Phosphorylated guanine nucleotide exchange factor C3G, induced by pervanadate and Src family kinases localizes to the Golgi and subcortical actin cytoskeleton
© Radha et al; licensee BioMed Central Ltd. 2004
Received: 22 April 2004
Accepted: 20 August 2004
Published: 20 August 2004
The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context.
C3G is phosphorylated in vivo on Y504 upon coexpression with Src or Hck, two members of the Src family tyrosine kinases. Here we have determined the subcellular localization of this protein using antibodies specific to C3G and Tyr 504 phosphorylated C3G (pY504 C3G). While exogenously expressed C3G was present mostly in the cytosol, pY504 C3G formed upon Hck or Src coexpression localized predominantly at the cell membrane and the Golgi complex. Tyrosine 504-phosphorylated C3G showed colocalization with Hck and Src. Treatment of Hck and C3G transfected cells with pervanadate showed an increase in the cytosolic staining of pY504 C3G suggesting that tyrosine phosphatases may be involved in dephosphorylating cytosolic phospho-C3G. Expression of Src family kinases or treatment of cells with pervanadate resulted in an increase in endogenous pY504 C3G, which was localized predominantly at the Golgi and the cell periphery. Endogenous pY504 C3G at the cell periphery colocalized with F-actin suggesting its presence at the subcortical actin cytoskeleton. Disruption of actin cytoskeleton by cytochalasin D abolished phospho-C3G staining at the periphery of the cell without affecting its Golgi localization.
These findings show that tyrosine kinases involved in phosphorylation of C3G are responsible for regulation of its localization in a cellular context. We have demonstrated the localization of endogenous C3G modified by tyrosine phosphorylation to defined subcellular domains where it may be responsible for restricted activation of signaling pathways.
Guanine nucleotide exchange factors (GNEFs) are components of signaling pathways that link transmembrane receptors to intracellular GTPase family members regulating a wide variety of cellular functions such as proliferation, differentiation, adhesion and apoptosis. C3G (RapGEF1) is an ubiquitously expressed GNEF for Ras family proteins that particularly targets Rap1, Rap2 and R-Ras [1–4]. It has been shown to mediate signals received from B and T cell receptor activation, growth factors, cytokines, G protein coupled receptors and also adhesion [5–15]. C3G is present in the cytoplasm in a complex with members of the Crk family of small adapter molecules. In response to stimuli, this complex is recruited to the cell membrane involving association of Crk with phosphotyrosine containing proteins like receptor tyrosine kinases, p130 Cas, IRS-1 and paxillin [16–18]. Following translocation from cytosol to cell membrane, C3G activates downstream signaling. Its activation has been shown to lead to an activation of mitogen activated protein kinase and Jun N-terminal kinase [9, 12, 19–21]. Studies involving overexpression of membrane targeted C3G or dominant negative forms have shown that C3G is involved in both growth suppression as well as transformation [22–24]. C3G appears to play an important role in mammalian development because C3G-/- mice die before embryonic day 7.5. These studies have shown that C3G is required for vascular myogenesis and for cell adhesion and spreading [25, 26].
The C-terminus of C3G, which shows homology to CDC25, harbors the catalytic domain. The central region of C3G, which spans about 300 residues, has polyproline tracts with the ability to bind to SH3 domains of various proteins like Crk, p130 Cas, Grb2 and Hck [1, 2, 9, 18, 27]. No function has particularly been attributed to the N-terminal sequences, which do not show homology to any defined protein sequences. The non-catalytic domain of C3G has been shown to negatively regulate its catalytic activity. Deletion of the N-terminal sequences or its association through its proline sequences to Crk leads to its activation . Integrin mediated cell adhesion causes tyrosine phosphorylation of C3G . It has been shown that overexpression of c-Crk1 or stimulation of cells with growth hormone leads to specific phosphorylation of Y504 [21, 29]. This modification results in an increase in C3G catalytic activity towards Rap1. Src and JAK have been implicated in Y504 phosphorylation of C3G. More recently we have used site – specific antibodies to show that the activation of Src family kinase Hck, leads to C3G phosphorylation on Y504 suggesting that Src family kinases can directly regulate C3G activity and function .
