Ca2+/Calmodulin-dependent Protein Kinase II Regulates Tiam1 by Reversible Protein Phosphorylation*

A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105–33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Cα and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Cα and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factorin vitro.

The Rho family of small GTPases plays an important role in the regulation of several key cellular functions. Rho is involved in the formation of actin stress fibers and focal adhesions (1)(2)(3), Rac is required in actin polymerization associated with membrane ruffling and lamellipodia formation in fibroblasts (3,4), and Cdc42 is important in the formation of filopodia in fibroblasts (3). Moreover, Rho family GTPases are involved in cell cycle progression (5), stimulate gene transcription through activation of the serum response factor (6), activate the Jun kinase and p38 mitogen-activated protein kinase signaling cascades (7)(8)(9)(10), enhance Ras-triggered transformation of NIH3T3 fibroblasts (11,12), and are required in the NADPH oxidasemediated phagocytic response in neutrophils (13).
During the past few years, a number of guanine nucleotide exchange factors for Rho family GTPases have been identified (14). These exchange factors promote binding of GTP by facilitating the release of GDP from Rho proteins. Nucleotide exchange factors which act on Rho proteins contain two key conserved domains: a Dbl homology domain, which is believed to be responsible for catalyzing GDP/GTP exchange; and a pleckstrin homology domain, which seems to be important for cellular localization through interaction with lipids and/or proteins (14). Relatively little is known concerning the specificity of these exchange factors in vivo, although it has been demonstrated that Tiam1 acts as a Rac1-specific exchange factor in NIH3T3 fibroblasts, stimulating membrane ruffling and Jun kinase (15,16), Lbc acts as a Rho-specific exchange factor, inducing stress fiber formation in Swiss 3T3 cells and foci in NIH3T3 cells (17), and Dbl stimulates Jun kinase in HeLa cells (8).
The mechanism(s) of activation of Rho family nucleotide exchange factors is not yet evident. It has been demonstrated that membrane localization of Tiam1 is required for Rac-dependent membrane ruffling and Jun kinase activation in NIH3T3 cells (16), and that the N-terminal pleckstrin homology domain and an adjacent protein interaction domain are required for membrane localization of the exchange factor (16,18). Phospholipids may play an important role in determining the cellular localization of Tiam1, since both PIP 2 1 and PIP 3 bind to its N-terminal pleckstrin homology domain (19), and phosphoinositide 3-kinase activity is required for activation of Rac1 by Tiam1 (20). Reversible protein phosphorylation may also be involved in the regulation of Rho family exchange factors. It has been shown that Dbl (21) and Ost (22) both exist as phosphoproteins in cells. Significantly, tyrosine phosphorylation of the oncogenes Vav (23) and Vav2 (24) by Lck results in increased GDP/GTP nucleotide exchange on Rac1 and RhoAlike GTPases, respectively, and PIP 3 may enhance both phosphorylation and activation of Vav (25). In addition, we have recently demonstrated that lysophosphatidic acid (LPA), platelet-derived growth factor, and several other agonists stimulate phosphorylation of Tiam1 in Swiss 3T3 fibroblasts, via activation of protein kinase C (PKC) (26,27), indicating that Rho exchange factors can also be phosphorylated on serine/threonine residues by a regulated mechanism.
In this study we demonstrate that Tiam1 is phosphorylated by several PKC isozymes in vitro, but is selectively phosphorylated by a classical PKC isoform, PKC␣, when Swiss 3T3 cells are treated with LPA. In addition, we present strong evidence that Ca 2ϩ /calmodulin-dependent protein kinase II (CamKII) also phosphorylates Tiam1 in Swiss 3T3 fibroblasts in response to LPA treatment and that this phosphorylation produces electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Finally, we show that phosphorylation of Tiam1 by Ca 2ϩ /calmodulin-dependent protein kinase II, but not protein kinase C␣, enhances its nucleotide exchange rate toward Rac1, and that this can be abrogated by treatment with protein phosphatase 1.
