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

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 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.

The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2

“…GST fusion proteins were expressed in bacteria, purified by affinity chromatography using glutathione-Sepharose (GE Healthcare), and quantified as described elsewhere (85,86).…”

“…Protein binding assays were performed as described previously (85,86). Quantification of binding was performed on a Typhoon TRIOϩ Imager using phosphorimaging mode.…”

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…S1C). These cytoskeletal JNK substrate proteins include those associating with microtubules (DCX [260][261][262], MAP1B [180,263], MAP2 [264], Tau [265], and WDR62 [213]), the actin cytoskeleton (MARCKSL1 [266] and SMTL2 [267]), or focal adhesions (paxillin [268,269] and ␤-catenin [270][271][272]). In addition, JNK-mediated phosphorylation of additional proteins may also direct vesicular transport (through phosphorylation of JIP1 and ␤-APP [104,217]), as well as the exocytosis of specialized, Glut4-containing vesicles (through insulin-stimulated phosphorylation of IRS1 and IRS2 [273,274]).…”

Bioinformatics Approaches for Predicting Kinase–Substrate Relationships

“…Trained with 20 experimentally identified D-sites for a c-Jun N-terminal kinase (JNK), they identified 394 proteins with putative D-sites, and experimentally validated some of them [59]. In order to prioritize the predictions made by D-finder, this was combined with a predictor of JNK phosphorylation sites based on position weight matrices, achieving the identification and experimental validation of one additional substrate of JNK [60].…”