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GRB2 couples RhoU to epidermal growth factor receptor signaling and cell migration.

Zhang JS, Koenig A, Young C, Billadeau DD - Mol. Biol. Cell (2011)

Bottom Line: The mechanisms regulating RhoU activation, as well as its downstream effectors, are not fully characterized.Moreover, RhoU physically associates with activated EGFR in a GRB2-dependent manner through specific proline-rich motifs within its N-terminus.Taken together, the data suggest a unique regulatory mechanism by which RhoU interaction with SH3 adaptor proteins might serve to integrate growth factor receptor signaling with RhoU activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology Research and Schulze Center for Novel Therapeutics.

ABSTRACT
RhoU is an atypical Rho family member with high homology to CDC42 but containing unique N- and C-terminal extensions. The mechanisms regulating RhoU activation, as well as its downstream effectors, are not fully characterized. We show that after epidermal growth factor (EGF) stimulation RhoU colocalizes with EGF receptor (EGFR) on endosomes, which requires both its N- and C-terminal extension sequences. Moreover, RhoU physically associates with activated EGFR in a GRB2-dependent manner through specific proline-rich motifs within its N-terminus. Mutation of these proline-rich sequences or suppression of GRB2 by RNA interference abrogates the interaction of RhoU with activated EGFR, as well as EGF-stimulated RhoU GTP binding. In addition, RhoU is involved in EGFR-mediated signaling, leading to AP1 transcriptional activity and cell migration in pancreatic cancer cells, events that require its interaction with the Grb2-EGFR complex. Taken together, the data suggest a unique regulatory mechanism by which RhoU interaction with SH3 adaptor proteins might serve to integrate growth factor receptor signaling with RhoU activation.

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Related in: MedlinePlus

Proline-rich motifs in the N-terminus of RhoU mediate its interaction with GRB2 and the EGFR. (A) Lysates from Flag-RhoU–transfected HeLa cells were used for GST pull-down assays with either the N- or C-terminal SH3 domain of GRB2 (top). The same blot stained with Ponceau S documents the fusion protein input (bottom). (B). The amino acid sequence of mouse RhoU N-terminal extension. The two class II SH3-binding consensuses (PXXPXR) are underlined in bold. The mutations by substitution of prolines with alanines in each mutant are specified. (C) Lysates from WT and mutant Flag-RhoU–transfected cells were used for GST pull-down assays with GRB2 N- and C-terminal SH3 fusion proteins, designated GST–SH3(N) and GST–SH3(C), respectively. Top two panels show Flag–RhoU bound to GST–SH3 fusion proteins as detected by anti-Flag antibodies (l.e. and s.e. indicate longer and shorter exposure, respectively). Ponceau S staining documents the input of recombinant GST fusion proteins. The expression level of Flag–RhoU constructs is shown in the bottom panel. (D) GST SH3(N) was used in pull-down assay with in vitro–translated [35S]methionine-labeled RhoU containing indicated deletion/mutations. A representative result of RhoU proteins bound to GST SH3(N) is shown in the top panel followed by the Coomassie staining of the same gel to the indicated input of GST–SH3 fusion proteins. The input (20%) of in vitro–translated RhoU proteins is shown at the bottom. The average band intensity for GST-SH3–precipitated RhoU from three independent experiments along with SD is shown in the histogram (right).
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Figure 5: Proline-rich motifs in the N-terminus of RhoU mediate its interaction with GRB2 and the EGFR. (A) Lysates from Flag-RhoU–transfected HeLa cells were used for GST pull-down assays with either the N- or C-terminal SH3 domain of GRB2 (top). The same blot stained with Ponceau S documents the fusion protein input (bottom). (B). The amino acid sequence of mouse RhoU N-terminal extension. The two class II SH3-binding consensuses (PXXPXR) are underlined in bold. The mutations by substitution of prolines with alanines in each mutant are specified. (C) Lysates from WT and mutant Flag-RhoU–transfected cells were used for GST pull-down assays with GRB2 N- and C-terminal SH3 fusion proteins, designated GST–SH3(N) and GST–SH3(C), respectively. Top two panels show Flag–RhoU bound to GST–SH3 fusion proteins as detected by anti-Flag antibodies (l.e. and s.e. indicate longer and shorter exposure, respectively). Ponceau S staining documents the input of recombinant GST fusion proteins. The expression level of Flag–RhoU constructs is shown in the bottom panel. (D) GST SH3(N) was used in pull-down assay with in vitro–translated [35S]methionine-labeled RhoU containing indicated deletion/mutations. A representative result of RhoU proteins bound to GST SH3(N) is shown in the top panel followed by the Coomassie staining of the same gel to the indicated input of GST–SH3 fusion proteins. The input (20%) of in vitro–translated RhoU proteins is shown at the bottom. The average band intensity for GST-SH3–precipitated RhoU from three independent experiments along with SD is shown in the histogram (right).