The effectiveness and precision of intracellular signal transduction depends on protein-protein interactions that regulate enzyme activity as well as subcellular localization. Cell surface receptor activation leads to assembly of adaptor protein complexes at the plasma membrane, which serve to localize guanine nucleotide exchange proteins. Earlier, both endogenous as well as exogenously expressed C3G has been shown to localize to the cytoplasm and not to associate with plasma membrane [22, 30]. Since activation of C3G occurs primarily through phosphorylation at Tyr 504 and membrane recruitment, we undertook a detailed study of the subcellular localization of both exogenously expressed and endogenous Y504 phosphorylated C3G (pY504 C3G). Expression of Src family kinases or pervanadate treatment of cells, which mimics stimulation by growth factors, resulted in marked tyrosine phosphorylation of C3G at Y504. Conventional as well as optical sectioning microscopy revealed that pY504 C3G was predominantly located at the Golgi complex and the subcortical actin cytoskeleton unlike non-phosphorylated C3G, which was largely cytosolic.
Colocalization of C3G with Hck
Src family kinases phosphorylate C3G and phospho-C3G localizes to the Golgi and plasma membrane
Phosphorylation of endogenous C3G and its localization to the Golgi and subcortical actin cytoskeleton
The localization of endogenous pY504 C3G to the Golgi was examined in Cos-1 cells transfected with Hck and VSVG-GFP. Immunostaining for pY504 C3G was seen predominantly at the cell periphery and the Golgi (Fig. 4C), which was confirmed by colocalization with VSVG-GFP. The effect of Golgi perturbing drugs on the localization of pY504 C3G was examined by treatment of cells with nocadazole for depolymerization of microtubules and concomitant Golgi fragmentation. Under these conditions pY504 C3G was detected as dispersed vesicles scattered in the cytoplasm and remained colocalized with VSVG-GFP (4C) confirming that endogenous pY504 C3G localized to the Golgi complex.
We have observed that pervanadate treatment increased tyrosine phosphorylation of endogenous C3G. Since overexpression of Src as well as Hck results in phosphorylation of C3G, it was of interest to determine whether pervanadate induced tyrosine phosphorylation of C3G was mediated by Src family kinases within the cell. Cells were treated with PP2, a specific SFK inhibitor prior to PV treatment . As shown in Fig. 6B, pY504 C3G staining of cells stimulated with pervanadate was considerably reduced, but not totally abolished when they were pretreated with PP2 suggesting the possibility of other tyrosine kinase family members activated by pervanadate contributing to C3G phosphorylation. To determine whether the increase in pY504 C3G staining was dependent on the presence of an intact cytoskeleton, we observed its localization in cells treated with cytochalasin D, a reagent that effectively disrupts the actin cytoskeletal network. Under these conditions there is a collapse of cell morphology and F-actin staining shows an irregular distribution at the cell cortex. As observed in Fig. 6B, the staining for pY504 C3G in the subcortical cytoskeleton was largely absent under conditions of moderate disruption of actin organization. Under these conditions, pY504 C3G staining at the Golgi complex, which shows a more dispersed morphology appeared not to be affected.
C3G is involved in a variety of signaling pathways and therefore its dynamic localization under normal and activated situations may be physiologically relevant. In this study we demonstrate the limited subcellular distribution of Y504 phosphorylated C3G, which is predominantly targeted to the Golgi apparatus and the subcortical actin cytoskeleton. This localization has been substantiated by colocalization with a Golgi marker protein and F-actin respectively.
