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Mutant huntingtin replaces Gab1 and interacts with C-terminal SH3 domain of growth factor receptor binding protein 2 (Grb2)
Neuroscience Research 87 (2014) 77–83
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Rapid Communication
Mutant huntingtin replaces Gab1 and interacts with C-terminal SH3 domain of growth factor receptor binding protein 2 (Grb2) Shounak Baksi, Sreetama Basu, Debashis Mukhopadhyay ∗ Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India
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Article history: Received 14 February 2014 Received in revised form 18 June 2014 Accepted 25 June 2014 Available online 18 July 2014 Keywords: Grb2 Htt Interaction SH3 domain MAPK signaling
a b s t r a c t Huntington’s disease (HD) is caused due to expansion of CAG repeats in the gene huntingtin (Htt). Adaptor protein Grb2, involved in Ras-MAP kinase pathway, is a known interactor of Htt. Mutant Htt–Grb2 interaction reduces Ras-MAPK signaling in HD models. In normal cells Grb2 forms Grb2–Sos1–Gab1 complex through its N-SH3 and C-SH3 domains respectively, essential for sustained activation of Ras. We found that C-SH3 of Grb2 mediates the interaction with mutant Htt and this interaction being stronger could replace Gab1, with mutant Htt becoming the preferred partner. This would have immense effect on downstream signaling events. © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Huntington’s disease (HD), an autosomal dominant neurodegenerative disorder, is caused due to expansion of CAG repeats (coding for glutamine) in the gene huntingtin (Htt) (The Huntington’s Disease Collaborative Research Group, 1993). Over the years, various cellular processes like excitotoxicity, oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, axonal transport, ubiquitin proteasome pathway, autophagy, transcriptional deregulation and apoptosis have been implicated in HD (Ross and Tabrizi, 2011; Seredenina and Luthi-Carter, 2012). Disease causing mutations in Htt often recruit many of its interacting partners of the mutant protein leading to the biological consequences of the disease (Basu et al., 2013). Growth factor receptor binding protein 2 (Grb2) is a wellknown adaptor protein involved in Ras-MAP kinase pathway. Grb2 signaling has been implicated in many human malignancies (Cheng et al., 1998; Dharmawardana et al., 2006; Lowenstein et al., 1992). Apart from its several signaling role, Grb2 is also known to act as a scavenger molecule in Alzheimer’s disease like condition (Raychaudhuri and Mukhopadhyay, 2010).
Abbreviations: HD, Huntington’s disease; Htt, huntingtin; Grb2, growth factor receptor binding protein 2; Sos1, Son of Sevenless homolog 1; Gab1, Grb2 associated binding protein 1; SH2, Src homology 2; SH3, Src homology 3; N-SH3, N-terminal SH3 domain; C-SH3, C-terminal SH3 domain; RTK, receptor tyrosine kinase; PRD, proline rich domain. ∗ Corresponding author. Tel.: +91 033 2337 5345–49; fax: +91 033 2337 4637. E-mail address: [email protected] (D. Mukhopadhyay).
The adaptor protein Grb2 is made up of a SH2 domain flanked by two SH3 domains. Through the SH2 domain Grb2 interacts with the receptor tyrosine kinases (RTKs) and non-RTKs having phospho tyrosine motifs (Dharmawardana et al., 2006; Lowenstein et al., 1992; Rozakis-Adcock et al., 1992, 1993). The N-terminal SH3 (NSH3) domain of Grb2 binds to SOS, Cbl, HYK1 and other proteins having the consensus proline rich motif (P-X-X-P-X-R) (Chardin et al., 1993; Giubellino and Arany, 2010; Lewitzky et al., 2001; Li et al., 1993; Odai et al., 1995; Schaeper et al., 2000; Seedorf et al., 1994; Vidal et al., 1998). However, a lack of consensus motif for the C-terminal SH3 (C-SH3) domain compromises its binding specificity and it has been reported that Grb2 C-SH3 can bind to peptides which even lack the core P-X-X-P sequence (Lewitzky et al., 2001), typically required by most SH3 domains. While Grb2 binds to the RTK’s via its SH2 domain, the SH3 domains remain available for binding a plethora of proline rich motif containing proteins to trigger different signaling (McDonald et al., 2010). The most common and well studied of these partners is Sos1 (Chardin et al., 1993; Li et al., 1993; McDonald et al., 2010; Schaeper et al., 2000), which upon recruitment to the inner membrane surface, facilitates GDP–GTP exchange within the membrane bound Ras GTPase and thereby turns on MAPK signaling cascade, crucial for cellular growth and proliferation. Further, the recruitment of Gab1 provides docking platforms for the Shp2 tyrosine phosphatase and the PI3K lipid kinase, which account for further amplification of Ras activity. For sustained activation of Ras, both Sos1 dependent and Gab1 dependent pathways (Araki et al., 2003; Cunnick et al., 2002; Gu and Neel, 2003; McDonald et al., 2010) have been implicated.
http://dx.doi.org/10.1016/j.neures.2014.06.009 0168-0102/© 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
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Fig. 1. (A) Representative confocal microscopy images of Htt deletion mutants cloned in GFP. Different clones are (I) N17-145Q, (II) PRD-145Q, (III) 1–17, (IV) P, (V) 1–17 25Q GFP, (VI) Grb2 Dsred in Neuro2A cell. Arrows show the aggregates of Htt GFP clones and vesicular structures of Grb2 Dsred. (B) Domain organization of Htt exon1 and the construction of deletion mutant clones. (C) Confocal images of co-transfected cells with Htt clones in A (I–V) and Grb2 Dsred. VI representative confocal image of neuro2A cell co-transfected with Htt 145Q exon1 GFP and Grb2 Dsred. Arrows show the colocalized structures
Grb2 is a known interactor of Htt and this interaction is known to be regulated by epidermal growth factor receptor (EGFR) activation (Liu et al., 1997). It is also reported that polyQ expansion leads to alteration in EGFR related trafficking (Lievens et al., 2005). Previously, we showed that Grb2 can interact with mutant Htt without any EGFR activation and that the interaction leads to downregulation of ERK signaling in striatal HD cell (Baksi et al., 2013). In this study, using biophysical tools, we tried to find out the mechanism of mutant Htt–Grb2 interaction leading to inhibition of ERK signaling in HD cell model.
