Abstract
Mixed-lineage kinase 3 (MLK3; also known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. Its Src homology 3 (SH3) domain has been implicated in MLK3 autoinhibition and interactions with other proteins, including those from viruses. The MLK3 SH3 domain contains a six-amino-acid insert corresponding to the n-Src insert, suggesting that MLK3 may bind additional peptides. Here, affinity selection of a phage-displayed combinatorial peptide library for MLK3's SH3 domain yielded a 13-mer peptide, designated “MLK3 SH3–interacting peptide” (MIP). Unlike most SH3 domain peptide ligands, MIP contained a single proline. The 1.2-Å crystal structure of the MIP-bound SH3 domain revealed that the peptide adopts a β-hairpin shape, and comparison with a 1.5-Å apo SH3 domain structure disclosed that the n-Src loop in SH3 undergoes an MIP-induced conformational change. A 1.5-Å structure of the MLK3 SH3 domain bound to a canonical proline-rich peptide from hepatitis C virus nonstructural 5A (NS5A) protein revealed that it and MIP bind the SH3 domain at two distinct sites, but biophysical analyses suggested that the two peptides compete with each other for SH3 binding. Moreover, SH3 domains of MLK1 and MLK4, but not MLK2, also bound MIP, suggesting that the MLK1–4 family may be differentially regulated through their SH3 domains. In summary, we have identified two distinct peptide-binding sites in the SH3 domain of MLK3, providing critical insights into mechanisms of ligand binding by the MLK family of kinases.
Highlights
Mixed-lineage kinase 3 (MLK3; known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues
A 1.5-Å structure of the MLK3 Src homology 3 (SH3) domain bound to a canonical proline-rich peptide from hepatitis C virus nonstructural 5A (NS5A) protein revealed that it and MLK3 SH3–interacting peptide” (MIP) bind the SH3 domain at two distinct sites, but biophysical analyses suggested that the two peptides compete with each other for SH3 binding
Experiments suggested that residues beyond the C terminus of the 12-mer peptide might contribute to binding, so we decided to characterize the binding properties of a 19-mer peptide, NH2-AIRINPNGTWSRQAETVES-COOH, named “MIP.” Truncation of MIP revealed that the entire 19-mer sequence is necessary for binding to GSTMLK3 SH3 (Fig. S2); for instance, deletion of Ala-1 (Fig. S2A) or deletion of several residues from the C terminus (Fig. S2B) resulted in lack of target binding
Summary
Mixed-lineage kinase 3 (MLK3; known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. MLK3, known as MAP3K11, is a serine/threonine protein kinase involved in regulating the JNK, p38, and ERK pathways [37,38,39,40,41,42] It is composed of several modules: an SH3 domain, a kinase domain, tandem leucine zippers, a Cdc42/Rac interactive binding (CRIB) motif, several linker regions, and a proline-rich C-terminal tail [39, 43]. The newly identified binding surface is composed of an extended n-Src loop (located between 2 and 3 strands) of the MLK3 SH3 domain, which opens up to bind a peptide ligand, named “MLK3 SH3– interacting peptide” (MIP), which lacks the canonical PXXP motif. As the extended n-Src loop is conserved among the four members of the MLK1– 4 protein family, we hypothesize that cellular interacting proteins exist that bind the SH3 domains of the MLK family of proteins in a unique MIP-like manner
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