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In vitro, Vav is a regulated guanine nucleotide dissociation inhibitor for Ras
Immunology Letters 80 (2002) 1 – 2 www.elsevier.com/locate/
Letter to the Editor In vitro, Vav is a regulated guanine nucleotide dissociation inhibitor for Ras Vav plays a pivitol role in the immune system by control of signals downstream of the T-cell receptor in part by regulation of Rac-related GTPases [1]. While Vav is structurally unrelated to the catalytic domains of the Ras-guanine nucleotide exchange factors (GEFs) (the CDC25-homology domain), a series of reports from Gulbins and co-workers reported Vav posesses a GEF activity directed towards Ras GTPases and this activity could be activated by phosphorylation by Lck [2 – 5]. Instead we observe a Lck-regulated GDI activity for Ras, such that tyrosine-phosphorylated Vav selelctively inhibited GTP release from Ras. Our findings support the notion that activated Vav helps maintain Ras in an active state in stimulated T cells. GEFs for GTPases of the Ras and Rac-families stimulate the release of nucleotides from these GTPases [6]. Two distinct assays have been used to monitor GEF activity, the first and more commonly used monitors the release of a labeled nucleotide (such as [3H]-GDP) from a GTPase [6]. The second assay monitors the association of free labeled-nucleotide (such as alpha[32P]-GTP) with a GTPase. To examine a possible GEF activity of purified Vav for human H-ras protein we used an assay that monitors the release of nucleotide from a preformed complex of histidine-tagged (His6)Ras and [3H]-GDP. Reactions were initiated by mixing equal molar amounts Vav and Ras at 30 °C. In the presence of the negative control, GST, approximately 50% of the starting substrate ((His6)-Ras-[3H]GDP) was found to have released the bound [3H]-GDP (Fig. 1A). This reflects the intrinsic release of nucleotide from Ras. In the presence of the positive control, GST-CDC25 [6], approximately 90% of the [3H]-GDP had dissociated from Ras, reflecting a dramatic stimulation of guanine nucleotide release (Fig. 1A). In contrast only 15% of the [3H]-GDP dissociated from Ras after 30 min in the presence of GST-Vav [7] (Fig. 1A). Under these conditions, a fragment of sos1 encompassing the Dbl-homology and pleckstrin-homology domains failed to affect the dissociation of GDP from Ras (unpublished data).
To examine the apparent GDI activity of Vav under conditions where the intrinsic dissociation of [3H]-GDP from Ras was accelerated, we monitored the release of
Fig. 1. (A) (His)6-H-Ras proteins (30 pmoles) were loaded with [3H]GDP and incubated for the indicated periods of time with 30 pmoles of GST, GST-Vav(L), or GST-CDC25 at 30 °C. At the indicated period of time the amount of [3H]GDP remaining bound to (His)6-H-Ras protein was determined by filter binding assay [6]. Data points reflect the average of two reactions which did not differ by more than 10%. (B) The stability of [3H]GDP bound to (His)6-H-Ras was again monitored as described above except the incubation temperature was 37 °C. Where indicated GST-Vav(L) was phosphorylated with p56lck (10 ng) before the GDI assay. Data points reflect the average of two reactions which did not differ by more than 5%. (C) (His)6-H-Ras proteins were preloaded with [3H]GTP and incubated for the indicated time periods in the absence or presence of Vav or p56lck-phosphorylated Vav. At the indicated periods of time, aliquots were taken and [3H]-GTP bound was measured by filter binding assay. Data points reflect the average of two reactions that did not differ by more than 5%.
