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Department of Biochemistry, Tufts University School of Medicine,1 Division of Hematology/Oncology, New England Medical Center, Boston, Massachusetts 021112
Received 24 October 2001/ Returned for modification 10 December 2001/ Accepted 25 February 2002
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ABSTRACT |
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INTRODUCTION |
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The Rac GTPase becomes activated in cells upon interaction with one of multiple Rac-specific guanine nucleotide exchange factors (Rac-GEFs), which promote the release of GDP from Rac, allowing GTP to take its place. Rac-GEFs all have similar Dbl homology (DH) catalytic domains (9). Individual Rac-GEF family members differ in their ability to be activated by distinct upstream signals. One ubiquitously expressed Rac-specific GEF is Tiam1, which was first identified in a screen for genes that promote invasion in murine T lymphoma cells (20). Overexpression of Tiam1 causes oncogenic transformation in fibroblasts and invasiveness in lymphocytes (27). However, in epithelial cells Tiam1 expression suppresses invasion and promotes adhesion through E-cadherin-mediated cell-cell contacts (21). Tiam1 has also been implicated in axon formation in neurons through regulation of growth cone actin organization (24). Finally, Tiam1 expression has been implicated in regulating apoptosis in human leukemia cell lines (23).
Which extracellular signals enhance Tiam1's ability to activate Rac in cells and how this activation is accomplished are not well characterized. Elevated calcium has been reported to increase the protein's intrinsic GEF activity (18), while platelet-derived growth factor has been shown to target Tiam1 to the plasma membrane (5), which could promote its interaction with Rac. The domain structure of Tiam1 implicates specific regions of the protein as mediators of responses to extracellular signals. Near the N terminus are an adjoining pleckstrin homology (PH) domain, a coiled-coil (CC) domain, and an undefined region termed Ex, which cooperatively function to localize Tiam1 to the membrane and which are required for Tiam1-mediated membrane ruffling and Jnk activation (28, 37). Tiam1 has been reported to interact with the hyaluronic acid receptor CD44 through this region, stimulating Tiam1-mediated Rac signaling and tumor cell migration (4). This cluster of motifs is strikingly similar to one found in Ras-GRF1 (36) (also referred to as CDC25Mm [26]), an exchange factor capable of activating both Ras (36) and Rac (22) GTPases. In Ras-GRF1, this region has been shown to play a role in targeting the protein to the membrane fraction of cells and in the activation of the protein by calmodulin binding in the presence of calcium (6).
The fact that Rac proteins can activate multiple downstream target proteins suggests that mechanisms exist to limit Rac target activation to generate signaling specificity. In fact, some evidence has implicated Rac-GEFs in this process since transfections of individual members of this family of GEFs generate different cellular responses even though they activate Rac similarly (46). In addition, one Rac-GEF, PIX, has been shown to bind directly to and help activate the Rac target protein PAK (13).
Another Rac target, MLK3, has been shown to complex with the IB/JIP family of scaffolds for the Jnk mitogen-activated protein (MAP) kinase cascade (45). JIP1/IB1 is required for proper Jnk kinase signaling in mice (43) and could potentially contribute to Rac signaling specificity. Here we show that two related Rac-GEFs, Tiam1 and Ras-GRF1, have unique N termini that allow them to bind to a member of this scaffold family, IB2/JIP2. Furthermore, we show that IB2/JIP2 functions as a scaffold for a p38 signaling cascade in mammalian cells. The binding of IB2/JIP2 to these Rac-GEFs connects a specific scaffolding protein to an upstream activator of its kinase components. This interaction influences Rac GTPase signaling by promoting p38 over Jnk activation in cells.
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MATERIALS AND METHODS |
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Plasmids.
