BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion

BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.     

TC-PTP dephosphorylates the guanine nucleotide exchange factor C3G (RapGEF1) and negatively regulates differentiation of human neuroblastoma cells

The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.     

EFA6A,a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin‐1 and regulates neurite outgrowth

The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6‐specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin‐1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two‐hybrid screening. The interaction was mediated by the C‐terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull‐down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post‐synaptic density and endosomes in dendritic spines. In Neuro‐2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co‐expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6‐mediated neurite formation through the interaction with SNX1.

     

Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells

The guanine nucleotide exchange factor (GEF), SmgGDS, promotes nucleotide exchange by several GTPases in both the Ras and Rho families, especially by RhoA. Because RhoA plays an important role in regulating the contraction of vascular smooth muscle cells (VSMC), we examined the expression and function of SmgGDS in VSMC. SmgGDS is expressed in primary rat aortic smooth muscle (ASM) cells, primary bovine coronary artery smooth muscle (BCASM) cells, and the immortalized A7r5 line of rat ASM cells. Down regulation of SmgGDS expression by siRNA transfection resulted in a decrease of RhoA-GTP levels, enhanced cell spreading, and loss of the characteristic elongated morphology of VSMC. A similar morphology was also observed following treatment with the Rho-kinase inhibitor, Y27632. In contrast, cells with reduced RhoA expression exhibit an elongated shape. Subsequent immunofluorescent staining revealed a disruption of the myosin filament organization in the cells with reduced SmgGDS expression. Further studies analyzed the effect of SmgGDS siRNA transfection on the contraction of A7r5 cells and BCASM cells, which is also a Rho-regulated pathway. Transfection of SmgGDS siRNA or RhoA siRNA resulted in an impaired ability of the A7r5 and BCASM cells to undergo contraction in a collagen gel matrix. However, phosphorylation of the myosin-binding subunit of myosin phosphatase (MYPT1) or the light chain of myosin II (MLC) was not altered by downregulating expression of either SmgGDS or RhoA GTPase. Taken together these results identify SmgGDS as a novel regulator of myosin organization and contraction in VSMC.     

Brain-specific potential guanine nucleotide exchange factor for Arf, synArfGEF (Po), is localized to postsynaptic density

We cloned from a rat brain cDNA library a novel cDNA and named it a potential synaptic guanine nucleotide exchange factor (GEF) for Arf (synArfGEF (Po)) (GenBank Accession no. AB057643) based on its domain structure and localization. The cloned gene was 7410 bases long with a 3585-bp coding sequence encoding a protein of 1194 amino acids. The deduced protein contained a coiled-coil structure in the N-terminal portion followed by Sec7 and Plekstrin homology (PH) domains. Thus, the protein was a member of the Sec7 family of proteins, GEFs. Conservation of the ADP-ribosylation factor (Arf)-binding sequence suggested that the protein was a GEF for Arf. The gene was expressed specifically in the brain, where it exhibited region-specific expression. The protein was highly enriched in the postsynaptic density (PSD) fraction prepared from the rat forebrain. Uniquely, the protein interacted with PSD-95, SAP97 and Homer/Vesl 1/PSD-Zip45 via its C-terminal PDZ-binding motif and co-localized with these proteins in cultured cortical neurons. These results supported its localization in the PSD. The postsynaptic localization was also supported by immunohistochemical examination of the rat brain. The mRNA for the synArfGEF was also localized to dendrites, as well as somas, of neuronal cells. Thus, both the mRNA and the protein were localized in the postsynaptic compartments. These results suggest a postsynaptic role of synArfGEF in the brain.     

GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factors,is localized to the cis-Golgi and involved in membrane association of the COPI coat

Formation of coated carrier vesicles, such as COPI-coated vesicles from the cis -Golgi, is triggered by membrane binding of the GTP-bound form of ADP-ribosylation factors. This process is blocked by brefeldin A, which is an inhibitor of guanine nucleotide exchange factors for ADP-ribosylation factor. GBF1 is one of the guanine nucleotide-exchange factors for ADP-ribosylation factor and is localized in the Golgi region. In the present study, we have determined the detailed subcellular localization of GBF1. Immunofluorescence microscopy of cells treated with nocodazole or incubated at 15 °C has suggested that GBF1 behaves similarly to proteins recycling between the cis -Golgi and the endoplasmic reticulum. Immunoelectron microscopy has revealed that GBF1 localizes primarily to vesicular and tubular structures apposed to the cis -face of Golgi stacks and minor fractions to the Golgi stacks. GBF1 overexpressed in cells causes recruitment of class I and class II ADP-ribosylation factors onto Golgi membranes. Furthermore, overexpressed GBF1 antagonizes various effects of brefeldin A, such as inhibition of membrane recruitment of ADP-ribosylation factors and the COPI coat, and redistribution of Golgi-resident and itinerant proteins. These observations indicate that GBF1 is involved in the formation of COPI-coated vesicles from the cis -Golgi or the pre-Golgi intermediate compartment through activating ADP-ribosylation factors.     

