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Previous Article | Next Article 
Molecular and Cellular Biology, November 2002, p. 7953-7966, Vol. 22, No. 22
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.22.7953-7966.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117 609, Singapore
Received 11 March 2002/ Returned for modification 22 April 2002/ Accepted 12 August 2002
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ABSTRACT |
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, which binds PtdIns(4,5)P2. The plasma membrane localization of hSpry2TD was abolished in ionomycin-treated MDCK cells or when PtdIns(4,5)P2 was specifically dephosphorylated by overexpression of an engineered, green fluorescent protein-tagged inositol 5-phosphatase. Similarly, Spred, a novel Ras/MAPK inhibitor recently found to contain the conserved cysteine-rich SpryTD, also translocated to peripheral membranes and bound to PtdIns(4,5)P2. Alignment of the Spry and Spred proteins led us to identify a translocation-defective point mutant, hSpry2 D252. Targeting of hSpry2 to PtdIns(4,5)P2 was shown to be essential for the down-regulation of Ras/MAPK signaling. |
INTRODUCTION |
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Spry isoforms specifically translocate to membrane ruffles upon RTK stimulation (11). Ruffles are cell peripheral-membrane protrusions enriched with a meshwork of filamentous actin (24). Rac1 is a key regulator in reorganizing actin cytoskeletal structures for membrane ruffle formation, while Cdc42 and RhoA activation results in the formation of microspikes and RhoA stress fibers, respectively (16).
There has been a paucity of details pertaining to the biochemistry of ruffle formation. Recently, the synergistic activation of phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K] by phosphatidic acid (PA) and Arf6 was reported to be important for membrane ruffling (6). The authors proposed a pathway whereby Rac1 activation leads to actin reorganization, in which the up-regulation of PI(4)P5K and resultant production of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] are necessary intermediate stages. In other studies PI(4)P5K was demonstrated to be the target of Rac1 in both pollen tube formation (10) and actin polymerization in platelets (27). The hydrolysis of PtdIns(4,5)P2 by phospholipase C
(PLC), the removal of phosphate by inositol 5-phosphatase (5P), phosphorylation at the 3 position by phosphatidylinositol 3-kinase (PI3K), and the reversible sequestration of the lipid by various membrane-located proteins keep the level of free PtdIns(4,5)P2 in the cells tightly regulated (26).
Several protein domains have been shown to target inositol phospholipids. FYVE (Fab1p, YOTB, Vac1p, and EEA1) and PX (Phox homology) domains play important roles in membrane trafficking of endosomes and lysosomes and generally bind to PtdIns lipids with a phosphate in the 3 position of the inositol ring (31). Pleckstrin homology (PH) domains, which are found mostly in signaling molecules, bind variably to inositol lipids with a wide range of affinity and specificity (1, 9). On the other hand, FERM (protein 4.1, ezrin, radixin, and moesin) and ENTH (epsin N-terminal homology) domains, which are involved in cytoskeletal organization and/or endocytosis, are believed to specifically bind PtdIns(4,5)P2 (8).
Recently, a novel class of Ras/MAPK inhibitor proteins named Spred (Sprouty-related EVH1 domain-containing protein) was identified (29). Both Spred-1 and Spred-2 contain a cysteine-rich domain related to the SpryTD. This domain most likely serves as a targeting domain in these proteins, as it appears to do with Spry isoforms. This finding indicates that the sequence has been conserved to perform a specific function in signaling modification, most likely the precise targeting of a select group of Ras/extracellular signal-related kinase (ERK)-inhibiting proteins.
Our aim was to identify the cellular target of the SpryTD. Previous evidence had indicated that the target "appeared" during membrane ruffle formation. We reasoned that the cellular target could be (i) a modified protein, as seen in the recruitment of Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains to the phosphotyrosine sites of RTKs or (b) a newly synthesized phospholipid, e.g., PtdIns(4,5)P2, which is responsible for targeting of PH and FERM domain-containing proteins (14, 21), or that (c) SpryTD could bind to a domain on an as yet unspecified protein that is involved in translocation to the membrane.