Plasmids encoding cDNAs for HA-Tiam C-1199, 
PCX-Tiam-1, Dbl, GST-p38, and GST-Jnk were kindly provided by J. Collard, M. Symons, R. Cerione, and T. Roberts. Plasmids encoding Flag-tagged human JIP1 and JIP2 were kindly provided by R. Davis. Plasmids encoding HA-tagged MKK3ß, MKK6ß, and MKK7 were kindly provided by G. Johnson, and HA-MLK3 was provided by K. Gallo. Ras-GRF1 cDNA has been previously described (36). To obtain plasmids encoding glutathione S-transferase (GST) fusions with amino acids 1 to 306 of rat IB2/JIP2 (rIB2), 127 to 202 of JIP1/IB1, and 1 to 229 of human IB2/JIP2, PCR products using appropriate primers on the respective scaffold cDNA templates were ligated into the pGEX5X3 vector. A similar method was used to derive GST-rIB2-511-835. rIB2 cDNAs containing deletions in the Tiam1-binding domain (398-422 and 434-522) were derived by overlap PCR and subcloned into the pJ3M vector. rIB2 cDNA with a deletion of the N-terminal 306 amino acids (rIB2-N306) was derived by PCR using appropriate primers on a full-length rIB2 cDNA template. rIB2-1-693, lacking the C-terminal 142 residues spanning the phosphortyrosine binding (PTB) domain, was derived by a similar method.
Antibodies and immunoblotting. Antibodies to Tiam1 (Santa Cruz), Ras-GRF1 (Santa Cruz), Dbl (Santa Cruz), HA-epitope tag (Covance), Myc-epitope (Santa Cruz), Flag-epitope (Sigma), GST (Sigma), p38, phospho-ATF2, phospho-Jun, and phospho-p38 (Cell Signaling) were used according to the manufacturers' instructions. Antibody to IB2 was kindly provided by M. Goldfarb. Secondary antibodies, immunoblotting, and the chemiluminescence protocol for developing blots have been previously described (6).
Cell culture and transfection. COS7 cells were grown in Dulbecco's modified Eagle's medium containing 10% iron-supplemented bovine calf serum (HyClone) in an incubator with humidified air (5% CO2) at 37°C. HEK 293T cells were grown as described above in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (HyClone). Cells were plated in 60-mm-diameter plastic dishes and transient transfections were performed using DEAE-dextran or Lipofectamine 2000 (Gibco BRL) according to the manufacturer's instructions. Cells were harvested 48 h after transfection. When indicated, cells were deprived of serum for 16 h prior to harvest.
Biochemical assays. (i) GST pull-down assay. Transiently transfected cells were washed with cold phosphate-buffered saline (PBS), pelleted, and lysed in 500 µl of buffer S (50 mM Tris [pH 8.0], 120 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing protease inhibitors (10 µg of aprotinin per ml, 20 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) and phosphatase inhibitors (50 µM sodium fluoride and 100 µM sodium orthovanadate). Lysates were cleared of unbroken cells and debris by centrifugation at 10,000 x g for 10 min and were incubated with equal amounts (usually 5 to 10 µg) of GST fusion proteins immobilized on S-hexyl-glutathione-agarose beads (Sigma) for 2 h at 4°C with constant agitation. Aliquots of cleared lysate were retained prior to precipitation for immunoblotting. After washing three times with ice-cold buffer S, bound proteins were eluted in 4x Laemmli buffer, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted.
(ii) IPs. Transfected cells were harvested, washed with cold PBS, pelleted, and lysed in buffer S containing protease and phosphatase inhibitors as described above. Cleared lysates were incubated with protein A-Sepharose beads (Pharmacia) and appropriate antibody (diluted according to the manufacturer's instructions) for 2 h at 4°C with constant agitation. Aliquots of cleared lysate were again retained prior to immunoprecipitation (IP) for immunoblotting. After washing three times with ice-cold buffer S, bound proteins were eluted in 4x Laemmli buffer, resolved by SDS-PAGE, and immunoblotted. For experiments on endogenous proteins, lysates were precleared on protein A-Sepharose beads loaded with rabbit immunoglobulin G (IgG). For experiments on scaffold-associated proteins, cells were lysed in buffer M (20 mM Tris [pH 7.4], 1% Triton X-100, 1 mM MgCl2, 125 mM NaCl). For quantification of the effects of exchange factor on scaffold binding activity, comparisons were made between signals from serial dilutions of control and experimental samples.