Htt exon1 145Q and 23Q clones used in our study, were kindly gifted by Patrick Lajoie, Albert Einstein College of Medicine, USA (Lajoie and Snapp, 2010). N17 GFP i.e. 145Q-PRD GFP: was cloned into GFPC1 vector sing Htt exon1 145Q as template and 5 -ACGCGTCGACGTATGCAGCAGCAGCAGCAGC-3 forward, 5 -TGGGATCCGGTC GGTGCAGCGGCTCCTCAGC-3 reverse primer Rest of the deletion mutant clones of Htt exon1 were kindly provided by Leslie Michels Thompson of University of California, Irvine (Rockabrand et al., 2007). The full length Grb2, NSH3 and C-SH3 domains of Grb2 were cloned into pET28A,
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Fig. 2. (A) Confocal images of 23Q, 145QHtt GFP, N-SH3 and C-SH3 Grb2 Dsred transfected in Neuro2A cells. (B) Confocal Images of Neuro2A cells transfected with (I) Grb2 C-SH3 Dsred and 23Q Htt GFP, (II) Grb2 N-SH3 Dsred and 23Q Htt GFP, (III) Grb2 N-SH3 Dsred and 145Q Htt GFP and (IV) Grb2 C-SH3 Dsred and 145Q Htt GFP. (C) Pearson correlation coefficients of images described in B (n = 20). (D) ICQ analysis of images described in B (n = 20).
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Fig. 3. (A) Representative non-linear and linear fits of Htt peptide binding with (i) Full length Grb2 and (ii) Grb2 C-SH3 (n = 3 for both). No binding was found with Grb2 N-SH3. (B) Co-immunoprecipitation of cell lysates from STHdhQ7/7 and STHdhQ111/111 cells pulled with anti Grb2 antibody, probed with anti Sos1 and anti Gab1 and anti Htt (n = 3) antibodies. (C) Fold changes of immunoprecipitated Sos1 vs. Gab1 levels in STHdhQ111/111 and STHdhQ7/7 cells. All being normalized to respective input levels.
with the following primers, with Nde1 and BamH1 restriction endonuclease. N-SH3 Forward: 5 -GGAATTCCATATGGAAGCCATCGCCAAATATGA C-3 ; N-SH3 Reverse: 5 -CGGGAT CCTTACATTTCTATGTAGTTCTTGGGAATG-3 ; C-SH3 Forward: 5 -GGAATTCCATAT GACATACGTCCAGGCCCTCTTTG-3 and in pGFPC1 (Clontech, USA) using Sal1 and BamH1 enzymes with N-SH3 Forward: 5 -ACGCGTCGACATGGAAGCCATCGCCAAATA TGAC-3 , C-SH3 Forward 5 ACGCGTCGACACATACGTCCAGGCCCTCTTTG-3 and same reverse primers as for pET28A. Full length Grb2 was cloned using N-SH3 forward and C-SH3 reverse primers. The His-tagged proteins were expressed with IPTG induction and purified using Ni-NTA beads (Qiagen, USA) according to manufacturer’s protocol and run on a 15% SDS PAGE to check the purity of the proteins. Fractions with pure proteins were used for further experiments. Neuro2A cells were procured from National Centre for Cell Science (NCCS), Pune, India and STHdhQ7/7 and STHdhQ111/111 cells were kindly gifted by Prof. Marcy McDonald of Massachusetts General Hospital, USA (Trettel et al., 2000). Confocal imaging and analysis was done as described previously (Baksi et al., 2013). Immunoprecipitation was described earlier (Baksi et al., 2013). For fluorescence measurements, steady-state fluorescence studies were performed using a Varian Cary Eclipse fluorescence
spectrophotometer. A 10-mer peptide PPPQLPQPPP, containing the SH3 binding motif P-X-X-P, was chosen from proline rich domain of Htt exon1 and was synthesized in vitro. The Grb2-N-SH3 domain possesses a tryptophan residue (Trp 36) at its peptide binding groove C-SH3 domain has two tryptophans (Trp 191,192). Our binding assay was based on the assumption that binding of the peptides to the Grb2-SH3 domains would reduce the surface accessibility of Trp, and therefore, a decay of intrinsic fluorescence should take place. The peptide binding assay was done as described before (Das et al., 2010; Ray and Chakrabarti, 2004). For theoretical modeling of protein-peptide binding Rosetta was used for flexible docking, Chiron for energy minimization, CMA: Contact Map Analysis (http://ligin.weizmann.ac.il/cma/) for identification of contact between residues and UCSF chimera for graphical representation of the structure (Pettersen et al., 2004). To understand the modalities of Grb2 interaction by the two different SH3 domains of Htt exon1, we used several deletion mutation clones of Htt exon1 and checked their sub cellular localization in Neuro2A cell. Httexon1 basically consists of first17 random amino acid stretch (N17), then the poly glutamine (poly Q) and finally proline rich domain (PRD). The clones we used were different combinations of these domains viz. (a) 145Q-P GFP (N17) this clone was expressed inside nucleus only and formed nuclear
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Fig. 4. Structural representations of interaction between Grb2 C-terminal SH3 domain and poly proline peptide of huntingtin exon1 complex. The molecular surface of Grb2 C-SH3 domain is shown with electrostatic potential painted on it and the poly proline domain is shown as a ribbon diagram. (A) Theoretical model of the complex of Grb2 C-SH3 with poly proline domain of Htt 23Q exon1 and (B) Theoretical model of the complex of Grb2 C-SH3 with poly proline domain of Htt 145Q exon1. (C and D) The lists of residue contacts for each complex are given at the representative images.