In this study we show that the plasma membrane localization of SpryTD is due to direct PtdIns(4,5)P2 binding and that this interaction is essential for the full function of human Spry2 (hSpry2) as an inhibitor of Ras/MAPK signaling.
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MATERIALS AND METHODS |
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and GFP-tagged 5P were obtained from Hitoshi Yagisawa (Himeji Institute of Technology, Himeji, Japan) and Tobias Meyer (Stanford University, Palo Alto, Calif.), respectively. Various constructs of Spry1, Spry2, and Spry4 were generated as described previously (11) by using pGEX4T1 as a cloning vector for GST fusion protein production. Point mutations were engineered with a site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) according to the manufacturer's instructions. Cell culture and transfection. PC12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (4.5 g of glucose/ml) supplemented with 10% fetal bovine serum, 5% horse serum, 2 mM L-glutamine, 10 mM HEPES (pH 7.4), 100 U of penicillin/ml, and 100 U of streptomycin/ml. COS-1 monkey kidney cells and Madin-Darby canine kidney (MDCK) cells were grown and maintained in DMEM with 1 g of glucose/ml; Swiss 3T3 fibroblast cells were grown and maintained in DMEM with 4.5 g of glucose/ml; and 293T human kidney epithelial cells were grown and maintained in RPMI medium. Culture medium was supplemented with 10% fetal bovine serum (HyClone Laboratories), 2 mM L-glutamine, 10 mM HEPES (pH 7.4), 100 U of penicillin/ml, and 100 U of streptomycin/ml. Cells were seeded in six-well plates containing glass coverslips for immunofluorescence studies or in 60-mm-diameter culture dishes for Western blot analyses. Cells at 80% confluency were transfected with various expression plasmids using LipofectAMINE 2000 reagent (Life Technologies Inc., Rockville, Md.) according to the manufacturer's recommendations. Four to six hours later, the transfection medium was aspirated, and the cells were washed twice with warm phosphate-buffered saline and incubated in 10% serum-containing medium for 24 h. The cells were then washed and maintained in serum-free medium overnight. Prior to being fixed or lysed, cells were either left untreated or stimulated with 50 ng of epidermal growth factor (EGF; Sigma Aldrich, St. Louis, Mo.)/ml for 10 min.
Lipid vesicle-binding assay. Phosphatidylcholine (PC; Sigma Aldrich) and the indicated lipids were solubilized in chloroform and dried under a stream of nitrogen. Lipids were then suspended in buffer A (170 mM sucrose buffered with 50 mM Tris to pH 7.3-100 mM sodium chloride) with PtdIns(4,5)P2 (Sigma) to a final concentration of 5% (vol/vol). Aliquots (0.5 ml) of 10 to 50 mM lipid were subjected to five freeze-thaw cycles and filtered repeatedly through 0.2-µm-pore-size filters. For "dose-dependent" binding studies, vesicles were made to contain increasing concentrations of PtdIns(4,5)P2 or phosphatidylserine (PS), as indicated. Vesicles were mixed with the sonicated COS-1 lysates in buffer A (with a cocktail of protease inhibitors [Roche Applied Science, Indianapolis, Ind.]), which had been precleared by centrifugation. The mixtures were immediately subjected to ultracentrifugation at 100,000 x g for 30 min at 4°C. One-tenth of the supernatant or the entire pellet of each sample was mixed with 2x Laemmli buffer before separation by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (SDS-12.5% PAGE) followed by Western-blotting with rabbit polyclonal anti-GFP (Santa Cruz, Santa Cruz, Calif.) or monoclonal anti-FLAG M2 (Sigma Aldrich). Western blots were developed by ECL (Amersham Biosciences Inc., Little Chalfont, Buckinghamshire, United Kingdom).
Amino acid sequence alignment. Partial amino acid sequences were extracted from the National Center for Biotechnology Information protein database. The GenBank accession numbers are as follows: hSpry2, NP_005833; hSpry1, XP_036349; hSpry4, XP_096029; hSpry3, O43610; mSpred1, BAB62848; mSpred2, BAB62849. Alignment was performed by using the DNASTAR Clustal method with a structural residue weight table.
