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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

RhoU, but not CDC42, physically associates with phosphorylated EGFR complex. (A, B) Flag-RhoU–transfected, serum-starved HeLa cells were stimulated with 20 ng/ml of EGF for the indicated time. Cells were lysed, immunoprecipitated, and immunoblotted with indicated antibodies. Arrows indicate tyrosine phosphorylated proteins coimmunoprecipitated with Flag–RhoU following EGF stimulation. Asterisk corresponds to nonspecific band from mouse immunoglobulin (Ig) G heavy chain. (C) HeLa cells were transfected with the empty vector, Flag–RhoU, and Flag–CDC42, respectively, immunoprecipitated, and immunoblotted with indicated antibodies. Asterisk denotes mouse IgG light chain.
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Figure 2: RhoU, but not CDC42, physically associates with phosphorylated EGFR complex. (A, B) Flag-RhoU–transfected, serum-starved HeLa cells were stimulated with 20 ng/ml of EGF for the indicated time. Cells were lysed, immunoprecipitated, and immunoblotted with indicated antibodies. Arrows indicate tyrosine phosphorylated proteins coimmunoprecipitated with Flag–RhoU following EGF stimulation. Asterisk corresponds to nonspecific band from mouse immunoglobulin (Ig) G heavy chain. (C) HeLa cells were transfected with the empty vector, Flag–RhoU, and Flag–CDC42, respectively, immunoprecipitated, and immunoblotted with indicated antibodies. Asterisk denotes mouse IgG light chain.

Mentions: We next tested whether RhoU localization and/or its function is linked to growth factor–mediated signaling. On the basis of using rhodamine-conjugated EGF (Rh-EGF), it was evident that RhoU colocalized with internalized EGFR in a time-dependent manner (Figure 1B) at both EEA1-positive early endosomes (Figure 1C) and Lamp1-positive late endosome/lysosome (Figure 1D). Because RhoU colocalized with internalizing EGFR, we further determined whether RhoU physically interacts with EGFR. Indeed, phosphorylated EGFR coimmunoprecipitates with RhoU after EGF stimulation in a time-dependent manner (Figure 2A). Because activation of EGFR is accompanied with its autophosphorylation as well as tyrosine phosphorylation of other downstream substrates, we also examined whether RhoU coprecipitated with additional tyrosine phosphorylated protein(s). Several distinct bands, besides the apparent pEGFR (∼180 kDa), were detected (Figure 2B), suggesting that RhoU associates with the phosphorylated EGFR signaling complex on endosomes (Figure 1, B and C). However, despite high sequence homology between CDC42 and RhoU, we did not detect association of CDC42 with the EGFR under the same conditions (Figure 2C).


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

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

RhoU, but not CDC42, physically associates with phosphorylated EGFR complex. (A, B) Flag-RhoU–transfected, serum-starved HeLa cells were stimulated with 20 ng/ml of EGF for the indicated time. Cells were lysed, immunoprecipitated, and immunoblotted with indicated antibodies. Arrows indicate tyrosine phosphorylated proteins coimmunoprecipitated with Flag–RhoU following EGF stimulation. Asterisk corresponds to nonspecific band from mouse immunoglobulin (Ig) G heavy chain. (C) HeLa cells were transfected with the empty vector, Flag–RhoU, and Flag–CDC42, respectively, immunoprecipitated, and immunoblotted with indicated antibodies. Asterisk denotes mouse IgG light chain.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113775&req=5

Figure 2: RhoU, but not CDC42, physically associates with phosphorylated EGFR complex. (A, B) Flag-RhoU–transfected, serum-starved HeLa cells were stimulated with 20 ng/ml of EGF for the indicated time. Cells were lysed, immunoprecipitated, and immunoblotted with indicated antibodies. Arrows indicate tyrosine phosphorylated proteins coimmunoprecipitated with Flag–RhoU following EGF stimulation. Asterisk corresponds to nonspecific band from mouse immunoglobulin (Ig) G heavy chain. (C) HeLa cells were transfected with the empty vector, Flag–RhoU, and Flag–CDC42, respectively, immunoprecipitated, and immunoblotted with indicated antibodies. Asterisk denotes mouse IgG light chain.
Mentions: We next tested whether RhoU localization and/or its function is linked to growth factor–mediated signaling. On the basis of using rhodamine-conjugated EGF (Rh-EGF), it was evident that RhoU colocalized with internalized EGFR in a time-dependent manner (Figure 1B) at both EEA1-positive early endosomes (Figure 1C) and Lamp1-positive late endosome/lysosome (Figure 1D). Because RhoU colocalized with internalizing EGFR, we further determined whether RhoU physically interacts with EGFR. Indeed, phosphorylated EGFR coimmunoprecipitates with RhoU after EGF stimulation in a time-dependent manner (Figure 2A). Because activation of EGFR is accompanied with its autophosphorylation as well as tyrosine phosphorylation of other downstream substrates, we also examined whether RhoU coprecipitated with additional tyrosine phosphorylated protein(s). Several distinct bands, besides the apparent pEGFR (∼180 kDa), were detected (Figure 2B), suggesting that RhoU associates with the phosphorylated EGFR signaling complex on endosomes (Figure 1, B and C). However, despite high sequence homology between CDC42 and RhoU, we did not detect association of CDC42 with the EGFR under the same conditions (Figure 2C).

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