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Grb2 Interacts with SGEF and Antagonizes the Ability of SGEF to Enhance EGF-Induced ERK1/2 Activation

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Grb2 Interacts with SGEF and Antagonizes the Ability of SGEF to Enhance EGF-Induced ERK1/2 Activation Mol Cell Biochem (2014) 389:239–247 DOI 10.1007/s11010-013-1945-7 Grb2 interacts with SGEF and antagonizes the ability of SGEF to enhance EGF-induced ERK1/2 activation Hongtao Wang • Shanhu Li • Hailiang Li • Peng Wang • Fang Huang • Yali Zhao • Lan Yu • Guolan Luo • Xiaoqing Zhang • Jian Wang • Jianguang Zhou Received: 22 September 2013 / Accepted: 18 December 2013 / Published online: 8 January 2014 Ó Springer Science+Business Media New York 2014 Abstract Previously, we demonstrated that SGEF enhan- Introduction ces EGFR stability; however, SGEF-mediated downstream signaling of EGFR is not well understood. Here, we show Epidermal growth factor receptor (EGFR), the best char- that SGEF enhances EGF-induced ERK1/2 activation inde- acterized member of the ErbB receptor tyrosine kinase pendent of its guanine nucleotide exchange (GEF) activity. (RTK) family, plays crucial roles in numerous cellular We further show that SGEF interacts with Grb2, a critical functions including cell proliferation, survival, differenti- downstream transducer of EGFR. Surprisingly, we found ation, and migration [1]. Aberrant EGFR signaling has that interaction of Grb2 to SGEF antagonizes the ability of been associated with human cancers in which high SGEF to enhance EGF-induced ERK1/2 activation. Taken expression and/or abnormal activation of EGFR are fre- together, this study reports a novel function of SGEF that quently observed [2]. After binding to EGF, EGFR excludes GEF and also provides important insights into the undergoes dimerization which promotes auto-phosphory- complex role of Grb2 in EGFR signal transduction. lation of multiple tyrosine residues in the cytoplasmic tail of the receptor. These phosphorylated tyrosines serve as Keywords SGEF Á Grb2 Á EGF Á EGFR Á ERK1/2 docking sites for specific downstream effectors, containing PTB or SH2 domains [3], such as PI3K and Src enzymes or Abbreviations Grb2 and Nck adaptor proteins which link EGFR to SGEF SH3-containing guanine nucleotide exchange downstream signaling pathways [4]. factor The adaptor protein Grb2 plays a critical role in EGF- EGFR Epidermal growth factor receptor activated Ras–ERK1/2 signaling pathway [5], which is one EGF Epidermal growth factor of the most important signaling cascades downstream of GEF Guanine nucleotide exchange factor EGFR. Grb2 contains a single SH2 domain flanked by two PTB Phosphotyrosine binding SH3 domains. Through its SH2 domain, which is a conserved SH2 Src homology 2 sequence of 100 amino acids, Grb2 recognizes and binds to SH3 Src homology 3 the phosphorylated residues Tyr-1068 and Tyr-1086 in the Pro Proline-rich domain cytoplasmic domain of EGFR [6]. The SH3 domain, which is a conserved sequence that binds to proline-rich sequences within interacting proteins, constitutively associates with SOS, a guanine nucleotide exchange factor (GEF), which H. Wang Á S. Li Á H. Li Á P. Wang Á F. Huang Á Y. Zhao Á L. Yu Á catalyzes GDP–GTP exchange on Ras [7, 8]. In this manner, G. Luo Á X. Zhang Á J. Wang (&) Á J. Zhou (&) Grb2 is recruited near