersinia pseudotuberculosis (CNFY) is a 114-kDa toxin protein that is similar to CNF1, which is prevalent among pathogenic and saprophytic Escherichia coli isolates. In 2002, the CNF of Y. pseudotuberculosis YPIII strain (United States) was first described. The Rho family small GTP-binding proteins (GTPases) are inactivated by CNFY acting on eukaryotic cells of humans and mammals, which causes the formation of large multinucleated cells. An analysis of Y. pseudotuberculosis toxins isolated in Russia showed that the thermolabile toxin TLT described earlier was identified as identical to CNFY except for three amino acid substitutions. The TLT’s cytotoxic activity is similar to that of CNFY. Screening of Y. pseudotuberculosis strains collection isolated during the outbreaks of Far East scarletlike fever (FESLF) and in sporadic cases from patients with pseudotuberculosis/FESLF, rodents, and the environment in the Far East, Siberia, and the northwest of the Russian Federation showed that most isolates carry the specific cnfY gene allele encoding the inactive CNFY isoform. This allele contains a stop codon and two large deletions that block the production of the full-sized toxin. Interestingly, all isolates obtained from patients with FESLF, which is a severe form of pseudotuberculosis and endemic to the Far East region, contain inactivated CNFY. Possible causes of CNFY inactivation in strains associated with Far East scarletlike fever are discussed.

Neutrophil-Derived Reactive Oxygen Orchestrates Epithelial Cell Signaling Events during Intestinal Repair

Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTP-binding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cell-signaling events functioning in intestinal repair.

Mechanochemical Coupling and Junctional Forces during Collective Cell Migration

Cell migration, a fundamental physiological process in which cells sense and move through their surrounding physical environment, plays a critical role in development and tissue formation, as well as pathological processes, such as cancer metastasis and wound healing. During cell migration, dynamics are governed by the bidirectional interplay between cell-generated mechanical forces and the activity of Rho GTPases, a family of small GTP-binding proteins that regulate actin cytoskeleton assembly and cellular contractility. These interactions are inherently more complex during the collective migration of mechanically coupled cells because of the additional regulation of cell-cell junctional forces. In this study, we adapted a recent minimal modeling framework to simulate the interactions between mechanochemical signaling in individual cells and interactions with cell-cell junctional forces during collective cell migration. We find that migration of individual cells depends on the feedback between mechanical tension and Rho GTPase activity in a biphasic manner. During collective cell migration, waves of Rho GTPase activity mediate mechanical contraction/extension and thus synchronization throughout the tissue. Further, cell-cell junctional forces exhibit distinct spatial patterns during collective cell migration, with larger forces near the leading edge. Larger junctional force magnitudes are associated with faster collective cell migration and larger tissue size. Simulations of heterogeneous tissue migration exhibit a complex dependence on the properties of both leading and trailing cells. Computational predictions demonstrate that collective cell migration depends on both the emergent dynamics and interactions between cellular-level Rho GTPase activity and contractility and multicellular-level junctional forces.

Developmental expression of the zebrafish Arf-like small GTPase paralogs arl13a and arl13b

Members of the Arf-like (Arl) family of small GTP-binding proteins regulate a number of cellular functions and play important roles in cilia structure and signaling. The small GTPase Arl13a is a close paralog to Arl13b, a small GTPase required for normal cilia formation that causes Joubert Syndrome when mutated. As mutation of arl13b causes a slow retinal degeneration in zebrafish (Song et al., 2016), we hypothesized that expression of arl13a may provide functional redundancy. We determined the expression domains of arl13a and arl13b during zebrafish development and examined subcellular localization by expression of fluorescence fusion proteins. Both genes are widely expressed during early cell division and gastrulation and Arl13a and Arl13b both localize to microtubules in ciliated and dividing cells of the early zebrafish embryo. Between 2 and 5 days post fertilization (dpf), arl13b is expressed in neural tissues while expression of arl13a is downregulated by 2 dpf and restricted to craniofacial structures. These results indicate that arl13a and arl13b have evolved different roles and that arl13a does not function in the zebrafish retina.