Fission yeast syt22 protein, a putative Arf guanine nucleotide exchange factor, is necessary for new end take off

In fission yeast Schizosaccharomyces pombe , the directions of cell growth change from monopolar to bipolar in character, which is known as 'new end take off' (NETO). We previously found that arf6p, a member (class III) of the ADP-ribosylation factor (Arf) family, is necessary for NETO in fission yeast. Here we report the characterization of an S. pombe gene, syt22 ⁺, encoding a putative Arf guanine nucleotide exchange factor (GEF). The syt22 protein contains a Sec7 domain and a PH domain conserved in the mammalian EFA6 GEF family, and has high similarity to Yel1p, which was identified as a GEF for Arf3p (class III Arf) in Saccharomyces cerevisiae. syt22 Δ cells, like arf6 Δ cells, completely failed to undergo NETO. Syt22p uniformly localizes to the cell periphery. Its localization is not dependent on microtubules, actin cytoskeletons or arf6p. We hypothesize that syt22p functions as a GEF for arf6p.     

SH3‐class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras‐like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras‐subfamily‐specific guanine nucleotide exchange factors (RasGEFs). Although Ras‐protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3‐, Ras guanyl nucleotide‐releasing protein [RasGRP]–, and LTE‐class), each with fungus‐specific attributes. Here, we show that the SH3‐class and RasGRP‐class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3‐class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3‐class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.     

The guanine nucleotide exchange factor CNrasGEF regulates melanogenesis and cell survival in melanoma cells

cAMP-dependent Ras activation has been demonstrated in numerous cell types, particularly of neuronal (including melanoma cells) and endocrine origin, but the Ras activator involved has not been identified. In B16 melanoma cells, cAMP activates the Ras/Erk pathway, leading initially to stimulation but subsequently to long term (>24-h) inhibition of melanogenesis (dendrite extension and melanin production). Here we identify CNrasGEF as the Ras guanine nucleotide exchange factor (GEF) involved. We demonstrate that CNrasGEF is expressed endogenously in B16 melanoma cells and that cAMP-mediated activation of Ras and Erk1/2 in these cells can be augmented by CNrasGEF overexpression and reduced by its knockdown by RNA interference. Moreover, we show that CNrasGEF participates in the regulation of melanogenesis. Knockdown of CNrasGEF leads to increased dendrite extension and melanin production observed approximately 50 h after forskolin/isobutylmethylxanthine treatment, suggesting that CNrasGEF inhibits melanogenesis in the long term. Independently, we find that overexpression of CNrasGEF leads to apoptosis, whereas its knockdown by RNAi enhances cell proliferation, independent of cAMP. Collectively, these results suggest that CNrasGEF regulates melanogenesis but that it also has a distinct role in regulating cell proliferation/apoptosis.     

Induction of axonal differentiation by silencing plasma membrane-associated sialidase Neu3 in neuroblastoma cells

A reduction of 70% of the plasma membrane-associated sialidase Neu3 activity, due to a corresponding reduction of the enzyme expression by transducing cells with a short hairpin RNA encoding a sequence target (complementary messenger of mouse Neu3), caused neurite elongation in Neuro2a murine neuroblastoma cells. The differentiation process was accompanied in parallel by an increase of the acetylcholinesterase activity, a moderate increase of the c-Src expression and by the presence of the axonal marker tau protein on the neurites. The sphingolipid pattern and turnover in transduced and control cells were characterized by thin layer chromatography, mass spectrometry and metabolic radiolabeling after feeding cells with tritiated sphingosine. Control cells contained about 2 nmol of gangliosides/mg cell protein. GM2 was the main compound, followed by GD1a, GM3 and GM1. In Neu3 silenced cells, the total ganglioside content remained quite similar, but GM2 increased by 54%, GM3 remain constant, and GM1 and GD1a decreased by 66% and 50%, respectively. Within the organic phase sphingolipids, ceramide decreased by 50%, whereas the sphingomyelin content did not change in Neu3 silenced cells.     

Biochemical characterisation of TCTP questions its function as a guanine nucleotide exchange factor for Rheb

Translationally controlled tumour protein (TCTP) is involved in malignant transformation and regulation of apoptosis. It has been postulated to serve as a guanine nucleotide exchange factor for the small G-protein Rheb. Rheb functions in the PI3 kinase/mTOR pathway. The study presented here was initiated to characterise the interaction between TCTP and Rheb biochemically. Since (i) no exchange activity of TCTP towards Rheb could be detected in vitro, (ii) no interaction between TCTP and Rheb could be detected by NMR spectroscopy, and (iii) no effect of TCTP depletion in cells on the direct downstream targets of Rheb could be observed in vivo, this study shows that TCTP is unlikely to be a guanine nucleotide exchange factor for Rheb.     