the cell membrane by binding to Laboratory of Medical Molecular Biology, Beijing Institute of phosphorylated EGFR, which also brings Sos to the mem- Biotechnology, 27 Taiping Road, Haidian, Beijing 100850, People’s Republic of China brane-bound Ras, resulting in the activation of Ras–ERK1/2 e-mail: [email protected] cascade, after EGFR activation [9]. J. Zhou Recently, we reported that SGEF, a RhoG-specific GEF, e-mail: [email protected] enhanced EGFR stability by delaying its lysosomal sorting 123 240 Mol Cell Biochem (2014) 389:239–247 and degradation [10]. We also demonstrated that SGEF pCDNA-3.1–SGEF and subcloned into pCMV-2B vector at was overexpressed during prostate cancer and therefore BamHI and EcoRI sites. The primers are as follows: contributed to cancer development and progression [11], For SGEF: most likely by enhancing EGFR stability. However, SGEF- 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 mediated downstream signaling of EGFR has not been (forward) elucidated. The various roles of SGEF, reported thus far, 50-GGGAATTCCTACACGTTGGTCTCCAGTCC-30 require the GEF activity of SGEF. These include various (reverse) physiologic and pathologic processes such as macropino- For SGEF-N terminus: cytosis, leukocyte transendothelial migration, and uptake 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 of Salmonella by epithelial cells [12–14]. In addition, (forward) recent studies have demonstrated that SGEF contributes to 50-GGGAATTCTTGGCTCCATGTGGATCTC-30 the invasive capacity of HPV-16 and HPV-18 transformed (reverse) cell [15]. For SGEFDSH3: In this study, we determined the role of SGEF in EGFR- 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 mediated signal transduction and showed that SGEF sig- (forward) nificantly enhances EGF-induced ERK1/2 activation inde- 50-GGGAATTCTCAGTGAGGTTCGGTCTG-30 pendent of its GEF activity. In addition, we demonstrated (reverse) that SGEF interacts with Grb2, a critical downstream For SGEFDDH: transducer of EGFR. By interacting with SGEF, Grb2 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 antagonizes the ability of SGEF to enhance EGF-induced (forward1) ERK1/2 activation. Taken together, our work not only 50-GGGAATTCCTACACGTTGGTCTCCAGTCC-30 identifies a new role for SGEF in EGFR-mediated ERK1/2 (reverse1) activation but also provides novel insights into the com- 50-GCCAGGAGGAAAGAAAGAGAAGCAAGTTGG plexity of Grb2 in EGFR-mediated signal transduction. TTCGACTATGC-30 (forward2) 50-GCATAGTCGAAC- CAACTTGCTTCTCTTTCTTTCCTCCTGGC-30 Materials and methods (reverse2) SGEFDPro: Antibodies and reagents 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 (forward1) Antibodies for Flag and GFP were obtained from Sigma 50-GGGAATTCCTACACGTTGGTCTCCAGTCC-30 (St. Louis, MO, USA). Anti-EGFR, anti-RhoG, anti-Grb2, (reverse1) and anti-tubulin antibodies were purchased from Santa 50-GAGTACAGGGCTGCCTCTACTG- Cruz (CA, USA). Anti-ERK1/2 and phospho-ERK1/2 CAAATGGCCTTGCC-30 (forward2) (Thr202/Tyr204) antibodies were obtained from Cell Sig- 50-GGCAAGGCCATTTGCAGTAGAGGCAGCCCTG- naling Technology (Beverly, MA, USA). Recombinant TACTC-30 (reverse2) EGF was obtained from ProSpec (Rehovot, Israel). SGEF–P1M: PD98058 was purchased from Cell Signaling Technology 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 (Beverly, MA, USA). (forward1) 50-GGGAATTCCTACACGTTGGTCTCCAGTCC-30 