Rap1, the substrate of C3G has been localized to the Golgi, lysosomal vesicles and cortical actin cytoskeleton . But, of the at least eight known exchange factors for Rap1, C3G is the only one that has been linked definitively to the tyrosine kinase signaling pathway. Src family members like Src and Hck have been shown to localize to the plasma membrane and other intracellular membranes with particular concentration in the Golgi [31, 42]. When endogenous C3G was phosphorylated by overexpressed Hck or Src, the localization of pY504 C3G matched that of the kinases suggesting that they may be part of the same molecular complexes. This is also evident from the staining pattern of pY504 C3G when C3G is expressed along with Hck, which is distinctly seen in the Golgi and plasma membrane. Since exogenously expressed C3G is predominantly cytosolic, it implies that, at any given time, only a small fraction of it is phosphorylated at Y504 at the plasma membrane and the Golgi.
We observed more exogenously expressed pY504 C3G in the cytoplasmic compartment under conditions of inhibition of tyrosine phosphatases suggesting that pY504 C3G may be targeted by cytosolic tyrosine phosphatases. This regulation may help in restricting the activity of C3G to specific compartments. We observe very little endogenous pY504 C3G in the cytosol when HeLa or Cos-1 cells are treated with pervanadate, which not only inactivates tyrosine phosphatases, but also activates tyrosine kinases. It is possible that upon PV treatment endogenous C3G present in the cells is phosphorylated at the sites of location of the activated kinases. Pervanadate treatment has been shown to increase phosphotyrosine staining at the cell periphery indicating activation of kinases present in this subcellular domain .
Recently c-Src and Jak2 have been implicated in the phosphorylation of C3G in response to growth hormone stimulation of NIH 3T3 cells because dominant negative mutants of these kinases inhibit C3G phosphorylation . It was suggested that this phosphorylation of endogenous C3G by c-Src occurs at Y504 because exogenously expressed Y504F mutant of C3G was not phosphorylated. Using a phosphospecific antibody we have directly shown the phosphorylation of endogenous C3G at Y504 upon overexpression of Hck  and c-Src (this report). c-Src is present in Cos-1 and HeLa cells which lack Hck. c-Src localizes to the cell membrane, focal adhesions and also to the Golgi [42, 44]. Since pervanadate is a good activator of Src (36,37), it is possible that pY504 C3G seen in pervanadate treated cells is because of C3G being a Src substrate.
Adhesion dependent Src activation leads to Rap-1 activation mediated by Crk and C3G . Fibroblasts lacking C3G are essentially compromised in adhesion-mediated responses [25, 26]. The localization of endogenous pY504 C3G at the subcortical actin cytoskeleton therefore suggests that this may be the site of action of C3G in mediating responses to cell adhesion. Modification of C3G by phosphorylation at defined subcellular domains may be important for restricted activation of C3G mediated signaling functions in the cells. Close structural and functional relationship is known to exist between the structural elements at the cell periphery and the signal transduction machinery. Several tyrosine kinases are known to be located in adherence junctions and the kinetics of phosphorylation and dephosphorylation appears to be controlled by structural molecules at the junctions. Our observation that disruption of actin cytoskeleton results in a loss of pY504 C3G staining at the cell periphery, but not at the Golgi complex reveals an important role for cytoskeletal network in the regulation of C3G.
The activity of guanine nucleotide exchange factor C3G is known to be regulated by tyrosine phosphorylation and membrane targeting. Using phospho-specific antibodies, we directly demonstrate that expression of Src family kinases or pervanadate treatment of cells induces phosphorylation of C3G on Y504. Unlike C3G, which is mostly cytosolic, pY504C3G locates to the Golgi and subcortical actin cytoskeleton. Demonstration of the localization of the active component of C3G to the Golgi and subcortical cytoskeleton provides evidence for a possible function for C3G at these cellular compartments.
Cell culture and treatment of cells
HeLa and Cos-1 cells were cultured in DMEM supplemented with 10% FCS. Transfections were performed on cells grown as a monolayer in either 35 mm dishes or glass coverslips using the cationic lipid DHDEAB as described . Briefly, 1 μl lipid diluted in 50 μl serum free DMEM was mixed with 1 μg DNA in 50 μl serum free DMEM. The mix was kept at room temperature for 30 min to allow complex formation before adding to the cell monolayer. Cells were fed with serum 5 hrs later and harvested 24–30 hrs after transfection.
