Elsevier

Cellular Signalling

Volume 25, Issue 12, December 2013, Pages 2518-2529
Cellular Signalling

Combining docking site and phosphosite predictions to find new substrates: Identification of smoothelin-like-2 (SMTNL2) as a c-Jun N-terminal kinase (JNK) substrate

https://doi.org/10.1016/j.cellsig.2013.08.004Get rights and content

Highlights

  • We searched the human genome for new MAP kinase substrates.

  • Our approach combined docking site and phosphosite predictions.

  • Smoothelin-like 2 (SMTNL2) is a novel JNK substrate.

  • SMTNL2 is phosphorylated by JNK in vitro and in cells.

  • SMTNL2 is highly expressed in skeletal muscle and several other tissues.

Abstract

Specific docking interactions between mitogen-activated protein kinases (MAPKs), their regulators, and their downstream substrates, are crucial for efficient and accurate signal transmission. To identify novel substrates of the c-Jun N-terminal kinase (JNK) family of MAPKs, we searched the human genome for proteins that contained (1), a predicted JNK-docking site (D-site); and (2), a cluster of putative JNK target phosphosites located close to the D-site. Here we describe a novel JNK substrate that emerged from this analysis, the functionally uncharacterized protein smoothelin-like 2 (SMTNL2). SMTNL2 protein bound with high-affinity to multiple MAPKs including JNK1-3 and ERK2; furthermore, the identity of conserved amino acids in the predicted docking site (residues 180–193) was necessary for this high-affinity binding. In addition, purified full-length SMTNL2 protein was phosphorylated by JNK1-3 in vitro, and this required the integrity of the D-site. Using mass spectrometry and mutagenesis, we identified four D-site-dependent phosphoacceptor sites in close proximity to the docking site, at S217, S241, T236 and T239. A short peptide comprised of the SMTNL2 D-site inhibited JNK-mediated phosphorylation of the ATF2 transcription factor, showing that SMTNL2 can compete with other substrates for JNK binding. Moreover, when transfected into HEK293 cells, SMTNL2 was phosphorylated by endogenous JNK in a D-site dependent manner, on the same residues identified in vitro. SMTNL2 protein was expressed in many mammalian tissues, with a notably high expression in skeletal muscle. Consistent with the hypothesis that SMTNL2 has a function in skeletal muscle, SMTNL2 protein expression was strongly induced during the transition from myoblasts to myotubes in differentiating C2C12 cells.

Introduction

Mitogen-activated protein kinase (MAPK) signaling cascades execute a number of critical functions in eukaryotic systems, including cell division, differentiation, and stress responses [1]. In mammalian cells, there are three major MAPK families, each containing multiple MAPK paralogs: the JNK (c-Jun N-terminal kinase) family, the ERK (extracellular-signal regulated kinase) family, and the p38 family [2]. JNK-family MAPKs are primarily activated by stress-associated signals, including UV radiation, ribotoxic stress, and cytokines, and are central to the regulation of stress responses and metabolic homeostasis [3]. Misregulated signal transduction involving the JNK cascade is linked to many human diseases including cancer, diabetes and muscular dystrophy, and to neurological disorders including stoke, Alzheimer's, Huntington's and Parkinson's diseases [4], [5], [6], [7].

JNKs and other MAPKs phosphorylate their substrates on serine or threonine residues that are immediately followed by proline residues. This S/T–P motif, however, is too degenerate to fully determine target specificity, as it is found in greater than 80% of all proteins. Thus, a critical feature of MAPK-substrate recognition is the binding of MAPKs to a short docking motif that resides on many substrates, distal to the target phosphorylation site(s) [8], [9]. For example, when JNK2 phosphorylates its substrate c-Jun, it first tethers itself to a docking site located within c-Jun residues 30–45, and then phosphorylates c-Jun on distal S–P sites such as Ser63 and Ser73. There are several classes of MAPK-docking sites, which differ in their sequence, and contact different regions of the MAPK. The abundant ‘D-site’ class of MAPK docking sites has a consensus sequence that consists of a cluster of basic residues, followed by a short spacer of 1–6 residues and a hydrophobic-X-hydrophobic submotif (K/R-X1–6-ϕ-X-ϕ). D-sites have been identified in a number of JNK substrates, including heterogeneous nuclear ribonucleoprotein K (hnRNP-K), insulin receptor substrate 1 (IRS-1), and transcription factors such as ATF2, c-Jun, Elk, Net, Gli1, Gli3, and NFAT4 [10]. D-sites are also found in the upstream MKKs that phosphorylate and activate JNK [11], [12], [13], in JNK scaffolding proteins such as JIP-1, and in MAPK phosphatases that dephosphorylate JNK [14], [15]. Docking sites facilitate the efficient and specific interaction of MAPKs with their substrates and binding partners [8]. Interfering with docking by mutagenesis, or by the use of blocking peptides, dramatically reduces the efficiency of substrate phosphorylation [16], [17], [18]. For these reasons, there is interest in targeting MAPK docking interactions as a therapeutic strategy. In this regard it is notable that a D-site peptide derived JIP-1 has been shown to have a protective effect in a rat model for stroke [19].

