Abstract
The Hippo tumor suppressor pathway regulates organ size and tissue homoeostasis in response to diverse signaling inputs. The core of the pathway consists of a short kinase cascade: MST1 and MST2 phosphorylate and activate LATS1 and LATS2, which in turn phosphorylate and inactivate key transcriptional coactivators, YAP1 and TAZ (gene WWTR1). The MOB1 adapter protein regulates both phosphorylation reactions firstly by concurrently binding to the upstream MST and downstream LATS kinases to enable the trans phosphorylation reaction, and secondly by allosterically activating the catalytic function of LATS1 and LATS2 to directly stimulate phosphorylation of YAP and TAZ. Studies of yeast Mob1 and human MOB1 revealed that the ability to recognize phosphopeptide sequences in their interactors, Nud1 and MST2 respectively, was critical to their roles in regulating the Mitotic Exit Network in yeast and the Hippo pathway in metazoans. However, the underlying rules of phosphopeptide recognition by human MOB1, the implications of binding specificity for Hippo pathway signaling, and the generality of phosphopeptide binding function to other human MOB family members remained elusive.Employing proteomics, peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly complementary to the substrate phosphorylation specificity of MST1 and MST2. We demonstrate that autophosphorylation of MST1 and MST2 on several threonine residues provides multiple MOB1 binding sites with varying binding affinities which in turn contribute to a redundancy of MST1-MOB1 protein interactions in cells. The crystal structures of MOB1A in complex with two favored phosphopeptide sites in MST1 allow for a full description of the MOB1A phosphopeptide-binding consensus. Lastly, we show that the phosphopeptide binding properties of MOB1A are conserved in all but one of the seven MOB family members in humans, thus providing a starting point for uncovering their elusive cellular functions.
Highlights
Employing proteomics, peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly
MOB1 Phosphorylation-dependent Interaction with MST1 is Direct and Maps to Regions C-terminal to the Kinase Domain—We previously demonstrated that MOB1 interacted in a phosphorylation-dependent manner with MST1, and that its association with MST1 and MST2 was abrogated by mutation of the MOB1B phosphate coordinating residues K153, R154 and R157
Because we surmised that the phospho-dependent binding of MST1 and MST2 to MOB1 could have functional consequences on Hippo pathway signaling and regulation, we set out to investigate the determinants for the MST1-MOB1 interaction in greater detail
Summary
Peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly. Consistent with our previous results indicating that coordination of the phosphate group by three basic amino acids in MOB1 (first identified in ref 22) was responsible for phosphorylation-induced interaction with MST1 [23], a recombinant MOB1A mutant bearing alanine substitutions at positions K153, R154, and R157 displayed no measurable binding to either of the phosphorylated peptides (Fig. 2B).
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