Abstract B12: The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis

Abstract The Ras family of small GTP-binding proteins is frequently activated by mutations, including NRAS (20%), KRAS (2%), and HRAS (1%), in melanoma. In addition to mutations, Ras isoforms can also be activated by the inactivation of Ras GTPase activating proteins (RasGAPs), such as NF1, RASA1, and RASA2. In our recent study, we observed that the inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform. We hypothesized that, although not mutated, R-Ras is activated in melanoma through the inactivation of RasGAPs and that RasGAP/R-Ras pathway activation cooperates with BRAF activation in melanoma tumorigenesis. In this study, we addressed the importance of R-Ras, a previously less-appreciated member of the Ras family, in melanoma tumorigenesis and investigated the molecular mechanisms underlying R-Ras signaling in BRAF mutant melanoma. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines. In addition, RNAi-mediated reduced expression of R-Ras suppressed anchorage-independent colony growth and tumor growth. Moreover, among the three major RAS effector pathways, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by RASA1 inactivation and subsequent R-Ras activation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacologic (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a RASA1 L/L; BRAF CA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in the expression of constitutively activated mutant BRAF and the deletion of RASA1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in RASA1 L/L BRAF CA/CA mice compared to RASA1 +/+ BRAF CA/CA littermates and the development of melanoma in RASA1 mutant mice. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways. Citation Format: Kristen S. Hill, Xue Wang, Evan R. Roberts, Youngchul Kim, Jane Messina, Minjung Kim. The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B12.

Molecular mechanisms and physiological relevance of RGK proteins in the heart.

The primary route of Ca2+ entry into cardiac myocytes is via 1,4-dihydropyridine-sensitive, voltage-gated L-type Ca2+ channels. Ca2+ influx through these channels influences duration of action potential and engages excitation-contraction (EC) coupling in both the atria and the myocardium. Members of the RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTP-binding proteins are potent, endogenously expressed inhibitors of cardiac L-type channels. Although much work has focused on the molecular mechanisms by which RGK proteins inhibit the CaV 1.2 and CaV 1.3 L-type channel isoforms that expressed in the heart, their impact on greater cardiac function is only beginning to come into focus. In this review, we summarize recent findings regarding the influence of RGK proteins on normal cardiac physiology and the pathological consequences of aberrant RGK activity.

Smuggling gold nanoparticles across cell types – A new role for exosomes in gene silencing

Once released to the extracellular space, exosomes enable the transfer of proteins, lipids and RNA between different cells, being able to modulate the recipient cells' phenotypes. Members of the Rab small GTP-binding protein family, such as RAB27A, are responsible for the coordination of several steps in vesicle trafficking, including budding, mobility, docking and fusion. The use of gold nanoparticles (AuNPs) for gene silencing is considered a cutting-edge technology. Here, AuNPs were functionalized with thiolated oligonucleotides anti-RAB27A (AuNP@PEG@anti-RAB27A) for selective silencing of the gene with a consequent decrease of exosomes´ release by MCF-7 and MDA-MB-453 cells. Furthermore, communication between tumor and normal cells was observed both in terms of alterations in c-Myc gene expression and transportation of the AuNPs, mediating gene silencing in secondary cells.

Distinct regulations of ARF1 by two Aplysia Sec7 isoforms

ABSTRACTSec7 protein is a guanine nucleotide exchange factor in the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. Aplysia Sec7 proteins (ApSec7s) play many roles in neurite outgrowth and synaptic facilitation in Aplysia neurons. However, the binding property of Aplysia ARF1 by ApSec7 isoforms has not been examined. In this study, we found that the cloned Aplysia ARF1 (ApARF1) protein only localized to the Golgi complex when it was expressed alone in HEK293T cells; however, if ApARF1 was co-expressed with plasma membrane-targeted ApSec7, it localized to both the plasma membrane and the Golgi complex via association with the Sec7 domain of ApSec7. Moreover, in HEK293T cells expressing both ApARF1 and another Sec7 isoform, ApSec7(VPKIS), the pleckstrin homology domain of ApSec7(VPKIS) associated with ApARF1, resulting in its localization to the Golgi complex. Overall, we propose a model in which ApSec7(VPKIS) activates ApARF1 in the Golgi complex, while ApSec7 recruits ApARF1 to the plasma membrane where it activates ApARF1/6 downstream signaling.

Knockdown of Rap2B Inhibits the Proliferation and Invasion in Hepatocellular Carcinoma Cells.

Rap2B, a member of the Ras family of small GTP-binding proteins, was found to be highly expressed in various human tumors and plays an important role in the development of tumors. However, the function of Rap2B in hepatocellular carcinoma (HCC) remains unclear. Therefore, in this study, we investigated the biological functions of Rap2B in HCC and the potential underlying mechanisms. Our results indicated that Rap2B was highly expressed in HCC tissues and cell lines. Rap2B silencing obviously inhibited the proliferation, migration, and invasion of HCC cells, as well as attenuated xenografted tumor growth in vivo. Furthermore, Rap2B silencing greatly reduced the expression levels of phosphorylated focal adhesion kinase (p-FAK), matrix metalloproteinase-2 (MMP-2), and MMP-9 in HCC cells. In conclusion, our data suggest that Rap2B silencing inhibits the proliferation and invasion in HCC cells. Thus, Rap2B may have potential as a treatment for HCC.

Dual roles of the N-terminal coiled-coil domain of an Aplysia sec7 protein: homodimer formation and nuclear export.