aggregates (Fig. 1A I and B); (b) 17-103Q GFP (PRD) (Fig. 1A II and B), this one showed cytoplasmic distribution and also formed aggregates in cell; (c) first 17 amino acids only (N17 GFP), which showed a uniform expression throughout cell (Fig. 1A III and B); (d) Only the proline rich domain (PRD GFP), also showed uniform expression across the cell (Fig. 1A IV and B) and (e) 17-25Q GFP (PRD) which showed expression similar to (c) and (d) (Fig. 1A V and B). Grb2 Dsred, when expressed in Neuro2A cell, was localized into big vesicular structures (Fig. 1A VI) and reported as late endosomal vesicles (Raychaudhuri and Mukhopadhyay, 2010). Mutant Htt exon1 (Htt145Qex1), when co-expressed with Grb2 Dsred, was colocalized within the late endosomal vesicular structures [see Fig. 1C VI also (Baksi et al., 2013)]. When co-expressed with Grb2 Dsred, N17 GFP (145Q-P) showed no colocalization since Grb2 was localized in endocytic vesicles and the mutant clone was localized in the nucleus (Fig. 1C I). PRD GFP (17-103Q) also showed no colocalization (Fig. 1C II), same was observed for N17 GFP (Fig. 1C III) and PRD GFP (17-25Q) (Fig. 1C V). Only with PRD GFP and Grb2 co-transfected cells showed colocalization in cell (Fig. 1C IV). 2–5% of the total PRD GFP colocalized with Grb2 Dsred. From these observations we could conclude that Grb2 might interact with the PRD of Htt exon1.
We transfected the N-SH3 and C-SH3 in neuro2A cells and both showed cytoplasmic distribution (Fig. 2A). When co-transfected with Htt 23Qexon1, both showed colocalization (Fig. 2B I, II) with similar Pearson’s correlation coefficients (N-SH3 = 0.89; CSH3 = 0.94) and ICQ values (N-SH3 = 0.39; C-SH3 = 0.41, Fig. 2C and D, n = 20). But when co-transfected with Htt 145Qexon1 C-SH3 (Rr: 0.90, ICQ: 0.40) showed higher Pearson’s correlation coefficient value with respect to that of N-SH3 (Rr: 0.74; ICQ: 0.34) (Fig. 2B III, IV, C and D, n = 20). To know the binding efficiency of different Grb2 SH3 domains with Htt we measured steady state fluorescence. We designed peptide containing P-X-X-P motif (PPPQLPQPPP) from Htt proline rich domain and checked binding with full length and the two individual SH3 domains of Grb2. Full length Grb2 showed binding with the peptide with a stoichiometry of 1 and KD 1.67 ± 0.08 M (Fig. 3A i, n = 3). While checking binding with N-terminal (N-SH3) SH3 and C-terminal SH3 (C-SH3) domains, no binding in case of N-SH3 and strong binding of KD 0.37 ± 0.03 M (Fig. 3A ii; n = 3) in case of C-SH3 was found. Stoichiometry in this case was also found to be 1. Previously we showed that Grb2 preferentially interacted with mutant Htt in STHdhQ111/111 cells but we could not detect any
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interaction in STHdhQ7/7 cells (Fig. 3B) (Baksi et al., 2013). Normally Grb2 is mostly associated with Sos1 and Gab1. So we pulled down Grb2 and checked the levels of both Sos1 and Gab1 associated with Grb2 in both STHdhQ7/7 and STHdhQ111/111 cells. In STHdhQ111/111 cells the SOS1 level was 30% downregulated compared to that of STHdhQ7/7 cells, whereas Gab1 levels in STHdhQ111/111 cells were found to be downregulated by 70% compared to that of STHdhQ7/7 cells (n = 3; P < 0.0001). After normalization of the immunoprecipitated Sos1 and Gab1 levels with those of the input levels in respective cell lysates, we found 1.8-fold higher levels of Sos1 relative to that of Gab1 in STHdhQ111/111 cell normalized to the levels of STHdhQ7/7 cells (Fig. 3B and C; P = 0.0006, n = 3). MAP kinase signaling pathway is an important signaling pathway that regulates the growth, survival and proliferation of the cell. Grb2 being a central adaptor protein in the pathway, interactions of Grb2 with its partners play a major regulatory effect on cellular survival. Disease causing mutant proteins in cells are known to disrupt many protein interactions. Mutant Htt vastly alters the protein interaction network in the cell due to altered protein interactions (Basu et al., 2013). Grb2-mutant Htt interaction is one such interaction that leads to alteration in growth and survival signaling in cell. Previously, we showed that ERK signaling in Huntington’s disease cell model was highly downregulated due to interaction between Grb2 and mutant Htt (Baksi et al., 2013). Transfection of mutant Htt exon1 shows time dependent reduction in ERK1/2 signaling in Neuro2A cell (unpublished data). In our present study we showed the specific binding between C-terminal SH3 domain of Grb2 with proline domain of Htt. We also measured the binding affinity between the two, which shows KD around 1 M. This actually explains why mutant Htt is able to destabilize the Grb2–Sos1–Gab1 ternary complex. Grb2 is associated with Sos1 via its N-terminal SH3 domain and Gab1 with its C-terminal SH3 domain to activate Ras-MAP kinase signaling. Mutant Htt preferentially interacts with the C-terminal SH3 of Grb2 and acts as a competitor of Gab1. Since the KD between Grb2 C-SH3 and Gab1 is around 50 M (McDonald et al., 2010), the affinity of C-SH3 for Htt proline domain is much higher. So, in the disease condition, Htt is able to replace Gab1 from the Grb2–Sos1–Gab1 complex and interact with Grb2. This is also evident from our immunoprecipitation data that in the disease model association of Grb2 with Sos1 is 1.8 times higher than Gab1 in the HD cells, compared to that in wild type. It suggests that mutant Htt mostly replaces Gab1 from the Grb2 complex. Probably due to disassembly of the ternary complex association of Grb2 with Sos1 is also compromised in the disease cell model, but not to the extent of that with Gab1. The preferential binding of Grb2 to mutant Htt however remains unexplained, especially due to lack of proper structure of Htt exon1 with extended