Phospho-MAPK assay and immunoprecipitation. The antibody against phospho-ERK was from New England Biolabs (Beverly, Mass.), and monoclonal antibodies against horseradish peroxidase-conjugated phosphotyrosine (PY20), pan-ERK, and ERK2 were from Transduction Laboratories (Lexington, Ky.). Cells were harvested at 40 h posttransfection and lysed in 1 ml of lysis buffer (20 mM HEPES [pH 7.4], 137 mM sodium chloride, 1.5 mM magnesium chloride, 1 mM EGTA, 10% [vol/vol] glycerol, 1% Triton X-100, a mixture of protease inhibitors, and 0.2 mM sodium orthovanadate). Protein concentrations of cell lysates were normalized by use of a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.) before incubation with various antibodies. FLAG-tagged proteins were immunoprecipitated by using mouse monoclonal anti-FLAG M2 agarose-conjugated beads for 2 h at 4°C. Immunoprecipitates were collected by centrifugation and washed three times with lysis buffer. Eluted proteins were resolved on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After being blocked in phosphate-buffered saline containing 1% bovine serum albumin and 0.1% Tween 20 for 1 h at room temperature, the membranes were probed with 1 µg of the primary antibody/ml followed by 0.5 µg of the horseradish peroxidase-conjugated secondary antibody/ml. Immunoreactive protein bands were visualized by ECL.
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RESULTS |
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We reasoned that the target for the SpryTD was therefore upstream of actin polymerization and downstream of Rac1 activation. Assessment of collated data and current models led to the suggestion that PtdIns(4,5)P2 is a likely target for the SpryTD in activated cells. PtdIns(4,5)P2 has also been shown to be a key factor in the formation of ruffles by Rac1 and is concentrated in actin-rich protrusions (25).
hSpry2 binds to PtdIns(4,5)P2. The likely binding of Spry proteins to PtdIns(4,5)P2 was assessed by using a lipid vesicle-binding assay coupled with ultracentrifugation. In the first experiment the ability of hSpry2 to bind to a range of phospholipids was assessed, and the binding to vesicles containing 5% of the indicated phospholipid is shown in Fig. 3A . There is essentially no binding to PC, PS, diacyl glycerol (DAG), or phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. There is differential binding to PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(4,5)P2, PtdIns(3,4)P2, PtdIns(3,5)P2, and PA. The highest level of binding is to PtdIns(4,5)P2. In the physiological context it has been shown previously (11) that PtdIns(3)P-substituted lipids are unlikely to play a major role in the translocation, because there is no prominent inhibition of membrane targeting when cells are treated with wortmannin (a PI3K inhibitor). Experiments utilizing an in vivo PA-binding probe have previously shown that PA is enriched in vesicles in stimulated cells and has scant presence on the cell membrane (19). We performed a similar experiment with the same probe and verified that PA is essentially localized to endosome-like structures (Fig. 3B). PA was therefore deemed not to be the target of hSpry2TD.
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) as a positive control and FLAG-tagged annexin II and the nonbinding GFP-PH mutant D40 as negative controls. GFP-PH (PLC) was enriched in the pellet after ultracentrifugation, while the GFP-PH D40 mutant and annexin II remained in the supernatant, confirming a lack of specific phospholipid binding (Fig. 3Ca and Cb). Full-length hSpry2 or hSpry2TD (hSpry2/TD) was detected in the pellet (Fig. 3Cb), whereas the N-terminal nonconserved region of hSpry2 was almost entirely present in the supernatant (data not shown). In dose-response experiments, increasing the amount of PtdIns(4,5)P2 in vesicles increased the abundance of hSpry2/TD in the pellet, whereas comparative increases in PS barely evoked any protein in the pellet fraction (Fig. 3Cc). As expected, a dose-dependent increase in the abundance of hSpry2/TD in the pellet and a concomitant decrease in the supernatant were apparent with the PH domain of PLC (Fig. 3Cd).