Cytohesin family proteins act as guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor family of small GTP-binding proteins. Aplysia Sec7 (ApSec7), a member of the cytohesin family in Aplysia, plays key roles in neurite outgrowth in Aplysia neurons. Although ApSec7 has a conserved coiled-coil (CC) domain, its role was not clear. In this study, we found that the CC domain of ApSec7 and ARNO/cytohesin 2 are involved in homodimer formation, leading to efficient plasma membrane targeting of ApSec7 and ARNO/cytohesin 2 in HEK293T cells. Therefore, deletion of the CC domain of ApSec7 and ARNO/cytohesin 2 may result in a loss of dimerization and reduce plasma membrane localization. In addition, the CC domains of ApSec7 and ARNO/cytohesin 2 have partially or fully CRM1-dependent nuclear export signals, respectively. Taken together, our results suggest that the CC domain of cytohesin family proteins, including ApSec7 and ARNO/cytohesin 2, has dual roles in intracellular targeting: increased plasma membrane targeting through homodimer formation and nuclear exclusion through either a CRM1-dependent or a CRM1-independent pathway.

Arl4c is a key regulator of tubulogenesis and tumourigenesis as a target gene of Wnt-β-catenin and growth factor-Ras signalling.

Epithelial tubular morphogenesis (tubulogenesis) is a fundamental morphogenetic process of many epithelial organs. In this developmental process, epithelial cells migrate, proliferate, polarize and differentiate towards surrounding mesenchymal tissue to form tubule structures. Although epithelial tissue structures are basically stable in the postnatal period, epithelial cells regain highly proliferative and invasive potentials within mesenchymal tissue during tumour formation (tumourigenesis). Therefore, there must be a common molecular basis orchestrating the cellular behaviours involved in both tubulogenesis and tumourigenesis. ADP-ribosylation factor (Arf)-like protein 4c (Arl4c), which belongs to the small GTP-binding protein family, is expressed by the simultaneous activation of Wnt-β-catenin and growth factor-Ras-mitogen-activated protein kinase signalling, was identified as an essential regulator of tubulogenesis. Arl4c expression was also involved in the tumour formation of colorectal and lung cancers. In this review, we focus on Arl4c as a novel Wnt signal target molecule that links epithelial tubulogenesis to tumourigenesis.

Characterizing PKA-Mediated Phosphorylation of Plexin Using Purified Proteins.

Protein phosphorylation is one of the widely used posttranslational modifications that alter protein function in vivo. We recently showed phosphorylation of Drosophila Plexin A by cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and subsequent inhibition of plexin-mediated repulsive guidance. This phosphorylation occurs in the active site of the plexin GTPase-activating protein (GAP) domain, which in turn inhibits endogenous GAP activity toward Ras/Rap family small GTP-binding proteins by recruiting the phospho-serine/threonine-binding protein 14-3-3ε. Here we describe how phosphorylation of Plexin A can be detected and quantitated using an in vitro kinase assay and radioactive [γ-P32] adenosine 5'-triphosphate (ATP).

Functional specialization of Arf paralogs in nodules of model legume, Medicago truncatula

ADP-ribosylation factors (Arf), a family of small GTP-binding proteins, play essential role in the regulation of housekeeping functions such as cellular trafficking in yeast, mammalian, and plants. Although this family of genes are strictly conserved both functionally and sequentially throughout Eukaryotae, putative lineage-specific members of these genes have been reported to be sub-or neofunctionalized in plant kingdom. In this study, we investigated the possible lineage-specific functional evolution of four Arf1 paralogous genes in the development and maintenance of nodular structures of Medicago truncatula. Even though all of MtArf1 paralogs were shown to have certain amount of expression in every tissue of M. truncatula, the expression of these Arf1 paralogs was significantly variable and higher in nodular tissues. To better gage the functional evolution of paralogous genes, the spatiotemporal expressional profiles of MtArf1 paralog genes have been investigated by measuring the level of variation in the amount of the transcripts during nodule formation immediately after the infection of roots of Medicago plants with Sinorhizobium melliloti. Of these previously described genes, MtrGB13597775, solely expressed at 72 hpi (hour post inoculation) and the expression level of MtrGB27524313 significantly hiked in the nodules at 6 dpi (days post inoculation) hinting possible subfunctionalization of Arf paralogs. To get more evidence about the possibility of nodule-specific functionilization of MtArf1 paralogs, the level of expression of these genes was also checked at protein level by Western blot analyses. Arf1 protein level was much higher in the infected roots and nodules in comparison to shoots. The findings in this study could suggest that Arf1 genes likely to play an important role in the nodule formation and maintenance in M. truncatula and the possibility of functional specialization of the duplicated Arf genes.