(iii) Immune complex kinase assays. (a) p38 activation. Transfected cells were washed with cold PBS, pelleted, and lysed in buffer M containing protease and phosphatase inhibitors as described above. Cleared lysates were incubated with S-hexyl-glutathione-agarose beads (Sigma) for 2 h at 4°C with constant agitation. Immobilized GST-p38 was washed three times with ice-cold PBS containing 1% Triton X-100 and one time with kinase buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl2). Kinase reactions proceeded for 15 min at room temperature in kinase buffer containing 1 µM dithiothreitol, 50 µM NaF, 100 µM orthovanadate, 50 µM GST-ATF2, and 25 µM ATP. Reactions were stopped with the addition of Laemmli buffer, resolved by SDS-PAGE, and immunoblotted. (b) Jnk activation. Transfected cells were processed as described above and kinase reactions were performed using purified GST-Jun as substrate.
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RESULTS |
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We then used transfected proteins to map the interaction sites on Tiam1 and rIB2. Myc-tagged rIB2 was transfected with either empty vector, a vector encoding Tiam1 (1,199 C-terminal amino acids lacking the destabilizing N-terminal PEST sequence [27]), or a vector encoding a Tiam1 mutant with an additional deletion of the N-terminal PH, CC, and Ex regions (PCX-Tiam1). Tiam1 was then immunoprecipitated and rIB2 was detected by immunoblotting with anti-Myc antibodies. rIB2 was detected in the immune complexes only when Tiam1 was transfected into cells (Fig. 3A, upper panel, compare lanes 2 and 4). Moreover, when the N-terminal PH, CC, and Ex domains used as bait in the two-hybrid experiments were deleted (PCX-Tiam1), there was little rIB2 observed in immune complexes (Fig. 3A, upper panel, lane 6). Lanes 3 through 6 of the lower panel show that the Tiam1 deletion mutant was expressed at even higher levels than wild-type Tiam1. Myc-rIB2 was also expressed at comparable levels in all experiments (Fig. 3A, upper panel, lanes 1, 3, and 5). These findings confirm that Tiam1 can bind to rIB2 in cells through the PH-CC-Ex motifs of the Rac-GEF.
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398-422) or C-terminal (434-522) residues of this region (see Fig. 1C). Whereas the wild type and rIB2398-422 bound Tiam1 in cells (Fig. 3B, upper panel, lanes 2 and 4), rIB2434-522 did not (Fig. 3B, upper panel, lane 6). Again, the mutant rIB2s (lanes 3 and 5) were present in cell extracts at levels comparable to wild-type rIB2 (lane 1) and Tiam1 was present at comparable levels in all immunoprecipitates (lower panel, lanes 2, 4, and 6). Taken together, these mutagenic studies argue that residues 434 to 522 of rIB2 interact with sequences in the N-terminal PH, CC, and Ex regions of Tiam1. Tiam1 binds to other IB/JIP-related proteins. The human proteins JIP1/IB1 and IB2/JIP2 were also tested for their ability to bind to Tiam1, as there is significant amino acid sequence similarity among IB/JIP family proteins in the Tiam-binding region of rIB2. Tiam1 was cotransfected into COS7 cells with either Myc-rIB2, Flag-huJIP1, or Flag-huIB2/JIP2. Tiam1 was immunoprecipitated and the IB/JIP-related proteins were detected by immunoblotting with appropriate epitope antibodies (Fig. 4). Human JIP1 and IB2/JIP2 were also detected in Tiam1 immune complexes as long as Tiam1 was transfected (Fig. 4a, b, and c, compare lanes 2 and 4). Despite being expressed at high levels, human JIP1 was consistently found at lower levels on Tiam1 immune complexes in growing cells, implying possible differences in binding affinity or regulation between Tiam1 interactions with IB2/JIP2 and JIP1.