Agonist Treatment and Preparation of Membrane Fraction-Serumstarved cultures, on 100-mm dishes, were treated with various concentrations of LPA, PMA, or ionomycin at 37°C for different times as noted in the experiments. The medium was removed, the cells washed three times with 5 ml of ice-cold PBS containing 500 M sodium orthovanadate, and scraped in 400 l/dish of lysis buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM MgCl 2 , 2 mM EDTA, 10 g/ml antipain and leupeptin, 1 mM phenylmethylsulfonyl fluoride, 500 M sodium orthovanadate, 10 mM pyrophosphate, 10 mM sodium fluoride, and 1 mM dithiothreitol). The cells were lysed by five passes through a 27-gauge needle (28) at 4°C. Lysates were centrifuged at 120,000 ϫ g for 45 min to prepare cytosolic and total particulate fractions. The membrane pellet was washed twice with lysis buffer to remove cytosolic proteins.
Protein determination was done by the method of Bradford (29).

SDS-Polyacrylamide Gel Electrophoresis and Western
Analysis-SDS-Polyacrylamide gel electrophoresis was performed on 6% or 4 -12% gradient polyacrylamide gels (Novel Experimental Corp) and proteins transferred onto polyvinylidene difluoride membranes (Millipore) for 1.5 h at 20 V using a Novex wet transfer unit. The membranes were blocked overnight with 5% (w/v) nonfat dried milk. Blots were incubated for 1 h with Tiam1 antibody (diluted 1:2000) in 1% bovine serum albumin, then for 1 h with a horseradish peroxidase-conjugated secondary antibody (Vector Laboratories), prior to development using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). Phosphothreonine Western blots were carried out essentially as described above, using a 1:500 primary antibody dilution.
Purified GST-Tiam1 (3 l) was incubated for 20 min at 30°C in the presence or absence of the indicated amounts of purified CamKII␣. Assays were carried out in 10 mM Tris buffer, pH 7.4, containing 0.1 mg/ml BSA, 1.25 mM CaCl 2 , 25 g/ml calmodulin, 15 mM MgCl 2 and 100 M ATP. Assays were carried out either using non-radiolabeled ATP and phosphorylation analysis by Western blotting or with [␥-32 P]ATP (specific activity 5 ϫ 10 6 dpm/nmol) and phosphorylation analysis by autoradiography.
Since the purified Tiam1 preparations contained detergent and some aggregated protein, the concentration of purified Tiam1 was estimated from silver-stained gels for the stoichiometry experiments, using BSA as a standard. 0.2 pmol of GST-Tiam1 was phosphorylated by PKC␣ (0.3 units) or CamKII␣ (4 g), for 1 h in the presence of [␥-32 P]ATP (specific activity 2 ϫ 10 7 dpm/nmol), as described above. The samples were separated by SDS-PAGE on 6% gels, the Tiam1 band excised from the gel and 32 P incorporation assessed by scintillation counting.
Dephosphorylation of Tiam1-Purified GST-Tiam1 (10 l) was phosphorylated with PKC␣ (0.3 units) or CamKII␣ (4 g), for 1 h in the presence of [␥-32 P]ATP (specific activity 2 ϫ 10 7 dpm/nmol), as described above. Phosphorylated GST-Tiam1 was incubated with 30 l of glutathione-Sepharose beads for 1 h at 30°C, and the beads collected by centrifugation (3,000 ϫ g for 5 min). The Tiam1-bound beads were washed three times with 200 l of 50 mM Tris buffer, pH 7.0, containing 0.5 mg/ml BSA to remove the kinase, resuspended in 50 l of the same buffer, and stored on ice until use.