Mentions: Having established that GRB2 is essential in coupling RhoU to the activated EGFR, we were interested in delineating which proline-rich motif within the N-terminus of RhoU is involved in mediating RhoU/GRB2 interactions and whether one or both GRB2 SH3 domains are involved. Although both GRB2 clones from YTH screening contained only the C-terminal SH3, we tested whether the N-terminal SH3 could also interact with RhoU. In fact, the N-terminal SH3 domain of GRB2 pulled down significantly more RhoU compared with the C-terminal domain (Figure 5A). We next examined the putative SH3-binding proline-rich sequences within the RhoU N-terminus. Mouse RhoU harbors two class II SH3-binding consensus (PXXPXR) motifs that are fully conserved in human RhoU (Figure 5B). We generated mutations in each of the potential binding sites designated as mutant 1 (M1), mutant 2 (M2), and a double mutant (DM) as shown in Figure 5B. To determine the effect of these mutations on RhoU interaction with GRB2 SH3 domains, we used recombinant GST fusion proteins of GRB2 N- and C-terminal SH3 domains for pull-down assays with lysate from cells transfected with the indicated RhoU constructs. For the N-terminal SH3, mutation of either site resulted in reduced binding, although the M2 had a more dramatic effect (Figure 5C, right). For the C-terminal SH3, M1 had only a moderate effect, whereas M2 largely abolished this interaction (Figure 5C, left). Of significance, neither SH3 domain bound to the DM, suggesting that both domains likely contribute to the interaction with GRB2 (Figure 5C). To further characterize the interactions between RhoU SH3-binding consensus and the SH3 domain, as well as to explore the potential effect of GTP-loading status on this interaction, we performed GST pull-down assays with in vitro–translated [35S]methionine-labeled Flag–RhoU with desired deletions/mutations. As shown in Figure 5D, mutation of either SH3 binding site markedly reduced the binding, whereas deletion of the N-terminal extension or mutation of both SH3-binding consensus sites within the N-terminus abolished the interaction (Figure 5D). However, deletion of the C-terminal extension did not reduce binding in this assay. These results suggest that both proline-rich sequences within the RhoU N-terminus and both GRB2 SH3 domains are involved in maintaining the optimal interaction between these two proteins.


GRB2 couples RhoU to epidermal growth factor receptor signaling and cell migration.

Zhang JS, Koenig A, Young C, Billadeau DD - Mol. Biol. Cell (2011)

Proline-rich motifs in the N-terminus of RhoU mediate its interaction with GRB2 and the EGFR. (A) Lysates from Flag-RhoU–transfected HeLa cells were used for GST pull-down assays with either the N- or C-terminal SH3 domain of GRB2 (top). The same blot stained with Ponceau S documents the fusion protein input (bottom). (B). The amino acid sequence of mouse RhoU N-terminal extension. The two class II SH3-binding consensuses (PXXPXR) are underlined in bold. The mutations by substitution of prolines with alanines in each mutant are specified. (C) Lysates from WT and mutant Flag-RhoU–transfected cells were used for GST pull-down assays with GRB2 N- and C-terminal SH3 fusion proteins, designated GST–SH3(N) and GST–SH3(C), respectively. Top two panels show Flag–RhoU bound to GST–SH3 fusion proteins as detected by anti-Flag antibodies (l.e. and s.e. indicate longer and shorter exposure, respectively). Ponceau S staining documents the input of recombinant GST fusion proteins. The expression level of Flag–RhoU constructs is shown in the bottom panel. (D) GST SH3(N) was used in pull-down assay with in vitro–translated [35S]methionine-labeled RhoU containing indicated deletion/mutations. A representative result of RhoU proteins bound to GST SH3(N) is shown in the top panel followed by the Coomassie staining of the same gel to the indicated input of GST–SH3 fusion proteins. The input (20%) of in vitro–translated RhoU proteins is shown at the bottom. The average band intensity for GST-SH3–precipitated RhoU from three independent experiments along with SD is shown in the histogram (right).
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Related In: Results  -  Collection