Plasmid constructs (reverse1) 50-GGTGACCTTGCCTGCGGCGGCGGCGGCGGC All vectors including pcDNA3–EGFR, pGEX-4T–Grb2, GGTTCTGCGCCCCCCGC-30 (forward2) pEGFP–Grb2, pEGFP–Grb2–W36, pEGFP–Grb2–R86K, 50-GCGGGGGGCGCAGAACCGCCGCCGCCGCCG and pEGFP–Grb2–W193A were gifts from Dr. Wannian CCGCAGGCAAGGTCACC-30 (reverse2) Yang (Weis Center for Research, Geisinger Clinic, Dan- SGEF–P2M: ville, PA, USA). Full-length, truncated, and point mutations 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 of SGEF were constructed by inserting PCR-amplified (forward1) fragments into the corresponding vectors. 50-GGGAATTCCTACACGTTGGTCTCCAGTCC-30 SGEF, SGEF-N terminus (1–420 amino acid), SGEF (reverse1) DSH3, SGEFDDH, SGEFDPro, SGEF–P1M, and SGEF– 50-CTGGGTTGACTGCCAGCGCGGTGGCTTCGCC- P2M were PCR or overlapping PCR amplified from CACTGCAAATGGCC-30 (forward2) 123 Mol Cell Biochem (2014) 389:239–247 241 50-GGCCATTTGCAGTGGGCGAAGCCACCGCGCT To suppress the expression of RhoG, HEK293T cells GGCAGTCAACCCAG-30 (reverse2) were infected with lentivirus carrying RhoG shRNA (GeneChem Co., Shanghai, China). 12 h later, virus-con- SGEF-(1–420 amino acid), SGEF-(1–200 amino acid), taining medium was replaced with fresh medium, and SGEF-(13–216 amino acid), SGEF-(216–420 amino acid), infected cells were then selected on 2 lg/ml puromycin SGEF–DH, SGEF–PH, and SGEF–SH3 were PCR ampli- after an additional 48 h. fied from pCDNA-3.1–SGEF and subcloned into pGEX– KG vector at BamHI and HindIII sites. The primers are as GST pull-down assay follows: For SGEF-(1–420 amino acid): GST or GST-tagged proteins expressed in bacteria were 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 immobilized on glutathione-Sepharose 4B beads (Amer- (forward) sham) and then incubated with cell lysates with gentle 50-CCAAGCTTTTGGCTCCATGTGGATCTC-30 rotation at 4 °C for 3 h. After washing three times with cell (reverse) lysis buffer, the beads were resolved on 10 % SDS-PAGE For SGEF-(1–200 amino acid): and subjected to Western blot analysis. 50-CGGGATCCGACGGCGAGAGCGAGGTGG-30 (forward) Western blotting and immunoprecipitation 50-CCAAGCTTCCGTTCGGGGTCCTTTGC-30 (reverse) Western blotting was performed as described previously For SGEF-(13–216 amino acid): [11]. 50-CGGGATCCAGCATAACCCCTTTGTG-30 For immunoprecipitation, precleared cell lysates were (forward) incubated with primary antibody at 4 °C for 2 h with gentle 50-CCAAGCTTGGGGAGTTTTTGTTCCG-30 (reverse) rotation followed by further incubation with protein A/G For SGEF-(216–420 amino acid): plus agarose beads for another 2 h at 4 °C. After washing 50-CGGGATCCCCCCTCCAAAGGCTGCC-30 three times with cell lysis buffer, the precipitated beads (forward) were eluted in SDS-PAGE loading buffer, resolved on 50-CCAAGCTTTTGGCTCCATGTGGATCTC-30 10 % SDS-PAGE, and subsequently used for western (reverse) blotting. For SGEF–DH: 50-CGGGATCCGCGGTGAAAAGAAAGGG-30 (forward) Results 50-CCAAGCTTAGTCCTTTCCATCTTCCG-30 (reverse) SGEF enhances EGF-induced ERK1/2 activation For SGEF–PH: independent of its GEF activity 50-CGGGATCCACAGCCTATGTTGAAGAC-30 (forward) Our previous studies demonstrated that SGEF attenuates 50-CCAAGCTTCTTCCCTCTGCTGTG-30 (reverse) EGFR degradation and enhances EGFR stability [10]. For SGEF–SH3: Given that the reduction in EGFR degradation is associated 50-CGGGATCCCCTGCAGACCGAACCTC-30 with
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