Tiam1 Exchange Assay-C1199-Tiam1 with an N-terminal hexahistidine tag was expressed in sf9 cells and purified using Talon metal affinity resin (CLONTECH) in a 25 mM Tris buffer, pH 8.0, containing 0.5 M ␤-mercaptoethanol and 100 mM NaCl. Tiam1 was eluted from the beads using 100 mM imidazole and dialyzed prior to freezing. GST-Rac1 was expressed in E. coli, and purified using glutathione-Sepharose beads in a 100 mM Tris buffer, pH 8.0, containing 250 mM NaCl and 0.1 mM dithiothreitol. GST-Rac1 was eluted from the beads with 10 mM glutathione, dialyzed, and frozen.
The Tiam1 exchange assay was carried out essentially as described (15). Purified GST-Rac1 (120 pmol) was preloaded with [ 3 H]GDP (30 M; 25 Ci/mmol) in 60 l of binding buffer. Eight l of the preloaded GTPase was added to 32 l of exchange mixture, which contained 5 pmol of Tiam1 or BSA, 1 mM GTP, and 75 M PIP 3 , in exchange buffer (15). At the indicated times, 8-l aliquots were pipetted into 1 ml stopping buffer (50 mM Tris, pH 7.4, 5 mM MgCl 2 , 50 mM NaCl), and [ 3 H]GDP bound to Rac1 analyzed by filtering through nitrocellulose. For some exchange experiments, Tiam1 (5 pmol) was prephosphorylated with 0.1 units of protein kinase C or 2 g of CamKII, as described above. In some experiments Tiam1 was assayed after prephosphorylation with 2 g of CamKII in the presence or absence of 0.2 units of PP1.

Role of Protein Kinase C Isozymes in Tiam1 Phosphorylation
in Vitro-In Swiss 3T3 fibroblasts, LPA stimulates threonine phosphorylation of Tiam1 through activation of PKC, and causes its electrophoretic retardation on SDS-PAGE (26). To understand further the mechanism of Tiam1 phosphorylation, we incubated purified GST-C1199-Tiam1 with several PKC isozymes to determine which isoform(s) phosphorylates the exchange factor. As shown in Fig. 1, all of the kinases tested phosphorylate Tiam1, indicating that PKC isozymes of the classical, novel, and atypical families can phosphorylate the protein in vitro. However, the different PKC isoforms phosphorylated Tiam1 to different extents. The exchange factor was preferentially phosphorylated by PKC␣, -␥, and -, moderately phosphorylated by PKC⑀, and only weakly phosphorylated by PKC␤1, -␤2, and -␦. Significantly, none of the PKC isozymes tested decreased the electrophoretic mobility of Tiam1, suggesting that this was probably caused by a kinase from a different family in vivo.
Role of Ca 2ϩ /Calmodulin-dependent Protein Kinase II in Tiam1 Phosphorylation-Down-regulation of non-atypical PKC isozymes by long term PMA pretreatment, or preincubation with the protein kinase C inhibitor Ro-31-8220, reduces LPA-or platelet-derived growth factor-stimulated Tiam1 phosphorylation by approximately 75% in Swiss 3T3 cells (26,27) suggesting that another protein kinase is also involved. Therefore, purified GST-C1199-Tiam1 was incubated with Ca 2ϩ /calmodulin-dependent protein kinase II (CamKII), a kinase with a very broad substrate specificity and widespread expression (30), to determine whether this kinase phosphorylated the exchange factor. Although some phosphorylation of Tiam1 was observed in the absence of CamKII ( Fig. 2A), perhaps due to a protein kinase which co-purifies with the GST-Tiam1 (26), addition of the kinase significantly enhanced 32 P phosphorylation of the exchange factor ( Fig. 2A), demonstrating that this kinase can phosphorylate Tiam1. Indeed, Western blotting with antibodies confirmed that CamKII stimulated phosphorylation of Tiam1 on threonine (Fig. 2B). Significantly, in addition to phosphorylating Tiam1, CamKII induced electrophoretic retardation of the exchange factor (Fig. 2), such as is observed upon stimulation of Swiss 3T3 cells with LPA (26). Ca 2ϩ /calmodulin-dependent protein kinase II induced the Tiam1 bandshift in a concentration-dependent (Fig. 2B) and time-dependent (Fig. 2C) manner, but only in the presence of Ca 2ϩ and calmodulin (data not shown). Intriguingly, the Tiam1 bandshift occurred in a gradual manner with time, and not as one step, suggesting that the exchange factor probably exists in several different phosphorylation states and has multiple phosphorylation sites which serve as substrates for CamKII. Indeed, when the Tiam1 protein concentration was estimated by silver staining, using BSA as a standard, stoichiometry experiments indicated that under maximal phosphorylating conditions, Tiam1 contains 10.1 Ϯ 2.7 PKC␣ and 3.7 Ϯ 0.6 CamKII phosphorylation sites.