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Figure 5: Proline-rich motifs in the N-terminus of RhoU mediate its interaction with GRB2 and the EGFR. (A) Lysates from Flag-RhoU–transfected HeLa cells were used for GST pull-down assays with either the N- or C-terminal SH3 domain of GRB2 (top). The same blot stained with Ponceau S documents the fusion protein input (bottom). (B). The amino acid sequence of mouse RhoU N-terminal extension. The two class II SH3-binding consensuses (PXXPXR) are underlined in bold. The mutations by substitution of prolines with alanines in each mutant are specified. (C) Lysates from WT and mutant Flag-RhoU–transfected cells were used for GST pull-down assays with GRB2 N- and C-terminal SH3 fusion proteins, designated GST–SH3(N) and GST–SH3(C), respectively. Top two panels show Flag–RhoU bound to GST–SH3 fusion proteins as detected by anti-Flag antibodies (l.e. and s.e. indicate longer and shorter exposure, respectively). Ponceau S staining documents the input of recombinant GST fusion proteins. The expression level of Flag–RhoU constructs is shown in the bottom panel. (D) GST SH3(N) was used in pull-down assay with in vitro–translated [35S]methionine-labeled RhoU containing indicated deletion/mutations. A representative result of RhoU proteins bound to GST SH3(N) is shown in the top panel followed by the Coomassie staining of the same gel to the indicated input of GST–SH3 fusion proteins. The input (20%) of in vitro–translated RhoU proteins is shown at the bottom. The average band intensity for GST-SH3–precipitated RhoU from three independent experiments along with SD is shown in the histogram (right).
Mentions: Having established that GRB2 is essential in coupling RhoU to the activated EGFR, we were interested in delineating which proline-rich motif within the N-terminus of RhoU is involved in mediating RhoU/GRB2 interactions and whether one or both GRB2 SH3 domains are involved. Although both GRB2 clones from YTH screening contained only the C-terminal SH3, we tested whether the N-terminal SH3 could also interact with RhoU. In fact, the N-terminal SH3 domain of GRB2 pulled down significantly more RhoU compared with the C-terminal domain (Figure 5A). We next examined the putative SH3-binding proline-rich sequences within the RhoU N-terminus. Mouse RhoU harbors two class II SH3-binding consensus (PXXPXR) motifs that are fully conserved in human RhoU (Figure 5B). We generated mutations in each of the potential binding sites designated as mutant 1 (M1), mutant 2 (M2), and a double mutant (DM) as shown in Figure 5B. To determine the effect of these mutations on RhoU interaction with GRB2 SH3 domains, we used recombinant GST fusion proteins of GRB2 N- and C-terminal SH3 domains for pull-down assays with lysate from cells transfected with the indicated RhoU constructs. For the N-terminal SH3, mutation of either site resulted in reduced binding, although the M2 had a more dramatic effect (Figure 5C, right). For the C-terminal SH3, M1 had only a moderate effect, whereas M2 largely abolished this interaction (Figure 5C, left). Of significance, neither SH3 domain bound to the DM, suggesting that both domains likely contribute to the interaction with GRB2 (Figure 5C). To further characterize the interactions between RhoU SH3-binding consensus and the SH3 domain, as well as to explore the potential effect of GTP-loading status on this interaction, we performed GST pull-down assays with in vitro–translated [35S]methionine-labeled Flag–RhoU with desired deletions/mutations. As shown in Figure 5D, mutation of either SH3 binding site markedly reduced the binding, whereas deletion of the N-terminal extension or mutation of both SH3-binding consensus sites within the N-terminus abolished the interaction (Figure 5D). However, deletion of the C-terminal extension did not reduce binding in this assay. These results suggest that both proline-rich sequences within the RhoU N-terminus and both GRB2 SH3 domains are involved in maintaining the optimal interaction between these two proteins.

Bottom Line: The mechanisms regulating RhoU activation, as well as its downstream effectors, are not fully characterized.Moreover, RhoU physically associates with activated EGFR in a GRB2-dependent manner through specific proline-rich motifs within its N-terminus.Taken together, the data suggest a unique regulatory mechanism by which RhoU interaction with SH3 adaptor proteins might serve to integrate growth factor receptor signaling with RhoU activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology Research and Schulze Center for Novel Therapeutics.

ABSTRACT
RhoU is an atypical Rho family member with high homology to CDC42 but containing unique N- and C-terminal extensions. The mechanisms regulating RhoU activation, as well as its downstream effectors, are not fully characterized. We show that after epidermal growth factor (EGF) stimulation RhoU colocalizes with EGF receptor (EGFR) on endosomes, which requires both its N- and C-terminal extension sequences. Moreover, RhoU physically associates with activated EGFR in a GRB2-dependent manner through specific proline-rich motifs within its N-terminus. Mutation of these proline-rich sequences or suppression of GRB2 by RNA interference abrogates the interaction of RhoU with activated EGFR, as well as EGF-stimulated RhoU GTP binding. In addition, RhoU is involved in EGFR-mediated signaling, leading to AP1 transcriptional activity and cell migration in pancreatic cancer cells, events that require its interaction with the Grb2-EGFR complex. Taken together, the data suggest a unique regulatory mechanism by which RhoU interaction with SH3 adaptor proteins might serve to integrate growth factor receptor signaling with RhoU activation.

Show MeSH
Related in: MedlinePlus