In order to better understand MAPK signaling pathways, and improve our ability to target these pathways to treat human disease, it is essential to identify additional MAPK-interacting proteins and substrates. One attractive strategy for the unbiased identification of novel substrates and regulators is to employ a computational bioinformatics approach to search protein sequence databases. Unlike phosphoproteomics and related biochemical methods, a computational approach is not biased against weakly-expressed proteins, or against proteins in little-studied cell types. While the MAPK target phosphorylation site S/T–P is too degenerate to be used as the basis of such a genome-wide search procedure, we have previously shown that D-sites can be used in this manner, provided that a sufficiently sophisticated search algorithm is employed. To this end, we developed D-finder, which uses a combination of expert knowledge and machine learning to predict D-sites in individual protein sequences or whole genomes [10]. D-finder identified previously unrecognized D-sites in known substrates (e.g., hnRNP-K) and it also uncovered new MAPK substrates of biomedical importance (e.g., Gli1 and Gli3), which we verified using rigorous and well-established biochemical assays [10].

Functionally verified D-sites in known substrates are typically located nearby (within 100 residues on the linear sequence) to the target phosphosites whose phosphorylation they promote [20]. Thus, while it is impractical to make potential phosphosites the focus of a genome-wide search, we hypothesized that the presence of nearby phosphosites might be used to augment and prioritize D-finder's predictions. Here we report an initial attempt to combine docking site and phosphosite prediction to identify new MAPK substrates. Furthermore, we provide a detailed investigation of SMTNL2, one of the high-priority hits obtained from this combined approach.

Section snippets

Construction of position weight matrices for the identification of potential JNK phosphorylation sites

We compiled known in vitro and in vivo JNK1/2/3 phosphosites, found in the current database of PhosphoSitePlus [21] (Table S2; 89, 42, and 11 peptides respectively), and used these data to create position weight matrices (PWMs). PWMs were computed for JNK1, JNK2 and JNK3, for phosphoserine and phosphothreonine sites independently, thus resulting in a total of 6 PWMs. These PWMs take as input 15-residue peptide substrings centered on the SP or TP residue being evaluated. We calculated background

Ranking of putative phosphoproteins with MAPK docking-sites

We started with a list of 394 human proteins with putative docking sites of the D-site class, identified by D-finder and published previously [10]. D-finder was developed using a training set of literature-verified D-sites found in JNK substrates and binding partners, and thus D-finder's predictions should be enriched for JNK substrates relative to ERK/p38 substrates.

Each protein on the list of D-finder predictions was scanned for SP and TP sites, as these constitute potential MAPK

Discussion

A common mechanism of interaction between MAP kinases and their substrates and binding partners involves the tethering of the MAPK to one or more docking sites on the binding partner [8]. In the case of MAPK substrates, after the MAPK docks to the substrate in question, it phosphorylates the substrate on one or more nearby serine or threonine residues. Here, with the goal of identifying new JNK substrates, we started with a list of human proteins that contained predicted JNK docking sites of

Acknowledgments

This work was supported by NIH National Institute of General Medical Sciences research grants P50 GM76516 and R01 GM86883 (L.B.), as well as by R01 GM74830-06A1 (L.H.). E.A.G. was supported in part by the Center for Regenerative Medicine training grant TG2-01152. W.M.G. was supported in part by Systems Biology of Development training grant T32-HD60555. The work of E.A.G., R.M.K., V.P., T.C.W., M.Z. and P.B. was supported in part by grants National Science Foundation IIS-0513376, NIH LM010235,

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    These authors contributed equally to this work.

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