PolyQ repeat. It has already been shown by NMR (Thakur et al., 2009), crystallographic (Kim, 2013; Kim et al., 2009) or bioinformatic (Lakhani et al., 2010) studies that with increase in polyQ number the region undergoes an ␣-helix to -sheet transition which is stabilized by the flexible flanking residues including the poly proline domain. The poly proline domain remains as a helix but with extremely flexible conformation switching between ‘kinked’ and ‘straight’ forms (Kim et al., 2009). To understand this variation we modeled the structure of Htt exon1 23Q and Htt exon1 145Q using Phyre2 server (Kelley and Sternberg, 2009) and the poly proline helices were turned out to be ‘extended’ and kinked respectively (Fig. 4A and B). The structures of the Grb2 C-SH3 domain: polyproline peptide complexes were now properly orientated and flexible docking of the peptide was done using Rosetta (https://www.rosettacommons.org/home) (Wang et al., 2007) and the models of the docked complexes were built using the loop modeling protocol of Modeller v.9.13. (Eswar et al., 2006; Fiser et al., 2000; Marti-Renom et al., 2000; Sali and Blundell, 1993), energy
minimized using CHIRON (Ramachandran et al., 2011). Although a theoretical model, the polyproline domain of Httexon1 145Q shows much higher contact at the protein–peptide interface especially with the residues Phe 165, Asp 166, Trp 193, Asn 208, Tyr 209 of C-SH3 of Grb2 (Fig. 4D), giving an interpretation of the higher binding affinity of Htt exon1 145Q with the poly proline domain. Graphical representation was made using UCSF Chimera (Pettersen et al., 2004). This interaction explains alteration of an important signaling pathway in Huntington’s disease, which disrupts the survival balance of the cell. Further these results suggest possibilities for alternate drug targets for better survival of diseased individuals expressing mutant Htt. Conflict of interest The authors declare no conflict of interest. Acknowledgements The work was supported by funding from IBOP, Department of Atomic Energy, Government of India. The authors acknowledge Dr. Marcy E. MacDonald of Massachusetts General Hospital, USA, for providing STHdhQ7/7 and STHdhQ111/111 cells and Prof. Nitai Pada Bhattacharyya, SINP, Ms. Seema Nath, SINP for useful discussion. References Araki, T., Nawa, H., Neel, B.G., 2003. Tyrosyl phosphorylation of Shp2 is required for normal ERK activation in response to some, but not all, growth factors. J. Biol. Chem. 278 (43), 41677–41684. Baksi, S., Jana, N.R., Bhattacharyya, N.P., Mukhopadhyay, D., 2013. Grb2 is regulated by foxd3 and has roles in preventing accumulation and aggregation of mutant huntingtin. PLoS ONE 8 (10), e76792. Basu, M., Bhattacharyya, N.P., Mohanty, P.K., 2013. Comparison of modules of wild type and mutant huntingtin and TP53 protein interaction networks: implications in biological processes and functions. PLoS ONE 8 (5), e64838. Chardin, P., Camonis, J.H., Gale, N.W., van Aelst, L., Schlessinger, J., Wigler, M.H., BarSagi, D., 1993. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science 260 (5112), 1338–1343. Cheng, A.M., Saxton, T.M., Sakai, R., Kulkarni, S., Mbamalu, G., Vogel, W., Tortorice, C.G., Cardiff, R.D., Cross, J.C., Muller, W.J., Pawson, T., 1998. Mammalian Grb2 regulates multiple steps in embryonic development and malignant transformation. Cell 95, 781–793. Cunnick, J.M., Meng, S., Ren, Y., Desponts, C., Wang, H.G., Djeu, J.Y., Wu, J., 2002. Regulation of the mitogen-activated protein kinase signaling pathway by SHP2. J. Biol. Chem. 277 (11), 9498–9504. Das, S., Raychaudhuri, M., Sen, U., Mukhopadhyay, D., 2010. Functional implications of the conformational switch in AICD peptide upon binding to Grb2-SH2 domain. J. Mol. Biol. 414 (2), 217–230. Dharmawardana, P.G., Peruzzi, B., Giubellino, A., Burke, J., Bottaro, D.P., 2006. Molecular targeting of growth factor receptor-bound 2 (Grb2) as an anti-cancer strategy. Anticancer Drugs 17, 13–20. Eswar, N., Marti-Renom, M.A., Webb, B., Madhusudhan, M.S., Eramian, D., Shen, M., Pieper, U., Sali, A., 2006. Comparative protein structure modeling with MODELLER. Curr. Protoc. Bioinformatics 15, 5.6.1–5.6.30. Fiser, A., Do, R.K., Sali, A., 2000. Modeling of loops in protein structures. Protein Sci. 9, 1753–1773. Giubellino, A., Arany, P.R., 2010. Grb2 and other adaptor proteins in tumor metastasis. Signal Transduct. Cancer Metastasis 15, 77–102. Gu, H., Neel, B.G., 2003. The “Gab” in signal transduction. Trends Cell Biol. 13 (3), 122–130. Kelley, L.A., Sternberg, M.J., 2009. Protein structure prediction on the web: a case study using the Phyre server. Nat. Protoc. 4 (3), 363–371. Kim, M., 2013. Beta conformation of polyglutamine track revealed by a crystal structure of huntingtin N-terminal region with insertion of three histidine residues. Prion 7 (3), 221–228. Kim, M.W., Chelliah, Y., Kim, S.W., Otwinowski, Z., Bezprozvanny, I., 2009. Secondary structure of huntingtin amino-terminal region. Structure 17 (9), 1205–1212. Lajoie, P., Snapp, E.L., 2010. Formation and toxicity of soluble polyglutamine oligomers in living cells. PLoS ONE 5 (12), e15245. Lakhani, V.V., Ding, F., Dokholyan, N.V., 2010. Polyglutamine induced misfolding of huntingtin exon1 is modulated by the flanking sequences. PLoS Comput. Biol. 6 (4), e1000772. Lewitzky, M., Kardinal, C., Gehring, N.H., Schmidt, E.K., Konkol, B., Eulitz, M., Birchmeier, W., Schaeper, U., Feller, S.M., 2001. The C-terminal SH3 domain of the
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Rapid Communication
Mutant huntingtin replaces Gab1 and interacts with C-terminal SH3 domain of growth factor receptor binding protein 2 (Grb2) Shounak Baksi, Sreetama Basu, Debashis Mukhopadhyay ∗ Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India