Assignment of the 1H, 13C and 15N resonances of the catalytic domain of guanine nucleotide exchange factor SopE2 from Salmonella dublin

Abbreviation: GEF – guanine nucleotide exchange factor.     

PKCε, Via its Regulatory Domain and Independently of its Catalytic Domain, Induces Neurite-like Processes in Neuroblastoma Cells

To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCalpha, betaII, delta, and epsilon fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCepsilon-EGFP induced cell processes whereas the other isoforms did not. The effect of PKCepsilon-EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCepsilon regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCepsilon and neurites induced during retinoic acid- and growth factor-induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCepsilon- PSC1V3 protein contained alpha-tubulin, neurofilament-160, and F-actin, but the PKCepsilon-PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y. These data suggest that PKCepsilon, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation.     

C3G is required for c-Abl-induced filopodia and its overexpression promotes filopodia formation

The Rap1 guanine nucleotide exchange factor, C3G (also known as Rap1GEF-1) is involved in signaling from growth factors, cytokines and integrins and plays a role in cell adhesion and migration, but the mechanism by which C3G regulates various cellular functions is poorly understood. We, therefore, investigated the ability of C3G to affect actin cytoskeleton-dependent morphological changes in cells. Using RNA interference, we provide evidence that C3G is required for c-Abl-induced filopodia during cell spreading on fibronectin. C3G expression induces actin cytoskeletal reorganization and promotes filopodia formation independent of its catalytic activity. It showed enrichment at filopodia tips characteristic of molecules involved in filopodia dynamics. C3G-induced filopodia were not inhibited by dominant negative mutants of Rho, Rac and Cdc42, but required Abl catalytic activity. Coexpression of N-Wasp-Crib inhibited C3G induced as well as c-Abl-induced filopodia and wiskostatin, a pharmacological inhibitor of N-Wasp attenuates C3G-induced filopodia. Cellular C3G interacts with c-Abl and C3G expression results in enhanced localization of endogenous c-Abl in the cytoplasm. We suggest that C3G and c-Abl function in an interdependent manner, in linking external signals to remodeling the cytoskeleton to induce filopodia.     

Localization of EFA6A,a guanine nucleotide exchange factor for ARF6, in spermatogenic cells of testes of adult mice

ADP ribosylation factors (ARFs) of small GTPase are molecular switches regulating various membrane dynamics. Among them, ARF6 has recently been highlighted because of its function to facilitate the interaction between the cytoskeleton and the plasma membrane. Each ARFs has its preferable or even specific guanine nucleotide exchange factors (GEFs) as its activators. According to our previous RT-PCR analysis, EFA6A, a guanine nucleotide exchange factor for ARF6, was restrictedly expressed in the brain, retina and testis. Different from previous studies on neurons, EFA6A, a guanine nucleotide exchange factor for ARF6, was first shown to be localized intensely in nuclei of spermatocytes of adult mouse testes in the present immunohistochemical study. This suggests a possible involvement of EFA6A-ARF6 signaling in the karyokinesis and cytokinesis.     

VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the Rho family of small GTPases. As a member of this family, Rac1 promotes VEGF-induced endothelial cell migration by stimulating the formation of lamellipodia and membrane ruffles. To form these membrane protrusions, Rac1 is activated by guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP. The goal of this study was to identify the GEF responsible for activating Rac1 in response to VEGF stimulation. We have found that VEGF stimulates biphasic activation of Rac1 and for these studies we focused on the peak of activation that occurs at 30 min. Inhibition of VEGFR-2 signaling blocks VEGF-induced Rac1 activation. Using a Rac1 nucleotide-free mutant (G15ARac1), which has a high affinity for binding activated GEFs, we show that the Rac GEF Vav2 associates with G15ARac1 after VEGF stimulation. Additionally, we show that depleting endothelial cells of endogenous Vav2 with siRNA prevents VEGF-induced Rac1 activation. Moreover, Vav2 is tyrosine phosphorylated upon VEGF treatment, which temporally correlates with Rac1 activation and requires VEGFR-2 signaling and Src kinase activity. Finally, we show that depressing Vav2 expression by siRNA impairs VEGF-induced endothelial cell migration. Taken together, our results provide evidence that Vav2 acts downstream of VEGF to activate Rac1.     

Phosphorylation of the exchange factor DENND3 by ULK in response to starvation activates Rab12 and induces autophagy

Unc-51-like kinases (ULKs) are the most upstream kinases in the initiation of autophagy, yet the molecular mechanisms underlying their function are poorly understood. We report a new role for ULK in the induction of autophagy. ULK-mediated phosphorylation of the guanine nucleotide exchange factor DENND3 at serines 554 and 572 upregulates its GEF activity toward the small GTPase Rab12. Through binding to LC3 and associating with LC3-positive autophagosomes, active Rab12 facilitates autophagosome trafficking, thus establishing a crucial role for the ULK/DENND3/Rab12 axis in starvation-induced autophagy.