Role of PKC and CamKII in Tiam1 Phosphorylation in Vivo-Swiss 3T3 cells were stimulated with LPA, in the presence and absence of the intracellular Ca 2ϩ chelator BAPTA/ AM, to investigate the importance of this metal ion in Tiam1 phosphorylation. The results (Fig. 3A) show that Tiam1 phosphorylation is totally abolished in the presence of the chelator, indicating that Ca 2ϩ plays an essential role in this pathway. Together with the results obtained using protein kinase inhibitors, and PKC down-regulation (26), this suggests that LPA stimulates Tiam1 phosphorylation through activation of a classical PKC isoform and another Ca 2ϩ -dependent enzyme. Therefore, since Swiss 3T3 cells only contain PKC␣, -␦, -⑀, and -(31), LPA must stimulate Tiam1 phosphorylation through activation of PKC␣, which is the only classical Ca 2ϩ -dependent enzyme present. Significantly, BAPTA treatment also inhibited the LPA-stimulated Tiam1 bandshift (Fig. 3B), indicating that Ca 2ϩ is required for this effect. However, the selective PKC inhibitors bisindolylmaleimide I (Fig. 3C) and Ro-31-8220 (data not shown) had no effect on the LPA-induced Tiam1 bandshift, providing further evidence that PKC does not cause this.
To confirm that CamKII is involved in LPA-induced Tiam1 phosphorylation, Swiss 3T3 cells were preincubated with the CamKII inhibitor KN93 (20 M) for 24 h, in the presence and absence of the PKC inhibitor Ro-31-8220 (5 M) for 1 h. As expected (26), Ro-31-8220 greatly reduced LPA-stimulated Tiam1 phosphorylation (Fig. 3D). KN93 also significantly reduced LPA-induced Tiam1 phosphorylation, and the two inhibitors together almost completely eliminated the phosphorylation (Fig. 3D). Therefore, these data strongly suggest that CamKII and PKC both contribute to the phosphorylation studied here.
To provide additional evidence that PKC␣ and CamKII phosphorylate Tiam1 in vivo, Swiss 3T3 cells were treated with the Ca 2ϩ ionophore ionomycin, in the presence and absence of PMA. PMA (1 M) alone induced limited threonine phosphorylation of Tiam1 ( Fig. 3E; Ref. 26). Ionomycin (1 M) alone stimulated Tiam1 phosphorylation to a greater extent (Fig. 3E), and enhanced the PMA-stimulated Tiam1 phosphorylation. Similar results were obtained with the ionophore A23187 (data not shown). Therefore, the observation that PMA and a Ca 2ϩ ionophore are sufficient to stimulate Tiam1 phosphorylation is consistent with a classical PKC isozyme and CamKII phosphorylating the exchange factor in vivo.
Dephosphorylation of Tiam1-We have previously established that LPA-stimulated Tiam1 phosphorylation is maximal

FIG. 1. Several protein kinase C isozymes phosphorylate purified GST-Tiam1 in vitro.