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Article history: Received 14 February 2014 Received in revised form 18 June 2014 Accepted 25 June 2014 Available online 18 July 2014 Keywords: Grb2 Htt Interaction SH3 domain MAPK signaling
a b s t r a c t Huntington’s disease (HD) is caused due to expansion of CAG repeats in the gene huntingtin (Htt). Adaptor protein Grb2, involved in Ras-MAP kinase pathway, is a known interactor of Htt. Mutant Htt–Grb2 interaction reduces Ras-MAPK signaling in HD models. In normal cells Grb2 forms Grb2–Sos1–Gab1 complex through its N-SH3 and C-SH3 domains respectively, essential for sustained activation of Ras. We found that C-SH3 of Grb2 mediates the interaction with mutant Htt and this interaction being stronger could replace Gab1, with mutant Htt becoming the preferred partner. This would have immense effect on downstream signaling events. © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Huntington’s disease (HD), an autosomal dominant neurodegenerative disorder, is caused due to expansion of CAG repeats (coding for glutamine) in the gene huntingtin (Htt) (The Huntington’s Disease Collaborative Research Group, 1993). Over the years, various cellular processes like excitotoxicity, oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, axonal transport, ubiquitin proteasome pathway, autophagy, transcriptional deregulation and apoptosis have been implicated in HD (Ross and Tabrizi, 2011; Seredenina and Luthi-Carter, 2012). Disease causing mutations in Htt often recruit many of its interacting partners of the mutant protein leading to the biological consequences of the disease (Basu et al., 2013). Growth factor receptor binding protein 2 (Grb2) is a wellknown adaptor protein involved in Ras-MAP kinase pathway. Grb2 signaling has been implicated in many human malignancies (Cheng et al., 1998; Dharmawardana et al., 2006; Lowenstein et al., 1992). Apart from its several signaling role, Grb2 is also known to act as a scavenger molecule in Alzheimer’s disease like condition (Raychaudhuri and Mukhopadhyay, 2010).
Abbreviations: HD, Huntington’s disease; Htt, huntingtin; Grb2, growth factor receptor binding protein 2; Sos1, Son of Sevenless homolog 1; Gab1, Grb2 associated binding protein 1; SH2, Src homology 2; SH3, Src homology 3; N-SH3, N-terminal SH3 domain; C-SH3, C-terminal SH3 domain; RTK, receptor tyrosine kinase; PRD, proline rich domain. ∗ Corresponding author. Tel.: +91 033 2337 5345–49; fax: +91 033 2337 4637. E-mail address: [email protected] (D. Mukhopadhyay).
The adaptor protein Grb2 is made up of a SH2 domain flanked by two SH3 domains. Through the SH2 domain Grb2 interacts with the receptor tyrosine kinases (RTKs) and non-RTKs having phospho tyrosine motifs (Dharmawardana et al., 2006; Lowenstein et al., 1992; Rozakis-Adcock et al., 1992, 1993). The N-terminal SH3 (NSH3) domain of Grb2 binds to SOS, Cbl, HYK1 and other proteins having the consensus proline rich motif (P-X-X-P-X-R) (Chardin et al., 1993; Giubellino and Arany, 2010; Lewitzky et al., 2001; Li et al., 1993; Odai et al., 1995; Schaeper et al., 2000; Seedorf et al., 1994; Vidal et al., 1998). However, a lack of consensus motif for the C-terminal SH3 (C-SH3) domain compromises its binding specificity and it has been reported that Grb2 C-SH3 can bind to peptides which even lack the core P-X-X-P sequence (Lewitzky et al., 2001), typically required by most SH3 domains. While Grb2 binds to the RTK’s via its SH2 domain, the SH3 domains remain available for binding a plethora of proline rich motif containing proteins to trigger different signaling (McDonald et al., 2010). The most common and well studied of these partners is Sos1 (Chardin et al., 1993; Li et al., 1993; McDonald et al., 2010; Schaeper et al., 2000), which upon recruitment to the inner membrane surface, facilitates GDP–GTP exchange within the membrane bound Ras GTPase and thereby turns on MAPK signaling cascade, crucial for cellular growth and proliferation. Further, the recruitment of Gab1 provides docking platforms for the Shp2 tyrosine phosphatase and the PI3K lipid kinase, which account for further amplification of Ras activity. For sustained activation of Ras, both Sos1 dependent and Gab1 dependent pathways (Araki et al., 2003; Cunnick et al., 2002; Gu and Neel, 2003; McDonald et al., 2010) have been implicated.
http://dx.doi.org/10.1016/j.neures.2014.06.009 0168-0102/© 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
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Fig. 1. (A) Representative confocal microscopy images of Htt deletion mutants cloned in GFP. Different clones are (I) N17-145Q, (II) PRD-145Q, (III) 1–17, (IV) P, (V) 1–17 25Q GFP, (VI) Grb2 Dsred in Neuro2A cell. Arrows show the aggregates of Htt GFP clones and vesicular structures of Grb2 Dsred. (B) Domain organization of Htt exon1 and the construction of deletion mutant clones. (C) Confocal images of co-transfected cells with Htt clones in A (I–V) and Grb2 Dsred. VI representative confocal image of neuro2A cell co-transfected with Htt 145Q exon1 GFP and Grb2 Dsred. Arrows show the colocalized structures
Grb2 is a known interactor of Htt and this interaction is known to be regulated by epidermal growth factor receptor (EGFR) activation (Liu et al., 1997). It is also reported that polyQ expansion leads to alteration in EGFR related trafficking (Lievens et al., 2005). Previously, we showed that Grb2 can interact with mutant Htt without any EGFR activation and that the interaction leads to downregulation of ERK signaling in striatal HD cell (Baksi et al., 2013). In this study, using biophysical tools, we tried to find out the mechanism of mutant Htt–Grb2 interaction leading to inhibition of ERK signaling in HD cell model.