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Binding of rIB2 to MAP kinases. Since human IB1/JIP1 and IB2/JIP2 have been reported to act as scaffold proteins for components of the Jnk signaling cascade, rIB2/JIP2 binding properties were assessed. As previously reported (16), a fusion protein of GST and the N-terminal sequences of JIP1 (GSTnJIP1) clearly precipitated Jnk from lysates of transiently transfected COS7 cells (Fig. 5A, lane 4). However, we were unable to demonstrate Jnk binding to a fusion protein of GST with the corresponding region of rIB2 (GSTn-rIB2) (Fig. 5A, lane 3). Moreover, a similar fusion construct of huIB2/JIP2 (GSTn-huIB2) also failed to precipitate Jnk from cell lysates (Fig. 5A, lane 5).
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from cell lysates of transiently transfected COS7 cells. As previously reported, GST-JIP1 failed to precipitate p38 (Fig. 5A, lane 9). In contrast, the analogous N terminus of rIB2, which could not precipitate Jnk, did precipitate transiently transfected p38 kinase (Fig. 5A, lane 8). The comparable N terminus of human IB2/JIP2 also precipitated p38 (Fig. 5A, lane 10). To confirm that rIB2 and p38 can interact in vivo, epitope-tagged scaffold proteins (rIB2, human IB1/JIP1, or human IB2/JIP2) and MAP kinases (GST-p38, GST-Jnk, or empty vector) were transfected into COS7 cells. MAP kinases were precipitated on glutathione beads and then tested for the presence of scaffold protein (Fig. 5B). As predicted from the experiments for Fig. 5A, rIB2 was clearly present on beads when GST-p38 was transfected but not when empty vector was transfected (compare upper panel, lanes 2 and 6). In contrast, only a faint rIB2 signal was observed when GST-Jnk was precipitated from cells (upper panel, lane 4), despite the fact that similar amounts of HA-rIB2 were expressed in cell lysates (compare lanes 1 and 3). Consistent with the results described for Fig. 5A, human IB2/JIP2 behaved like rIB2 and was clearly precipitated with GST-p38 (middle panel, lane 2), only faintly with GST-Jnk (middle panel, lane 4) and not at all when empty vector was transfected (middle panel, lane 6). Finally, as previously reported, human JIP1 was precipitated with GST-Jnk (bottom panel, lane 4) and not with GST-p38 (bottom panel, lane 2) or empty vector (lane 6). These results indicate that both rat and human IB2/JIP2 are p38 binding proteins.
Binding of rIB2 to kinases upstream of MAP kinase. In addition to binding to MAP kinases such as Jnk, IB/JIP family scaffolds also bind to MAP kinase kinases (MKK) and MAP kinase kinase kinases (MKKK). Since p38 is activated by a different set of upstream kinases than Jnk kinases, the binding specificity of rIB2 was investigated. Amino acids 511 to 835 of rIB2, which are analogous to the MKK binding site of JIP1, were expressed as a fusion protein with GST and tested for the ability to precipitate HA-tagged MKKs, such as MKK3 and MKK6, which activate p38, as well as MKK7, which is known to activate Jnk. As would be expected for a scaffolding protein for p38, this region of rIB2 clearly precipitated the p38-activating MKK3 (Fig. 6A, lane 3) but only weakly precipitated the Jnk-activating MKK7 (Fig. 6A, lane 9). Interestingly, rIB2 does not interact with all p38-activating MKKs because it did not precipitate MKK6 (Fig. 6A, lane 6). In all cases the MKKs were not precipitated by GST itself, confirming the specificity of these binding experiments (Fig. 6A, lanes 2, 5, and 8).
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Rat and human IB2/JIP2 facilitate Tiam1 activation of p38 but not Jnk.