The SpryTD colocalizes with the PtdIns(4,5)P2-binding PH domain (PLC) following various experimental procedures in different cell types. (i) Cytosol to membrane translocation.
We next investigated whether hSpry2/TD and the PH domain of PLC colocalize in cells. Free PtdIns(4,5)P2 has been variously reported to appear in the membranes of stimulated cells and sometimes in nonstimulated cells. We set up experiments to test the location of hSpry2/TD in various cells and with various agonists in comparison to the disposition of the PH domain of PLC. First, COS-1 cells were transfected with FLAG-hSpry2TD or GFP-PH (PLC) and the cells were either left unstimulated or stimulated with EGF (50 ng/ml) for 10 min. From the images shown in Fig. 4A, it is apparent that both hSpry2TD (a and b) and GFP-PH (PLC) (c and d) were translocated to ruffles at the cell periphery upon stimulation. Next, the cellular localizations of PtdIns(4,5)P2, GFP-PH, and the D40 mutant of PLC were assessed following their microinjection into Swiss 3T3 cells with combinations of Rac1 and hSpry2/TD. As previously shown, hSpry2/TD localized to membrane ruffles (Fig. 4Ba and Bd). GFP-PH (PLC) (Fig. 4Bb) but not GFP-PH (PLC) D40 (Fig. 4Be) was localized to membrane ruffles induced by active Rac1. In all of the many experiments performed, hSpry2/TD colocalized with GFP-PH (PLC), as exemplified by the image shown in Fig. 4Bc.
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) translocate into membrane ruffles from the cytosol when resting COS-1 cells are stimulated with EGF. Conversely, Stauffer et al. (21) and Várnai and Balla (28), similarly using the PH domain of PLC to monitor the dynamics of PtdIns(4,5)P2 levels, found that the probe moved from the plasma membrane to the cytosol in response to receptor activation. To test whether hSpry2 behaves similarly in similar circumstances, we overexpressed GFP-PH (PLC) or FLAG-tagged hSpry2 in MDCK cells, a line previously shown to exhibit plasma membrane localization of the PH domain of PLC in unstimulated cells (3). Indeed, both tagged molecules were observed to localize in the plasma membranes of unstimulated cells (Fig. 5Aa and Ad). It is therefore apparent that the disposition of PtdIns(4,5)P2 that can be detected by these specific probes is cell type and stimulation dependent.
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is responsible for the hydrolysis of PtdIns(4,5)P2 to Ins(1,4,5)P3 and DAG in the plasma membrane in a calcium-dependent manner (28). To assess whether membrane targeting of hSpry2 is abolished when PtdIns(4,5)P2 is depleted, we treated MDCK cells with the calcium ionophore ionomycin to induce a calcium influx. We observed a relocation of hSpry2 from the plasma membrane to the cytosol (Fig. 5Ae). It should be noted that although a high proportion of the previously membrane-located probes was now in the cytosol, the transition was not complete and some weak membrane staining is apparent. The reverse effect was observed when hSpry2-transfected MDCK cells were sequentially treated by ionomycin and the cell-permeant, calcium-chelating reagent BAPTA (Fig. 5Ac and Af). Following the BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] treatment, both probes show a more definitive membrane staining than was apparent in unstimulated cells. An alternative method was employed to specifically reduce the level of PtdIns(4,5)P2 in cell peripheral membranes. We utilized a constitutively active, membrane-targeting, GFP-tagged yeast 5P which has previously been shown to greatly decrease levels of PtdIns(4,5)P2 in target cells (18). We cotransfected this construct with hSpry2 into MDCK cells. When the 5-phosphatase was overexpressed in cells (Fig. 5Ba), the plasma membrane distribution of hSpry2 was almost completely abolished (Fig. 5Bb). Moreover, the microtubule staining was enhanced and was as prominent as that observed with unstimulated COS-1 cells (11).