Htt exon1 145Q and 23Q clones used in our study, were kindly gifted by Patrick Lajoie, Albert Einstein College of Medicine, USA (Lajoie and Snapp, 2010). N17 GFP i.e. 145Q-PRD GFP: was cloned into GFPC1 vector sing Htt exon1 145Q as template and 5 -ACGCGTCGACGTATGCAGCAGCAGCAGCAGC-3 forward, 5 -TGGGATCCGGTC GGTGCAGCGGCTCCTCAGC-3 reverse primer Rest of the deletion mutant clones of Htt exon1 were kindly provided by Leslie Michels Thompson of University of California, Irvine (Rockabrand et al., 2007). The full length Grb2, NSH3 and C-SH3 domains of Grb2 were cloned into pET28A,
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Fig. 2. (A) Confocal images of 23Q, 145QHtt GFP, N-SH3 and C-SH3 Grb2 Dsred transfected in Neuro2A cells. (B) Confocal Images of Neuro2A cells transfected with (I) Grb2 C-SH3 Dsred and 23Q Htt GFP, (II) Grb2 N-SH3 Dsred and 23Q Htt GFP, (III) Grb2 N-SH3 Dsred and 145Q Htt GFP and (IV) Grb2 C-SH3 Dsred and 145Q Htt GFP. (C) Pearson correlation coefficients of images described in B (n = 20). (D) ICQ analysis of images described in B (n = 20).
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Fig. 3. (A) Representative non-linear and linear fits of Htt peptide binding with (i) Full length Grb2 and (ii) Grb2 C-SH3 (n = 3 for both). No binding was found with Grb2 N-SH3. (B) Co-immunoprecipitation of cell lysates from STHdhQ7/7 and STHdhQ111/111 cells pulled with anti Grb2 antibody, probed with anti Sos1 and anti Gab1 and anti Htt (n = 3) antibodies. (C) Fold changes of immunoprecipitated Sos1 vs. Gab1 levels in STHdhQ111/111 and STHdhQ7/7 cells. All being normalized to respective input levels.
with the following primers, with Nde1 and BamH1 restriction endonuclease. N-SH3 Forward: 5 -GGAATTCCATATGGAAGCCATCGCCAAATATGA C-3 ; N-SH3 Reverse: 5 -CGGGAT CCTTACATTTCTATGTAGTTCTTGGGAATG-3 ; C-SH3 Forward: 5 -GGAATTCCATAT GACATACGTCCAGGCCCTCTTTG-3 and in pGFPC1 (Clontech, USA) using Sal1 and BamH1 enzymes with N-SH3 Forward: 5 -ACGCGTCGACATGGAAGCCATCGCCAAATA TGAC-3 , C-SH3 Forward 5 ACGCGTCGACACATACGTCCAGGCCCTCTTTG-3 and same reverse primers as for pET28A. Full length Grb2 was cloned using N-SH3 forward and C-SH3 reverse primers. The His-tagged proteins were expressed with IPTG induction and purified using Ni-NTA beads (Qiagen, USA) according to manufacturer’s protocol and run on a 15% SDS PAGE to check the purity of the proteins. Fractions with pure proteins were used for further experiments. Neuro2A cells were procured from National Centre for Cell Science (NCCS), Pune, India and STHdhQ7/7 and STHdhQ111/111 cells were kindly gifted by Prof. Marcy McDonald of Massachusetts General Hospital, USA (Trettel et al., 2000). Confocal imaging and analysis was done as described previously (Baksi et al., 2013). Immunoprecipitation was described earlier (Baksi et al., 2013). For fluorescence measurements, steady-state fluorescence studies were performed using a Varian Cary Eclipse fluorescence
spectrophotometer. A 10-mer peptide PPPQLPQPPP, containing the SH3 binding motif P-X-X-P, was chosen from proline rich domain of Htt exon1 and was synthesized in vitro. The Grb2-N-SH3 domain possesses a tryptophan residue (Trp 36) at its peptide binding groove C-SH3 domain has two tryptophans (Trp 191,192). Our binding assay was based on the assumption that binding of the peptides to the Grb2-SH3 domains would reduce the surface accessibility of Trp, and therefore, a decay of intrinsic fluorescence should take place. The peptide binding assay was done as described before (Das et al., 2010; Ray and Chakrabarti, 2004). For theoretical modeling of protein-peptide binding Rosetta was used for flexible docking, Chiron for energy minimization, CMA: Contact Map Analysis (http://ligin.weizmann.ac.il/cma/) for identification of contact between residues and UCSF chimera for graphical representation of the structure (Pettersen et al., 2004). To understand the modalities of Grb2 interaction by the two different SH3 domains of Htt exon1, we used several deletion mutation clones of Htt exon1 and checked their sub cellular localization in Neuro2A cell. Httexon1 basically consists of first17 random amino acid stretch (N17), then the poly glutamine (poly Q) and finally proline rich domain (PRD). The clones we used were different combinations of these domains viz. (a) 145Q-P GFP (N17) this clone was expressed inside nucleus only and formed nuclear
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Fig. 4. Structural representations of interaction between Grb2 C-terminal SH3 domain and poly proline peptide of huntingtin exon1 complex. The molecular surface of Grb2 C-SH3 domain is shown with electrostatic potential painted on it and the poly proline domain is shown as a ribbon diagram. (A) Theoretical model of the complex of Grb2 C-SH3 with poly proline domain of Htt 23Q exon1 and (B) Theoretical model of the complex of Grb2 C-SH3 with poly proline domain of Htt 145Q exon1. (C and D) The lists of residue contacts for each complex are given at the representative images.