0165-2478/02/$ - see front matter © 2002 Published by Elsevier Science B.V. PII: S 0 1 6 5 - 2 4 7 8 ( 0 1 ) 0 0 3 0 9 - 1
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Letter to the Editor
[3H]-GDP from Ras at 37 °C. As seen in Fig. 1B, after 30 min, nearly 80% of the [3H]-GDP had dissociated from Ras. In contrast, in the presence of GST-Vav, less than 20% of the [3H]-GDP had dissociated from Ras. The src-related tyrosine kinase, Lck, has been reported to phosphorylate Vav and regulate its cellular and biochemical activities [1,8]. As seen in Fig. 1B, relative to GST-Vav, GST-Vav that was first phosphorylated by Lck was markedly less effective in inhibiting the release of [3H]-GDP from Ras. In dramatic contrast to assays described above monitoring the dissociation of GDP from Ras in the presence of GST-Vav, the dissociation of [3H]GTP from Ras was unaffected by GST-Vav, relative to a control reaction with GST. Further, phosphorylation of GST-Vav by Lck resulted in a dramatic activation of a GDI activity for Ras-[3H]-GTP. Thus, unphosphorylated Vav posesses a GDI activity that has a strong preference for Ras-GDP as opposed to RasGTP. Phosphorylation of Vav results in a dramatic reversal for the preference of substrates such that phosphorylated Vav displays a GDI activity specific for Ras-GTP. In vitro, in the presence of a GDI that blocks the dissociation of GTP from Ras, accumulation of GTP-bound Ras would occur relative to a control reaction where [32P]-GTP that binds to Ras would be free to dissociate from Ras. Thus, we suggest that the GEF activity reported by Gulbins and coworkers [2– 5] by use of an assay monitoring association of GTP and Ras could actually reflect a GDI activity. If the biochemical activities described here are working in the cell, then unphosphorylated Vav would maintain Ras in its inactive GDP-bound state, and phosphorylated Vav would hold active GTP-bound Ras in its active state. Several observations in the literature are consistent with Vav influencing Ras signaling. First, activation of H-Ras or activation of Vav in cells results in increased ERK phosphorylation [3,4,7,9,10], while other Dbl-related GEFs fail to activate ERKs. Second, dominant-interfering mutants of Ras block morphological transformation by oncogenic Vav [7,9], as well as ERK activation [10] suggesting Ras is downstream of Vav. Third, a moderate but consistent increase in Ras-GTP levels is observed in Vav-transformed cells [9– 11]. Forth, like Ras, Vav is sufficient to activate the transcription factor NF-AT [12], and ras is required for Vav-mediated activation of NF-AT [13]. Mutants of Vav that lack Rac-GEF activity are still able to activate NF-AT [12]. Fifth, Vav-deficient T-cells are defective in Erk activation [14]. Each of these observations is consistent with reports from the Altman group suggesting Vav can function to activate Ras.
Acknowledgements We are grateful to Amnon Altman for providing the Lck kinase used for these studies. This work was supported by NIH grant CA50261.
References [1] J. Han, et al., Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav, Science 279 (5350) (1998) 558 – 560. [2] E. Gulbins, et al., Tyrosine kinase-stimulated guanine nucleotide exchange activity of Vav in T cell activation, Science 260 (5109) (1993) 822 – 825. [3] E. Gulbins, et al., Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein, Mol. Cell. Biol. 14 (2) (1994) 906 – 913. [4] E. Gulbins, et al., Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides, Mol. Cell. Biol. 14 (7) (1994) 4749 – 4758. [5] E. Gulbins, et al., Molecular analysis of Ras activation by tyrosine phosphorylated Vav, Biochem. Biophys. Res. Commun. 217 (3) (1995) 876 – 885. [6] C.C. Lai, et al., Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins, Mol. Cell. Biol. 13 (3) (1993) 1345 – 1352. [7] X.R. Bustelo, et al., Vav cooperates with Ras to transform rodent fibroblasts but is not a Ras GDP/GTP exchange factor, Oncogene 9 (8) (1994) 2405 – 2413. [8] J. Han, et al., Lck regulates Vav activation of members of the Rho family of GTPases, Mol. Cell. Biol. 17 (3) (1997) 1346 – 1353. [9] R. Khosravi-Far, et al., Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras, Mol. Cell. Biol. 14 (10) (1994) 6848 – 6857. [10] M. Vallalba, et al., Vav modulation of the Ras/MEK/ERK signaling pathway plays a role in NFAT activation and CD69 up-regulation, Eur. J. Immunol. 30 (6) (2000) 1587 – 1596. [11] C.J. Der, personal communication. [12] M.R. Kuhne, et al., A guanine nucleotide exchange factor-independent function of Vav1 in transcriptional activation, J. Biol. Chem. 275 (3) (2000) 2185 – 2190. [13] J. Wu, et al., A functional T-cell receptor signaling pathway is required for p95vav activity, Mol. Cell. Biol. 15 (8) (1995) 4337 – 4346. [14] P.S. Costello, et al., The Rho-family GTP exchange factor Vav is a critical transducer of T cell receptor signals to the calcium, ERK and NF-kappaB pathways, Proc. Natl. Acad. Sci. USA 96 (6) (1999) 3035 – 3040.