Purified GST-Tiam1 was incubated with or without 0.06 units of the indicated protein kinase C isozymes for 20 min at 30°C, as described under "Experimental Procedures." Control experiments containing kinase, but no GST-Tiam1, were also done. Phosphorylation experiments were carried out using [␥-32 P]ATP and the results analyzed by autoradiography. Results are representative of two independent experiments.

FIG. 2. Phosphorylation of Tiam1 by Ca 2؉ /calmodulin-dependent protein kinase II reduces the electrophoretic mobility of the exchange factor.
Purified GST-Tiam1 was incubated with the indicated amounts of purified Ca 2ϩ /calmodulin-dependent protein kinase II for 20 min at 30°C (A and B), or for various times (C), as described under "Experimental Procedures." Control experiments containing kinase, but no GST-Tiam1, were also done. Phosphorylation experiments were carried out using [␥-32 P]ATP and the results analyzed by autoradiography (A), or with non-radiolabeled ATP and phosphorylation analyzed by Western blotting with the phosphothreonine antibody (B). Electrophoretic retardation of Tiam1 was also analyzed by Western blotting using the Tiam1 antibody (C). Results are representative of at least three independent experiments. at 2.5 min, begins to decrease after 10 min LPA treatment, but is still readily detectable after 60 min of LPA treatment (26). Further experiments showed that Tiam1 phosphorylation was still detectable after 3 h of LPA treatment, but that the stimulation was lost after 4 h (data not shown), presumably because of dephosphorylation. To elucidate further the mechanisms involved in controlling the level of Tiam1 phosphorylation, we investigated which phosphatases are involved in the dephosphorylation process. The results show that Tiam1 is preferentially dephosphorylated by the catalytic subunit of PP1 in vitro, when the exchange factor is phosphorylated by PKC␣ or CamKII (Fig. 4). Tiam1 was also dephosphorylated by protein phosphatase 2B in vitro, but at a much slower rate (Fig. 4). Interestingly, protein phosphatase 2A slowly dephosphorylated Tiam1 when it was phosphorylated by CamKII, but not when it was phosphorylated by PKC␣.
Effects of Phosphorylation on the GDP/GTP Exchange Activity of Tiam1-Since Tiam1 acts as a Rac1-specific exchange factor in NIH3T3 fibroblasts, stimulating membrane ruffling and Jun kinase activity (15,16), we investigated whether protein phosphorylation could affect Tiam1 GDP/GTP exchange activity toward Rac1. Purified hexahistidine-tagged Tiam1 protein was incubated with ATP in the presence or absence of purified CamKII, and the GDP/GTP exchange rate of Tiam1 assessed by following the dissociation of [ 3 H]GDP from Rac1. As expected (15), Tiam1 stimulated release of [ 3 H]GDP from GST-Rac1 in a concentration-dependent (data not shown) and time-dependent manner (Fig. 5, A and B). Importantly, preincubation of Tiam1 with CamKII stimulated the exchange ac-tivity of Tiam1 toward GST-Rac1 (Fig. 5A). CamKII alone had no effect on [ 3 H]GDP release from Tiam1. CamKII treatment stimulated the GDP/GTP exchange activity of Tiam1 toward Rac1 in a concentration-dependent manner (data not shown), enhancing the initial exchange activity of Tiam1 by approximately 2-fold. In contrast, preincubation of Tiam1 with PKC had no detectable effect on exchange activity (Fig. 5B), although a phosphorylation experiment carried out in parallel, in the presence of [␥-32 P]ATP, confirmed that the kinase strongly phosphorylated Tiam1 under the conditions used.