As a GEF for Rac GTPases, Tiam1 has the potential to activate Rac and thus promote the activation of each of the GTPase's many downstream effector proteins. One such Rac-binding protein is MLK3. Since we showed that Tiam1 can bind to rIB2, a putative scaffold protein with preference for kinases that activate p38 rather than Jnk, we tested whether expression of rIB2 could promote p38 activation by Tiam1. Cells were transfected with plasmids expressing GST-p38 and combinations of Tiam1 and rIB2. The stimulation of GST-p38 in cells was detected by the ability of precipitated p38 to phosphorylate the p38 target ATF2, which was detected with an antibody that specifically recognizes phospho-ATF2 (Fig. 7A). Transfection of Tiam1 alone (Fig. 7A, upper panel, lane 2) or rIB2 alone (Fig. 7A, upper panel, lane 3) did not stimulate p38 activity over control transfection (upper panel, lane 1). However, the presence of both rIB2 and Tiam1 consistently enhanced p38 activation (upper panel, lane 4) approximately threefold. For comparison, in this system an 
10-fold activation was observed for overexpressed MKK3, the direct upstream kinase activator of p38 (upper panel, lane 11). The rIB2-Tiam1 enhancement in p38 activation was not due to changes in expression levels of total p38 (middle panel) or Tiam1 (lower panel, compare lanes 2 and 4).
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n306), failed to fully promote p38 activation when expressed alone or when coexpressed with Tiam1 (Fig. 7A, upper panel, lanes 5 and 8). A rIB2 mutant lacking an MLK3 binding site (rIB2-1-693) also failed to activate p38 fully when transfected alone or when cotransfected with Tiam1 (upper panel, lanes 7 and 10). These findings show that the scaffold function of rIB2 is involved in augmentation of p38 activation. Finally, deletion of residues 434 to 522 (rIB2-434-522), which disrupts rIB2 binding to Tiam1 (Fig. 3B), also failed to activate p38 alone or together with Tiam1 (lanes 6 and 9), indicating that Tiam1 binding to rIB2 has functional significance in p38 regulation. Similar results were obtained with huIB2/JIP2 (Fig 7B). Thus, expression of either huIB2/JIP2 or Tiam1 alone had no effect on p38 activity (Fig. 7B, upper panel, compare lanes 1, 2, and 3), while their concurrent expression resulted in elevated p38 activity (lane 5). This result gave additional support to the notion that human IB2/JIP2 is a functional homologue of rat IB2 and is a scaffold protein for p38. JIP1 was then tested in the same assay. As expected for a protein that does not bind to p38, JIP1 did not activate p38 on its own (Fig. 7B, upper panel, lane 4). It also did not augment Tiam1 induction of p38 activity in cells (Fig. 7B, upper panel, lane 6) even though it was expressed at levels comparable to those of huIB2/JIP2 (lower panels).
To test the specificity of this phenomenon, the effect of rIB2 on Jnk activity was assessed. Unlike p38, no detectable increase in Jnk activity was observed when Tiam1 was transfected alone or together with rIB2 (Fig. 7C).
Thus, rat and human IB2/JIP2 exhibit identical binding preferences for the p38 MAP kinase over the Jnk MAP kinase as well as identical potential to enhance Tiam1-mediated p38 activation. They likely represent species variants of the same gene product which, despite its assigned name, functions to enhance a specific pathway involving p38.
Increased scaffold-kinase complex formation induced by Tiam1 and Ras-GRF1.