In short, the membrane-targeting behavior of hSpry2 was demonstrated to be most likely due to the specific interaction with PtdIns(4,5)P2, since reduction of PtdIns(4,5)P2 levels in the membrane by various means resulted in the relocalization of hSpry2/TD to the cytosol.
The SpryTD in Spred proteins also mediates translocation and PtdIns(4,5)P2 binding. In our initial studies with the SpryTD, we queried why this highly conserved domain was not found in other proteins outside the Spry family. Recent identification of the Spred proteins revealed that the cysteine-rich translocation domain is also present in these proteins (29). We therefore asked if Spred proteins behave in a manner analogous to that of Spry proteins when cells are stimulated with growth factors. The cellular disposition of Spred-1 was assessed by immunofluorescent staining combined with confocal microscopy. Not surprisingly, Spred-1 translocated to the membrane ruffles in EGF-stimulated COS-1 cells (Fig. 6A). Vesicle-binding assays were then performed, and Spred-1 was revealed to be enriched in the PtdIns(4,5)P2-containing vesicle pellets (Fig. 6B). These experiments clearly indicated that the SpryTD in Spred-1 also functions to direct the protein toward enhanced concentrations of free PtdIns(4,5)P2 in the plasma membrane.
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Taken together, these data demonstrate that with the D252 point mutation in SpryTD, there is substantially reduced binding or no binding to PtdIns(4,5)P2 and consequently no translocation to membrane ruffles in appropriately stimulated cells.
The hSpry2 D252 mutant has reduced binding to PtdIns(4,5)P2 compared to that of wild-type hSpry2 but binds similarly to associated proteins. Next we analyzed more closely the characteristics of the hSpry2 D252 mutant in terms of phospholipid binding, normality of folding, and its disposition in unstimulated MDCK cells. We subjected the hSpry2 D252 mutant to the vesicle-binding assay with the range of phospholipids previously used to characterize the binding of wild-type hSpry2/TD. From Fig. 8A it can be seen that the binding of the hSpry2 D252 mutant to the various inositol phospholipids is severely reduced from that of the wild-type protein in all cases.
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as a control, was performed to investigate this possibility. From the immunofluorescent images shown in Fig. 8B, it is apparent that while the PH domain (PLC) (Fig. 8Bb and Bd) and wild-type hSpry2 (Fig. 8Ba) locate to the plasma membrane, the D252 mutant is located in the cytosol (Fig. 8Bc) with little or no membrane binding. To ensure that the D252 mutant protein was being expressed in a native conformation, we assessed whether it could still associate with c-Cbl (30) and Grb2 (our unpublished data), previously characterized binding partners of the wild-type protein. We have shown that the N-terminal SH3 domain of Grb2 interacts directly with a canonical proline-rich sequence (PXXPXR) on the extreme C terminus of hSpry2 (unpublished data). COS-1 cells were transiently transfected with wild-type hSpry2 or the D252 mutant, the cells were lysed, and the lysates were subjected to a pulldown experiment with GST-Grb2 (N-terminal SH3 domain). Bound proteins were analyzed by Western blotting with anti-FLAG (Fig. 8Ca). Both full-length hSpry2 and the point mutant bound equally well to the N-terminal SH3 domain of Grb2.
The binding of hSpry2 and c-Cbl occurs distally to the C terminus, in the N-terminal region (30). Immunoprecipitation experiments were performed following transient expression of FLAG-tagged wild-type hSpry2 or its D252 mutant in COS-1 cells. Cell lysates were precipitated with anti-FLAG, and the associated proteins were analyzed by Western blotting using anti-c-Cbl (Fig. 8Cb). The blot was reprobed with anti-FLAG to demonstrate equality in expression and precipitation of the two forms of hSpry2. Taken together, these data lead to the conclusion that hSpry2 and its D252 mutant bind equally to Grb2 or c-Cbl, indicating that the D252 mutant behaves normally in terms of interaction with other proteins.