To verify that CamKII stimulates exchange activity through reversible protein phosphorylation, Tiam1 exchange activity was measured after pretreatment with CamKII, in the presence and absence of PP1. Under the conditions used, CamKII stimulated phosphorylation of Tiam1, and this was abrogated by the inclusion of PP1 (data not shown). PP1 had little effect on basal exchange activity (Fig. 5C), suggesting that the basal exchange activity is not due to serine/threonine phosphorylation of the protein during expression in Sf9 cells. In contrast, PP1 treatment eliminated the CamKII-stimulated activation of Tiam1 (Fig. 5C), causing the exchange activity to revert to the basal level and providing strong evidence that activation is due to phosphorylation of Tiam1.

DISCUSSION
The data presented here suggest that the classical PKC isozyme, PKC␣, and Ca 2ϩ /calmodulin-dependent protein kinase II, both phosphorylate the Rac1-specific exchange factor, Tiam1, in response to LPA treatment of Swiss 3T3 fibroblasts. Furthermore, this phosphorylation is likely to be functionally important, since CamKII treatment enhances the GDP/GTP exchange activity of Tiam1 (Fig. 5A). This is the first evidence that Rho family exchange factors can be activated by serine/ threonine phosphorylation, and it is likely to be a general regulatory mechanism for Tiam1, since it is phosphorylated by several different agonists in Swiss 3T3 cells (26).
While we have previously established that LPA stimulates Tiam1 phosphorylation through activation of PKC (26), the fact that neither PKC inhibitors nor long term PMA treatment could completely abrogate this effect suggested that a second protein kinase was also involved in this pathway. This hypothesis is supported by the observation that LPA-induced electrophoretic retardation of Tiam1 is partially inhibited by staurosporine (26), but not by the PKC-specific inhibitors bisindolylmaleimide I (Fig. 3C) and Ro-31-8220, and by the fact that none of the PKC isozymes tested decreased the electrophoretic mobility of the exchange factor (Fig. 1). Several lines of evidence indicate that the second kinase involved in Tiam1 phosphorylation is Ca 2ϩ /calmodulin-dependent protein kinase II. First, the LPA-induced Tiam1 bandshift (Fig. 3B) and the PKC-independent (26) threonine phosphorylation of Tiam1 in LPA-stimulated fibroblasts (Fig. 3A) are both inhibited by BAPTA/AM, indicating that Ca 2ϩ is essential for these effects.
The conclusion that CamKII and PKC coordinately phosphorylate Tiam1 is supported by the fact that the exchange factor contains approximately 10.1 Ϯ 2.7 PKC sites and 3.7 Ϯ 0.6 CamKII sites in vitro, and PKC catalyzes 70 -75% of the LPAinduced Tiam1 phosphorylation in vivo (26). Indeed, CamKII may account for the sustained nature of the Tiam1 bandshift and phosphorylation (26), since Ca 2ϩ stimuli can induce CamKII to autophosphorylate to a Ca 2ϩ -independent form, which retains kinase activity even after Ca 2ϩ levels decline (32). The finding that Tiam1 contains 3-4 CamKII phosphorylation sites is consistent with the observation that the kinase stimulates the Tiam1 bandshift in a gradual manner, and not in one step (Fig. 3). Furthermore, Tiam1 is particularly rich in serine and threonine residues (33) and contains several potential CamKII phosphorylation consensus sequences. Although Tiam1 contains approximately 3 times more PKC sites than CamKII sites, PKC does not alter the electrophoretic mobility of the exchange factor. These results suggest that the CamKII phosphorylation sites are located close together on the Tiam1 protein, and may cause a change in the conformation of the exchange factor.