Our results indicate that specific Rac-GEFs are also part of the p38 signaling complex recruited by IB2. We have begun to investigate how exchange factors and IB2 cooperate to potentiate p38 activation. Since the hypothesized function of a scaffolding protein is to bring together components of a signaling cascade, we tested whether the binding of Tiam1 to IB2 could enhance this function. In particular, the ability of Tiam1 expression to increase the association of each component of the p38 signaling cascade with IB2 was tested. Flag-IB2 was cotransfected with either MLK3, MKK3, or p38 and then the consequence of adding Tiam1 on the association of each kinase with IB2 was assessed (Fig. 8). The expression of Tiam1 increased the amount of MLK3 bound to IB2 approximately fourfold (Fig. 8A, upper panel, compare lanes 2 and 3), even though similar amounts of MLK3 were present in cell lysates (Fig. 8A, lower panel, compare lanes 2 and 3). Importantly, no increase in MLK3 association with IB2 was observed when the Tiam mutant, PCX-Tiam1, which does not bind to IB2, was used (Fig. 8A, lane 4). A similarity in association was observed when the association of MKK3 (approximately fourfold increase) with IB2 was studied (Fig. 8B) and when the association of p38 (approximately twofold increase) with IB2 was investigated (Fig. 8C, lanes 1 to 4).
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Interestingly, the ability of Ras-GRF1 to enhance the association of inactive p38 with IB2 was not dependent on Rac activation, as this effect was observed even with Ras-GRF1-DH(-), a mutant protein containing an inactivating point mutation in its Rac-activating DH domain (Fig. 8D, top panel, lanes 4 and 7) (19). However, this mutant did not activate scaffold-associated p38, demonstrating that this effect was dependent on Rac activation (Fig. 8D, middle IP panel, lanes 6 and 7).
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DISCUSSION |
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In all of our experiments the rat cDNA we cloned and human JIP2 displayed indistinguishable properties. Moreover, our cDNA is 86% homologous to human IB2 and 96% homologous to mouse IB2. Finally, no gene in the human database is closer in amino acid sequence to our cDNA than human IB2 and no other gene in the mouse database is closer to our cDNA than mouse IB2. These observations strongly argue that our rat cDNA clone and human IB2 are species variants of the same gene.
The p38 MAP kinases, p38, -ß, -
, and -
, were the second mammalian stress- and cytokine-activated MAP kinase family to be identified (for a review, see reference 25). They are homologous to HOG1, the osmo-sensing MAP kinase of S. cerevisiae. In most, but not all, cases p38 and Jnk are activated concomitantly; however, the kinase pathways that lead to their activation are distinct. Whereas Jnk is activated via phosphorylation by SEK1 and MKK7 (15, 34), p38 is activated by MKK3 and MKK6 (11, 12, 15, 32). Upstream kinases that activate the MKKs are not always as specific, however. For example, while MEKK1 can activate only SEK1 and MKK7 (44) and TAO can activate only MKK3 (10), MEKK3 can activate both Jnk- and p38-activating kinases (2, 14, 17). Once activated, p38 can activate other kinases such as MAPKAP-K2, PRAK, MSK, and MNK and transcription factors such as CHOP, MEF2, and ATF2. p38 activity can have diverse effects on cells, including regulation of cell cycle arrest, apoptosis, cell migration-invasion, and differentiation.
Our conclusion that IB2/JIP2 is a scaffold for the p38 cascade is based on the observation that both rat and human IB2/JIP2 bound more effectively to p38 and an activator of p38, MKK3, than to Jnk or an activator of Jnk, MKK7, both in vitro and in vivo. In addition, we found that the expression of rat or human IB2/JIP2 in cells potentiated p38 activation but not Jnk activation by the Tiam1 exchange factor. Finally, Tiam1 or Ras-GRF1 binding to IB2/JIP2 enhanced the binding of all three components of a p38 MAP kinase signaling pathway to IB2/JIP2. This conclusion was surprising at first because JIP2 and IB2 were originally described as Jnk-binding proteins (29, 45). However, in this earlier work Jnk binding to IB2/JIP2 was much weaker than to IB1/JIP1, suggesting that the scaffold might actually play a different role. In addition, the existence of a p38 scaffold in yeast (31) suggested that there was likely a counterpart in mammalian cells. Finally, recent work has shown that IB2 can bind to p38, but not to Jnk, in the presence of fibroblast growth factor homologous factor 1 (35).