Jaewon Han, Balaka Das, Daniel Broek Department of Biochemistry and Molecular Biology, Norris Comprehensi6e Cancer Center, Keck School of Medicine, Uni6ersity of Southern California, Los Angeles, CA 90033, USA
Letter to the Editor In vitro, Vav is a regulated guanine nucleotide dissociation inhibitor for Ras Vav plays a pivitol role in the immune system by control of signals downstream of the T-cell receptor in part by regulation of Rac-related GTPases [1]. While Vav is structurally unrelated to the catalytic domains of the Ras-guanine nucleotide exchange factors (GEFs) (the CDC25-homology domain), a series of reports from Gulbins and co-workers reported Vav posesses a GEF activity directed towards Ras GTPases and this activity could be activated by phosphorylation by Lck [2 – 5]. Instead we observe a Lck-regulated GDI activity for Ras, such that tyrosine-phosphorylated Vav selelctively inhibited GTP release from Ras. Our findings support the notion that activated Vav helps maintain Ras in an active state in stimulated T cells. GEFs for GTPases of the Ras and Rac-families stimulate the release of nucleotides from these GTPases [6]. Two distinct assays have been used to monitor GEF activity, the first and more commonly used monitors the release of a labeled nucleotide (such as [3H]-GDP) from a GTPase [6]. The second assay monitors the association of free labeled-nucleotide (such as alpha[32P]-GTP) with a GTPase. To examine a possible GEF activity of purified Vav for human H-ras protein we used an assay that monitors the release of nucleotide from a preformed complex of histidine-tagged (His6)Ras and [3H]-GDP. Reactions were initiated by mixing equal molar amounts Vav and Ras at 30 °C. In the presence of the negative control, GST, approximately 50% of the starting substrate ((His6)-Ras-[3H]GDP) was found to have released the bound [3H]-GDP (Fig. 1A). This reflects the intrinsic release of nucleotide from Ras. In the presence of the positive control, GST-CDC25 [6], approximately 90% of the [3H]-GDP had dissociated from Ras, reflecting a dramatic stimulation of guanine nucleotide release (Fig. 1A). In contrast only 15% of the [3H]-GDP dissociated from Ras after 30 min in the presence of GST-Vav [7] (Fig. 1A). Under these conditions, a fragment of sos1 encompassing the Dbl-homology and pleckstrin-homology domains failed to affect the dissociation of GDP from Ras (unpublished data).
To examine the apparent GDI activity of Vav under conditions where the intrinsic dissociation of [3H]-GDP from Ras was accelerated, we monitored the release of
Fig. 1. (A) (His)6-H-Ras proteins (30 pmoles) were loaded with [3H]GDP and incubated for the indicated periods of time with 30 pmoles of GST, GST-Vav(L), or GST-CDC25 at 30 °C. At the indicated period of time the amount of [3H]GDP remaining bound to (His)6-H-Ras protein was determined by filter binding assay [6]. Data points reflect the average of two reactions which did not differ by more than 10%. (B) The stability of [3H]GDP bound to (His)6-H-Ras was again monitored as described above except the incubation temperature was 37 °C. Where indicated GST-Vav(L) was phosphorylated with p56lck (10 ng) before the GDI assay. Data points reflect the average of two reactions which did not differ by more than 5%. (C) (His)6-H-Ras proteins were preloaded with [3H]GTP and incubated for the indicated time periods in the absence or presence of Vav or p56lck-phosphorylated Vav. At the indicated periods of time, aliquots were taken and [3H]-GTP bound was measured by filter binding assay. Data points reflect the average of two reactions that did not differ by more than 5%.