Cells were subjected to pervanadate treatment by the addition of a freshly prepared solution of pervanadate at 50 μM conc. for 10 min prior to harvesting. Pervanadate stock solution (50 mM) was prepared by mixing equal volumes of 100 mM solution of H2O2, with 100 mM solution of sodium orthovanadate. It was added to the cells within 5 mins of preparation. Golgi disruption was performed by treating the cells with 5 μg/ml of nocadazole for 30 min prior to fixation. To disrupt actin cytoskeleton, cells were treated with 1 μg/ml cytochalasin D for 20 mins. PP2 was added to cells 2 hrs before pervanadate treatment at a concentration of 10 μM to inhibit Src family kinases.
Full length human C3G cloned in pcDNA3-FLAG was kindly provided by Dr S Tanaka. Y504F mutant of C3G in which tyrosine 504 is mutated to phenylalanine cloned in a His tagged expression vector was provided by Dr M Matsuda. The wild type rat p59 Hck cDNA was cloned in the pCI plasmid (Promega) and has been described earlier . Expression plasmid for vesicular stomatitis virus glycoprotein as a GFP fusion protein (VSVG-GFP) was a kind gift from Jennifer Lippincott-Schwartz . c-Src-GFP expression vector expressing wild type c-Src fused to GFP at C-terminal was from Dr D L Anders . Wild type human Hck cDNA cloned into pCDNA6 expression vector was a kind gift of Dr Todd Miller .
Whole cell lysates were prepared by lysing cells directly in Laemli's sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. After transfer onto nitrocellulose membranes, they were processed for western blotting using the required primary antibodies. Detection was based on either color development using alkaline phosphatase conjugated secondary antibodies or on chemiluminescence using horse radish peroxidase conjugated secondaries.
Indirect immunoflourescence and microscopy
Cells were processed for immunoflourescence staining as described earlier . The primary antibodies used were rabbit polyclonal anti-C3G (Santa Cruz Biotechnology), rabbit polyclonal anti pY504-C3G (SC-12926 R from Santa Cruz) and anti-Hck (3E9 monoclonal) made in our laboratory . Dual labeling for Hck and C3G was performed by incubating the cells serially with C3G antibody, anti rabbit Cy3, monoclonal anti-Hck, and anti-mouse FITC. Cells were incubated with Oregon-green phalloidin after staining for pY504 with Cy3 to visualize F-actin. Cells transfected with vectors encoding GFP fusion proteins (GFP-Src or VSVG) were observed directly by fluorescent microscopy. Dual labeling for the C3G constructs and phospho-C3G was performed using the corresponding monoclonal tag antibodies (detected by FITC) and pY504 antibody (detected by Cy3). C3G was detected using Flag tag antibody (from Sigma) and Y504FC3G by His tag antibody (from Qiagen). Cells were examined using an Olympus microscope equipped with a cool SNAP color CCD camera. Images were captured using Image Pro Plus software. Immunoflurescence staining and colocalization was also observed using a Zeiss Axioplan 2 microscope fitted with an Apotome. The apotome (from Carl Zeiss Microimaging) is a new 3D imaging system for contrast enhancement in fluorescence microscopy. It uses structured illumination to reject signals belonging to regions of the sample that are outside the best focus position of the microscope. Images were captured using the Axiocam (Zeiss) CCD camera and processed using the Axiovision 4 software. Colocalization was also determined by observing the staining patterns using the LSM 510 Meta confocal microscope from Carl Zeiss.
guanine nucleotide exchange factor
Src family kinase
- pY504 C3G:
Tyrosine 504 phosphorylated C3G
Vesicular stomatitis virus glycoprotein
Dulbecco's modified Eagle's medium
We wish to acknowledge Ms Nandini R for help with confocal microscopy.
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