aggregates (Fig. 1A I and B); (b) 17-103Q GFP (PRD) (Fig. 1A II and B), this one showed cytoplasmic distribution and also formed aggregates in cell; (c) first 17 amino acids only (N17 GFP), which showed a uniform expression throughout cell (Fig. 1A III and B); (d) Only the proline rich domain (PRD GFP), also showed uniform expression across the cell (Fig. 1A IV and B) and (e) 17-25Q GFP (PRD) which showed expression similar to (c) and (d) (Fig. 1A V and B). Grb2 Dsred, when expressed in Neuro2A cell, was localized into big vesicular structures (Fig. 1A VI) and reported as late endosomal vesicles (Raychaudhuri and Mukhopadhyay, 2010). Mutant Htt exon1 (Htt145Qex1), when co-expressed with Grb2 Dsred, was colocalized within the late endosomal vesicular structures [see Fig. 1C VI also (Baksi et al., 2013)]. When co-expressed with Grb2 Dsred, N17 GFP (145Q-P) showed no colocalization since Grb2 was localized in endocytic vesicles and the mutant clone was localized in the nucleus (Fig. 1C I). PRD GFP (17-103Q) also showed no colocalization (Fig. 1C II), same was observed for N17 GFP (Fig. 1C III) and PRD GFP (17-25Q) (Fig. 1C V). Only with PRD GFP and Grb2 co-transfected cells showed colocalization in cell (Fig. 1C IV). 2–5% of the total PRD GFP colocalized with Grb2 Dsred. From these observations we could conclude that Grb2 might interact with the PRD of Htt exon1.
We transfected the N-SH3 and C-SH3 in neuro2A cells and both showed cytoplasmic distribution (Fig. 2A). When co-transfected with Htt 23Qexon1, both showed colocalization (Fig. 2B I, II) with similar Pearson’s correlation coefficients (N-SH3 = 0.89; CSH3 = 0.94) and ICQ values (N-SH3 = 0.39; C-SH3 = 0.41, Fig. 2C and D, n = 20). But when co-transfected with Htt 145Qexon1 C-SH3 (Rr: 0.90, ICQ: 0.40) showed higher Pearson’s correlation coefficient value with respect to that of N-SH3 (Rr: 0.74; ICQ: 0.34) (Fig. 2B III, IV, C and D, n = 20). To know the binding efficiency of different Grb2 SH3 domains with Htt we measured steady state fluorescence. We designed peptide containing P-X-X-P motif (PPPQLPQPPP) from Htt proline rich domain and checked binding with full length and the two individual SH3 domains of Grb2. Full length Grb2 showed binding with the peptide with a stoichiometry of 1 and KD 1.67 ± 0.08 M (Fig. 3A i, n = 3). While checking binding with N-terminal (N-SH3) SH3 and C-terminal SH3 (C-SH3) domains, no binding in case of N-SH3 and strong binding of KD 0.37 ± 0.03 M (Fig. 3A ii; n = 3) in case of C-SH3 was found. Stoichiometry in this case was also found to be 1. Previously we showed that Grb2 preferentially interacted with mutant Htt in STHdhQ111/111 cells but we could not detect any
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interaction in STHdhQ7/7 cells (Fig. 3B) (Baksi et al., 2013). Normally Grb2 is mostly associated with Sos1 and Gab1. So we pulled down Grb2 and checked the levels of both Sos1 and Gab1 associated with Grb2 in both STHdhQ7/7 and STHdhQ111/111 cells. In STHdhQ111/111 cells the SOS1 level was 30% downregulated compared to that of STHdhQ7/7 cells, whereas Gab1 levels in STHdhQ111/111 cells were found to be downregulated by 70% compared to that of STHdhQ7/7 cells (n = 3; P < 0.0001). After normalization of the immunoprecipitated Sos1 and Gab1 levels with those of the input levels in respective cell lysates, we found 1.8-fold higher levels of Sos1 relative to that of Gab1 in STHdhQ111/111 cell normalized to the levels of STHdhQ7/7 cells (Fig. 3B and C; P = 0.0006, n = 3). MAP kinase signaling pathway is an important signaling pathway that regulates the growth, survival and proliferation of the cell. Grb2 being a central adaptor protein in the pathway, interactions of Grb2 with its partners play a major regulatory effect on cellular survival. Disease causing mutant proteins in cells are known to disrupt many protein interactions. Mutant Htt vastly alters the protein interaction network in the cell due to altered protein interactions (Basu et al., 2013). Grb2-mutant Htt interaction is one such interaction that leads to alteration in growth and survival signaling in cell. Previously, we showed that ERK signaling in Huntington’s disease cell model was highly downregulated due to interaction between Grb2 and mutant Htt (Baksi et al., 2013). Transfection of mutant Htt exon1 shows time dependent reduction in ERK1/2 signaling in Neuro2A cell (unpublished data). In our present study we showed the specific binding between C-terminal SH3 domain of Grb2 with proline domain of Htt. We also measured the binding affinity between the two, which shows KD around 1 M. This actually explains why mutant Htt is able to destabilize the Grb2–Sos1–Gab1 ternary complex. Grb2 is associated with Sos1 via its N-terminal SH3 domain and Gab1 with its C-terminal SH3 domain to activate Ras-MAP kinase signaling. Mutant Htt preferentially interacts with the C-terminal SH3 of Grb2 and acts as a competitor of Gab1. Since the KD between Grb2 C-SH3 and Gab1 is around 50 M (McDonald et al., 2010), the affinity of C-SH3 for Htt proline domain is much higher. So, in the disease condition, Htt is able to replace Gab1 from the Grb2–Sos1–Gab1 complex and interact with Grb2. This is also evident from our immunoprecipitation data that in the disease model association of Grb2 with Sos1 is 1.8 times higher than Gab1 in the HD cells, compared to that in wild type. It suggests that mutant Htt mostly replaces Gab1 from the Grb2 complex. Probably due to disassembly of the ternary complex association of Grb2 with Sos1 is also compromised in the disease cell model, but not to the extent of that with Gab1. The preferential binding