Proteomic analyses of the SMYD family interactomes identify HSP90 as a novel target for SMYD2

doi:10.1093/jmcb/mjr025 Journal of Molecular Cell Biology (2011), 3, 301– 308 | 301 Article Proteomic analyses of the SMYD family interactomes identify HSP90 as a novel target for SMYD2 Mohamed Abu-Farha 1,2,†, Sylvain Lanouette 1,†, Fred Elisma 1, Véronique Tremblay 1, Jeffery Butson 1, Daniel Figeys 1, *, and Jean-François Couture 1,* 1 Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Roger-Guindon Hall, Ottawa, ON, K1H8M5, Canada 2 Biochemistry and Molecular Biology Unit, Dasman Diabetes Institute, Dasman 15462, Kuwait * Correspondence to: Daniel Figeys, E-mail: ; Jean-François Couture, E-mail: The SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in various cellular processes, including gene expression regulation and DNA damage response. Initially identified as genuine histone methyltransferases, specific members of this family have recently been shown to methylate non-histone proteins such as p53, VEGFR, and the retinoblastoma tumor suppressor (pRb). To gain further functional insights into this family of KMTs, we generated the protein interaction network for three different human SMYD proteins (SMYD2, SMYD3, and SMYD5). Characterization of each SMYD protein network revealed that they associate with both shared and unique sets of proteins. Among those, we found that HSP90 and several of its co-chaperones interact specifically with the tetratrico peptide repeat (TPR)-containing SMYD2 and SMYD3. Moreover, using proteomic and biochemical techniques, we provide evidence that SMYD2 methylates K209 and K615 on HSP90 nucleotide-binding and dimerization domains, respectively. In addition, we found that each methylation site displays unique reactivity in regard to the presence of HSP90 co-chaperones, pH, and demethylation by the lysine amine oxidase LSD1, suggesting that alternative mechanisms control HSP90 methylation by SMYD2. Altogether, this study highlights the ability of SMYD proteins to form unique protein complexes that may underlie their various biological functions and the SMYD2-mediated methylation of the key molecular chaperone HSP90. Keywords: SMYD proteins, HSP90, lysine methylation, SET domain, histone methylation Introduction Lysine methylation by the SET-domain-containing lysine methyltransferases (KMTs) has emerged as an important regulator of protein functions (Huang and Berger, 2008) and nuclear processes (Martin and Zhang, 2005). Initially characterized as histone KMTs (HMKTs), SET domain enzymes have recently been shown to methylate a much broader repertoire of protein substrates. For example, VEGFR1 di-methylation by SMYD3 increases its kinase activity (Kunizaki et al., 2007). The sensitivity of ER to estrogens is increased by the mono-methylation of K302 which thereby stimulates the expression of estrogen-responsive genes (Subramanian et al., 2008). Methylation of the elongation factor E2F1 by SET7/9 decreases its lifespan whereas methylation of both p53 and pRb by the same enzyme protects their pro-apoptotic function (Chuikov et al., 2004; Munro et al., 2010). Conversely, methylation of different target lysine residues † These authors contributed equally to this work. Received August 12, 2011. Revised August 18, 2011. Accepted August 21, 2011. # The Author (2011). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. on pRb and p53 by SMYD2 represses their apoptotic activity (Huang et al., 2006; Saddic et al., 2010). Based on sequence homology, SET-domain-containing KMTs are divided into seven groups (Dillon et al., 2005). Among these, the SMYD (SET and MYND domain) group is characterized by the insertion of a MYND zinc finger (Spadaccini et al., 2006) within the SET domain (Xiao et al., 2003). SMYD1, SMYD2, and SMYD3 share a high degree of sequence homology and, with the exception of SMYD5, human SMYD proteins harbor at least one C-terminal tetratrico peptide repeat (TPR) domain. Both TPR and MYND domains are important protein –protein interaction domains (Sims et al., 2002; Abu-Farha et al., 2008; Yamamoto et al., 2011). The SMYD proteins have been found to be linked to various cancers (Hamamoto et al., 2004, 2006; Wang et al., 2008; Hu et al., 2009; Komatsu et al., 2009; Zou et al., 2009) and biological processes (Gottlieb et al., 2002; Brown et al., 2006; Huang et al., 2006; Tan et al., 2006; Thompson and Travers, 2008; Li et al., 2009a). Correlatively, the interactions of SMYD proteins with different complexes were shown to modulate their cellular localization sites, functions, and KMT activities 302 | Journal of Molecular Cell Biology (Gottlieb et al., 2002; Hamamoto et al., 2004; Tan et al., 2006; Abu-Farha et al., 2008; Yamamoto et al., 2011). HSP90 is a homodimeric, ubiquitous, and essential chaperone composed of three functional domains, namely the nucleotidebinding domain (NBD), the middle domain (MD), and the dimerization domain (DD). HSP90 is involved in a large variety of biological processes, including, but not limited to, heat-shock response, signal transduction, steroid signaling, and tumorigenesis (Taipale et al., 2010). These roles are finely regulated through the binding of co-chaperones and client proteins as well as post-translational modifications (PTMs), including phosphorylation (Scroggins and Neckers, 2007), ubiquitylation (Kundrat and Regan, 2010), acetylation (Scroggins et al., 2007; Yang et al., 2008), and nitrosylation (Martinez-Ruiz et al., 2005; Retzlaff et al., 2009). In this study, we generated the protein interaction network for SMYD2, SMYD3, and SMYD5. Characterization of each SMYD protein network revealed that they associate with both unique and shared subsets of proteins. Mass spectrometry and biochemical data also revealed that SMYD2 methylates HSP90a on lysine residues located on its NBD and DD, respectively. In addition, we found that HSP90 methylation by SMYD2 is partially reversed by LSD1 and inhibited by the presence of the co-chaperone HOP, suggesting that it can be dynamically regulated in vivo. Overall, our data provide insights into novel regulatory networks for three members of the SMYD family of KMTs and evidence that HSP90a is a novel substrate for SMYD2. Results SMYD proteins have non-overlapping protein networks As shown in Figure 1A, all human SMYD proteins include the SET and MYND domain. In addition, the highly homologous SMYD1, SMYD2 (data not shown), and SMYD3 share a TPR-like domain found on their C-termini (Figure 1B). Comparatively, SMYD4 has an additional region of 240 amino acids on its N-terminus while SMYD5 lacks any clear domain other than the SET and MYND domain, but has a C-terminal glutamate-rich extension. To investigate whether the structural differences between SMYD2, SMYD3, and SMYD5 underlie the formation of alternative protein complexes, we have undertaken to establi (...truncated)