The translocation-defective hSpry2 D252 mutant loses its MAPK-inhibitory function. It has previously been shown that in a fibroblast growth factor (FGF)-signaling context, the phosphorylation of ERK was inhibited in cells overexpressing hSpry2 (33). To examine the role of the SpryTD in this inhibitory function, we transfected PC12 cells with FLAG-tagged wild-type hSpry2 or its D252 mutant and subsequently performed a phospho-ERK Western blot analysis. The ERK protein was potently phosphorylated in response to FGF stimulation, while its phosphorylation was reduced to almost basal levels in hSpry2-transfected cells. There was little or no inhibition of ERK phosphorylation in cells transfected with the D252 mutant (Fig. 9). Similar results were obtained by transfection studies in 293T cells (data not shown), indicating that the assay was not cell line specific.
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DISCUSSION |
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Although the single point mutants E183 and E185 did not disrupt hSpry2 translocation, deletion of amino-acids 178 to 194 (which include K183, K185, K187, and R193 in hSpry2) resulted in failure of the protein to translocate to membrane ruffles in EGF-stimulated cells (11) and abolished the inhibitory effect of hSpry2 on cell migration in COS-7 cells (32). The various conserved basic residues within the SpryTD could contribute synergistically to inositol lipid binding. We have worked mainly with the hSpry2/TD sequence. It is possible that subtle variations in the amino acid sequences of the various translocation domains could confer different binding specificities on the Spry and Spred family members.
In the studies of Honda et al. (6) and other groups (17, 20), Arf6 activation was instrumental in the formation of membrane ruffles. During the course of these studies in which we compared the dispositions of the PLC PH domain and hSpryTD, we employed the constitutively active form of Arf6 (L67 mutant), either by transfection or by microinjection. We were unable to induce ruffle formation either with the Arf6 mutant or with PI5K constructs, the products of which are postulated to be downstream of active Rac and instrumental to the ruffle formation cascade. We postulate that Rac is the key molecule in signaling for membrane ruffle formation, and it is likely that other targets of Rac, besides Arf6 and PI5K, are needed synergistically to drive actin reorganization.
We suggest that Spry proteins bind to various partner proteins via their respective N termini, targeting these proteins to a peripheral membrane location via PtdIns(4,5)P2 interaction following RTK activation, and are thus able to fulfill their respective physiological functions. The C terminus of hSpry2 is capable of translocating upon cellular stimulation and binds to PtdIns(4,5)P2 but does not inhibit the Ras/ERK pathway (33). This indicates that hSpry2TD is not by itself able to cause inhibition of the Ras/ERK pathway; it suggests, rather, that hSpryTD is a targeting domain and that critical proteins capable of controlling the Ras/ERK pathway bind to the N-terminal sequence. So far we have characterized c-Cbl as being one of these proteins, but there is currently no evidence to suggest that c-Cbl binding is responsible for the inhibition of the Ras/ERK pathway in the context of FGF signal transduction.
There is no compelling evidence to propose that binding of the SpryTD to PtdIns(4,5)P2 has any profound effect on the metabolism of the lipid or any downstream physiological function of transfected cells. In hSpry2-transfected cells, neither Ins(1,4,5)P3 production nor tyrosine phosphorylation of PLC was affected (data not shown). PtdIns(4,5)P2, like the other lipid messengers DAG and PA, enables the translocation of various catalytically active molecules such as PLC, protein kinase C, and Raf, respectively, in a diffusible, highly regulated, and rapidly reversible manner. This mode of localization-activation provides a sensitive response to rapid changes whereby subtle changes at the surface of the cell can be rapidly translated into a cytosolic signaling response.
It appears that inhibitory proteins of the Spry and Spred families utilize a novel and common PtdIns(4,5)P2-binding domain that targets the proteins to a cellular localization that maximizes their inhibitory potential. These inhibitory proteins seem to have arisen in evolution to control complex organ patterning. They are not found in Caenorhabditis elegans, and only one isoform of Spry is present in Drosophila, compared to at least four isoforms in mammals. There is considerable variation in the N termini of the various Spry proteins, but the SpryTD is highly conserved during evolution, which indicates a close link between sequence fidelity and specificity of function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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