of Grb2 to mutant Htt however remains unexplained, especially due to lack of proper structure of Htt exon1 with extended PolyQ repeat. It has already been shown by NMR (Thakur et al., 2009), crystallographic (Kim, 2013; Kim et al., 2009) or bioinformatic (Lakhani et al., 2010) studies that with increase in polyQ number the region undergoes an ␣-helix to -sheet transition which is stabilized by the flexible flanking residues including the poly proline domain. The poly proline domain remains as a helix but with extremely flexible conformation switching between ‘kinked’ and ‘straight’ forms (Kim et al., 2009). To understand this variation we modeled the structure of Htt exon1 23Q and Htt exon1 145Q using Phyre2 server (Kelley and Sternberg, 2009) and the poly proline helices were turned out to be ‘extended’ and kinked respectively (Fig. 4A and B). The structures of the Grb2 C-SH3 domain: polyproline peptide complexes were now properly orientated and flexible docking of the peptide was done using Rosetta (https://www.rosettacommons.org/home) (Wang et al., 2007) and the models of the docked complexes were built using the loop modeling protocol of Modeller v.9.13. (Eswar et al., 2006; Fiser et al., 2000; Marti-Renom et al., 2000; Sali and Blundell, 1993), energy
minimized using CHIRON (Ramachandran et al., 2011). Although a theoretical model, the polyproline domain of Httexon1 145Q shows much higher contact at the protein–peptide interface especially with the residues Phe 165, Asp 166, Trp 193, Asn 208, Tyr 209 of C-SH3 of Grb2 (Fig. 4D), giving an interpretation of the higher binding affinity of Htt exon1 145Q with the poly proline domain. Graphical representation was made using UCSF Chimera (Pettersen et al., 2004). This interaction explains alteration of an important signaling pathway in Huntington’s disease, which disrupts the survival balance of the cell. Further these results suggest possibilities for alternate drug targets for better survival of diseased individuals expressing mutant Htt. Conflict of interest The authors declare no conflict of interest. Acknowledgements The work was supported by funding from IBOP, Department of Atomic Energy, Government of India. The authors acknowledge Dr. Marcy E. MacDonald of Massachusetts General Hospital, USA, for providing STHdhQ7/7 and STHdhQ111/111 cells and Prof. Nitai Pada Bhattacharyya, SINP, Ms. Seema Nath, SINP for useful discussion. References Araki, T., Nawa, H., Neel, B.G., 2003. Tyrosyl phosphorylation of Shp2 is required for normal ERK activation in response to some, but not all, growth factors. J. Biol. Chem. 278 (43), 41677–41684. Baksi, S., Jana, N.R., Bhattacharyya, N.P., Mukhopadhyay, D., 2013. Grb2 is regulated by foxd3 and has roles in preventing accumulation and aggregation of mutant huntingtin. PLoS ONE 8 (10), e76792. Basu, M., Bhattacharyya, N.P., Mohanty, P.K., 2013. Comparison of modules of wild type and mutant huntingtin and TP53 protein interaction networks: implications in biological processes and functions. PLoS ONE 8 (5), e64838. Chardin, P., Camonis, J.H., Gale, N.W., van Aelst, L., Schlessinger, J., Wigler, M.H., BarSagi, D., 1993. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science 260 (5112), 1338–1343. Cheng, A.M., Saxton, T.M., Sakai, R., Kulkarni, S., Mbamalu, G., Vogel, W., Tortorice, C.G., Cardiff, R.D., Cross, J.C., Muller, W.J., Pawson, T., 1998. Mammalian Grb2 regulates multiple steps in embryonic development and malignant transformation. Cell 95, 781–793. Cunnick, J.M., Meng, S., Ren, Y., Desponts, C., Wang, H.G., Djeu, J.Y., Wu, J., 2002. Regulation of the mitogen-activated protein kinase signaling pathway by SHP2. J. Biol. Chem. 277 (11), 9498–9504. Das, S., Raychaudhuri, M., Sen, U., Mukhopadhyay, D., 2010. Functional implications of the conformational switch in AICD peptide upon binding to Grb2-SH2 domain. J. Mol. Biol. 414 (2), 217–230. Dharmawardana, P.G., Peruzzi, B., Giubellino, A., Burke, J., Bottaro, D.P., 2006. Molecular targeting of growth factor receptor-bound 2 (Grb2) as an anti-cancer strategy. Anticancer Drugs 17, 13–20. Eswar, N., Marti-Renom, M.A., Webb, B., Madhusudhan, M.S., Eramian, D., Shen, M., Pieper, U., Sali, A., 2006. Comparative protein structure modeling with MODELLER. Curr. Protoc. Bioinformatics 15, 5.6.1–5.6.30. Fiser, A., Do, R.K., Sali, A., 2000. Modeling of loops in protein structures. Protein Sci. 9, 1753–1773. Giubellino, A., Arany, P.R., 2010. Grb2 and other adaptor proteins in tumor metastasis. Signal Transduct. Cancer Metastasis 15, 77–102. Gu, H., Neel, B.G., 2003. The “Gab” in signal transduction. Trends Cell Biol. 13 (3), 122–130. Kelley, L.A., Sternberg, M.J., 2009. Protein structure prediction on the web: a case study using the Phyre server. Nat. Protoc. 4 (3), 363–371. Kim, M., 2013. Beta conformation of polyglutamine track revealed by a crystal structure of huntingtin N-terminal region with insertion of three histidine residues. Prion 7 (3), 221–228. Kim, M.W., Chelliah, Y., Kim, S.W., Otwinowski, Z., Bezprozvanny, I., 2009. Secondary structure of huntingtin amino-terminal region. Structure 17 (9), 1205–1212. Lajoie, P., Snapp, E.L., 2010. Formation and toxicity of soluble polyglutamine oligomers in living cells. PLoS ONE 5 (12), e15245. Lakhani, V.V., Ding, F., Dokholyan, N.V., 2010. Polyglutamine induced misfolding of huntingtin exon1 is modulated by the flanking sequences. PLoS Comput. Biol. 6 (4), e1000772. Lewitzky, M., Kardinal, C., Gehring, N.H., Schmidt, E.K., Konkol, B., Eulitz, M., Birchmeier, W., Schaeper, U., Feller, S.M., 2001. The C-terminal SH3 domain of the
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