Another point of interest regarding the p38 scaffold IB2/JIP2 is its ability to bind to MLK3, since this MKKK has been associated primarily with activation of the Jnk kinase cascade. In fact, overexpression of MLK3 has been reported to activate Jnk much more efficiently than p38 in cells (33). Nevertheless, MLK3 has been shown to stimulate the kinase activity of the p38-activating MKK3 (40). Our finding that IB2 expression enhances p38 activation in cells suggests that MLK3 may require the presence of a scaffolding protein for efficient activation of the p38 signaling cascade. Interestingly, the expression pattern of IB2 is restricted to neurons and pancreatic ß-cells, possibly explaining the failure to observe p38 activation by MLK3 in many cell types.
The precise function of scaffolds like the IB/JIP family is still not completely clear. Their overexpression in cells can enhance the magnitude of signaling through a particular MAP kinase cascade, as was shown previously for JIP1 and the Jnk kinase cascade (42), and as we showed here for IB2 and the p38 cascade. However, this may not be their primary function, since changing the ratio of expression of the scaffold to kinases in the cascade can make the scaffolds appear as inhibitors rather than activators of the cascade (16). Alternatively, they may function in directing signaling specificity, rather than as modifiers of signal amplitude. At one level this could occur by promoting the interaction of the correct MKKKs, MKKs, and MAPKs to make a functional cascade. However, in many cases the kinases themselves already have binding sites that recognize their substrates with high affinity (for a review, see reference 25).
Our findings suggest that a function of these scaffolds is to contribute to signaling specificity by coupling Rac activation to one of its many downstream target proteins. This is based on our observation that another component of the IB/JIP scaffold protein complex in cells is a Rac-GEF, either Tiam1 or Ras-GRF1. One of the many target proteins of Rac GTPases is MLK3, the initiating kinase in the cascade that leads to p38 activation (8, 39). In fact, we have found that activation of scaffold-bound p38 by Tiam1 or Ras-GRF1 was dependent upon the Rac-GEF domain of the exchange factors. We also found that the binding of Tiam1 or Ras-GRF1 to IB2/JIP2 contributed to the enhancement of p38 activation through its ability to increase the association of kinases with the scaffold. These findings argue that scaffolds can potentially contribute to signaling specificity by coupling individual upstream activators of Rac to one of the many downstream signaling cascades initiated by Rac. Supporting this notion is our observation that Tiam1 expression did not lead to significant activation of p38 in HEK 293 cells, which do not express IB2/JIP2. Apparently, in the absence of this scaffold activated Rac is targeted toward other effector pathways. However, ectopically expressed scaffold formed a complex with Tiam1 or Ras-GRF1, directing activated Rac toward the components of a p38 kinase cascade rather than a Jnk kinase cascade (Fig. 9).
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Although we have focused the experiments in this paper on the potential role of IB2/JIP2 in mediating signaling specificity downstream of GEFs and Rac, the properties of JIP-related proteins suggest that they may also participate in coupling Tiam1 and Ras-GRF1 to upstream signals. That is because IB2/JIP2 and IB1/JIP1 contain virtually identical PTB domains at their C termini, which have the capacity to bind to various cell surface proteins. One documented IB1/JIP1 and IB2/JIP2 binding partner is ApoER2, a receptor for neuronal Reelin that plays a key role in neuronal development (38). Thus, the PTB domain of IB2/JIP2 or IB1/JIP1 may target Tiam1 or Ras-GRF1 to specific membrane components that can contribute to their activation in cells. For the Rac and Ras GEF, Ras-GRF1, we have already shown that the IB2/JIP2-binding PH-CC-IQ motif is required for its activation by calcium in cells (6). In neurons, where Ras-GRF1 and IB2 are highly enriched, calcium is known to enter cells through a variety of channels which generate distinct intracellular signaling cascades (41). By its ability to bind to cell surface components and Ras-GRF1, it is possible that IB2/JIP2 may couple specific calcium channels to both the Ras/Erk and Rac/p38 signaling cascades.
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ACKNOWLEDGMENTS |
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This work was supported by a PHS grant to L.A.F. from the NCI.
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FOOTNOTES |
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