0165-2478/02/$ - see front matter © 2002 Published by Elsevier Science B.V. PII: S 0 1 6 5 - 2 4 7 8 ( 0 1 ) 0 0 3 0 9 - 1
2
Letter to the Editor
[3H]-GDP from Ras at 37 °C. As seen in Fig. 1B, after 30 min, nearly 80% of the [3H]-GDP had dissociated from Ras. In contrast, in the presence of GST-Vav, less than 20% of the [3H]-GDP had dissociated from Ras. The src-related tyrosine kinase, Lck, has been reported to phosphorylate Vav and regulate its cellular and biochemical activities [1,8]. As seen in Fig. 1B, relative to GST-Vav, GST-Vav that was first phosphorylated by Lck was markedly less effective in inhibiting the release of [3H]-GDP from Ras. In dramatic contrast to assays described above monitoring the dissociation of GDP from Ras in the presence of GST-Vav, the dissociation of [3H]GTP from Ras was unaffected by GST-Vav, relative to a control reaction with GST. Further, phosphorylation of GST-Vav by Lck resulted in a dramatic activation of a GDI activity for Ras-[3H]-GTP. Thus, unphosphorylated Vav posesses a GDI activity that has a strong preference for Ras-GDP as opposed to RasGTP. Phosphorylation of Vav results in a dramatic reversal for the preference of substrates such that phosphorylated Vav displays a GDI activity specific for Ras-GTP. In vitro, in the presence of a GDI that blocks the dissociation of GTP from Ras, accumulation of GTP-bound Ras would occur relative to a control reaction where [32P]-GTP that binds to Ras would be free to dissociate from Ras. Thus, we suggest that the GEF activity reported by Gulbins and coworkers [2– 5] by use of an assay monitoring association of GTP and Ras could actually reflect a GDI activity. If the biochemical activities described here are working in the cell, then unphosphorylated Vav would maintain Ras in its inactive GDP-bound state, and phosphorylated Vav would hold active GTP-bound Ras in its active state. Several observations in the literature are consistent with Vav influencing Ras signaling. First, activation of H-Ras or activation of Vav in cells results in increased ERK phosphorylation [3,4,7,9,10], while other Dbl-related GEFs fail to activate ERKs. Second, dominant-interfering mutants of Ras block morphological transformation by oncogenic Vav [7,9], as well as ERK activation [10] suggesting Ras is downstream of Vav. Third, a moderate but consistent increase in Ras-GTP levels is observed in Vav-transformed cells [9– 11]. Forth, like Ras, Vav is sufficient to activate the transcription factor NF-AT [12], and ras is required for Vav-mediated activation of NF-AT [13]. Mutants of Vav that lack Rac-GEF activity are still able to activate NF-AT [12]. Fifth, Vav-deficient T-cells are defective in Erk activation [14]. Each of these observations is consistent with reports from the Altman group suggesting Vav can function to activate Ras.
Acknowledgements We are grateful to Amnon Altman for providing the Lck kinase used for these studies. This work was supported by NIH grant CA50261.
References [1] J. Han, et al., Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav, Science 279 (5350) (1998) 558 – 560. [2] E. Gulbins, et al., Tyrosine kinase-stimulated guanine nucleotide exchange activity of Vav in T cell activation, Science 260 (5109) (1993) 822 – 825. [3] E. Gulbins, et al., Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein, Mol. Cell. Biol. 14 (2) (1994) 906 – 913. [4] E. Gulbins, et al., Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides, Mol. Cell. Biol. 14 (7) (1994) 4749 – 4758. [5] E. Gulbins, et al., Molecular analysis of Ras activation by tyrosine phosphorylated Vav, Biochem. Biophys. Res. Commun. 217 (3) (1995) 876 – 885. [6] C.C. Lai, et al., Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins, Mol. Cell. Biol. 13 (3) (1993) 1345 – 1352. [7] X.R. Bustelo, et al., Vav cooperates with Ras to transform rodent fibroblasts but is not a Ras GDP/GTP exchange factor, Oncogene 9 (8) (1994) 2405 – 2413. [8] J. Han, et al., Lck regulates Vav activation of members of the Rho family of GTPases, Mol. Cell. Biol. 17 (3) (1997) 1346 – 1353. [9] R. Khosravi-Far, et al., Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras, Mol. Cell. Biol. 14 (10) (1994) 6848 – 6857. [10] M. Vallalba, et al., Vav modulation of the Ras/MEK/ERK signaling pathway plays a role in NFAT activation and CD69 up-regulation, Eur. J. Immunol. 30 (6) (2000) 1587 – 1596. [11] C.J. Der, personal communication. [12] M.R. Kuhne, et al., A guanine nucleotide exchange factor-independent function of Vav1 in transcriptional activation, J. Biol. Chem. 275 (3) (2000) 2185 – 2190. [13] J. Wu, et al., A functional T-cell receptor signaling pathway is required for p95vav activity, Mol. Cell. Biol. 15 (8) (1995) 4337 – 4346. [14] P.S. Costello, et al., The Rho-family GTP exchange factor Vav is a critical transducer of T cell receptor signals to the calcium, ERK and NF-kappaB pathways, Proc. Natl. Acad. Sci. USA 96 (6) (1999) 3035 – 3040.
Jaewon Han, Balaka Das, Daniel Broek Department of Biochemistry and Molecular Biology, Norris Comprehensi6e Cancer Center, Keck School of Medicine, Uni6ersity of Southern California, Los Angeles, CA 90033, USA