Since Ca 2ϩ and PMA are intimately involved in Tiam1 phosphorylation (Fig. 3), it seems likely that it is stimulated via the PLC pathway, which generates diacylglycerol and inositol 1,4,5-trisphosphate, second messengers that activate PKC and mobilize Ca 2ϩ respectively. This agrees with the facts that nanomolar concentrations of LPA activate Tiam1 phosphorylation, via a pertussis toxin-insensitive mechanism, and that Tiam1 phosphorylation is stimulated by LPA, platelet-derived growth factor, endothelin-1, bombesin, and bradykinin (26), agonists which activate PLC and PKC (34 -36), but not by epidermal growth factor, which produces barely detectable phosphoinositide hydrolysis in Swiss 3T3 cells (34). Indeed, PLC-␥1 is required for platelet-derived growth factor-induced phosphorylation of Tiam1 (27), and the PLC inhibitor U-73122 abolishes Tiam1-induced cell invasion in T-lymphoma cells (37), indicating that PLC is functionally important in regulation of this exchange factor. Indeed, since CamKII is activated via the PLC pathway in many cell types (32), and CamKII activates Tiam1 (Fig. 5A), PLC probably regulates Tiam1 through stimulation of this kinase.
The Rho exchange factors Vav and Vav2 are also regulated through protein phosphorylation (23,24). However, these proteins are activated through tyrosine phosphorylation by Lck (23,24), whereas Tiam1 is activated via threonine phosphorylation (26), by CamKII (Fig. 5A). Interestingly, while Vav is totally inactive until phosphorylated (23), both Vav2 (24) and Tiam1 (Fig. 5) have a low basal rate of exchange activity. This basal activity may partially explain why phosphorylation does not stimulate the exchange activity of Tiam1 (Fig. 5A) or Vav2 (24), as much as Vav (23). Alternatively, Tiam1 may be regulated by additional factors. However, phosphorylation by PKC does not appear to be this signal, since it does not affect Tiam1 GDP/GTP exchange activity (Fig. 5B). Protein phosphatase 1 treatment had no significant effect on control Tiam1 activity (Fig. 5C), suggesting that the basal exchange activity is not due to serine/threonine phosphorylation of the protein in Sf9 cells. This eliminates the possibility that PKC does not regulate Tiam1 activity in vitro because of prior phosphorylation of a key PKC regulatory site. On the other hand, PP1 treatment eliminated the CamKII-stimulated activation of Tiam1 (Fig.  5C), returning Tiam1 exchange activity to basal levels. Therefore, unlike the activation of p115 by G␣13 (38), Tiam1 activation is due to reversible phosphorylation rather than a direct protein-protein interaction.
The function of Tiam1 phosphorylation by PKC is not yet apparent. It remains possible that phosphorylation plays a role in the regulated membrane localization of the exchange factor (16). Alternatively, this phosphorylation may regulate the activity of Tiam1 against other potential target GTPases. It is clear that CamKII and PKC phosphorylate different sites on Tiam1, since only CamKII causes the electrophoretic retardation (Fig. 2) and activation (Fig. 5A) of Tiam1. However, a possible interaction between the two kinases in the regulation of the phosphorylation and activation of Tiam1 has not been explored. It is also not yet apparent how CamKII activates Tiam1, but it seems likely that the phosphorylation causes a key change in the conformation of Tiam1. This could involve reorientation of the Dbl homology domain and pleckstrin homology domains (39), perhaps allowing the GTPase easier access to its binding site, or enhancing the GDP/GTP exchange reaction by another mechanism.
Rac1 affects many cellular processes, including gene transcription activated by the serum response factor (6), membrane ruffling and lamellipodia formation (3,4), activation of the Jun kinase pathway (7-9), cell cycle progression (5), and phospholipase D activity (40). Moreover, LPA regulates several signaling pathways that involve Rho family GTPases including stress fiber formation (1), gene transcription through activation of the serum response factor (6), and phospholipase D (41). The work of Collard and associates indicates that Tiam1 produces the same cytoskeletal changes as induced by Rac1 and also activates the Jun kinase pathway (15,16). However, it is not yet clear if the other pathways are regulated by Tiam1 in vivo, or if other exchange factors are involved in the effects of Rac1 on the cytoskeleton and these other signaling processes. Further work will also be required to determine the role of PKC in Tiam1 regulation, and elucidate the molecular mechanism by which CamKII activates Tiam1.