DataSets / Project home (Regulation of interactions of the core Hippo pathway)
Proteomic and structural investigation of phosphorylation-dependent interactions in the core mammalian Hippo pathway
First Author: Amber Couzens (proteomics) and Shawn Xiong (structural biology)
Abstract:
  * These datasets are associated with two manuscripts in press at Molecular and Cellular Proteomics

Manuscript 1: MOB1 mediated phospho-recognition in the core mammalian Hippo pathway (Couzens, Xiong et al.)

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 co-activators, 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.


Manuscript 2: Regulation of protein interactions by MOB1 phosphorylation (Xiong, Couzens et al.)

MOB1 is a multifunctional protein best characterized for its integrative role in regulating Hippo and NDR pathway signaling in metazoans and the Mitotic Exit Network in yeast. Human MOB1 binds both the upstream kinases MST1 and MST2 and the downstream AGC group kinases LATS1, LATS2, NDR1 and NDR2. Binding of MOB1 to MST1 and MST2 is mediated by its phosphopeptide-binding infrastructure, the specificity of which matches the phosphorylation consensus of MST1 and MST2. On the other hand, binding of MOB1 to the LATS and NDR kinases is mediated by a distinct interaction surface on MOB1. By assembling both upstream and downstream kinases into a single complex, MOB1 facilitates the activation of the latter by the former through a trans-phosphorylation event. Binding of MOB1 to its upstream partners also renders MOB1 a substrate, which serves to differentially regulate its two protein interaction activities (at least in vitro).

Our previous interaction proteomics analysis revealed that beyond associating with MST1 (and MST2), MOB1A and MOB1B can associate in a phosphorylation-dependent manner with at least two other signaling complexes, one containing the Rho guanine exchange factors (DOCK6–8) and the other containing the serine/threonine phosphatase PP6. Whether these complexes are recruited through the same mode of interaction as MST1 and MST2 remains unknown.

Here, through a comprehensive set of biochemical, biophysical, mutational and structural studies, we quantitatively assess how phosphorylation of MOB1A regulates its interaction with both MST kinases and LATS/NDR family kinases in vitro. Using interaction proteomics, we validate the significance of our in vitro studies and also discover that the phosphorylation-dependent recruitment of PP6 phosphatase and Rho guanine exchange factor protein complexes differ in key respects from that elucidated for MST1 and MST2.

Together our studies confirm and extend previous work to delineate the intricate regulatory steps in key signaling pathways.


* Datasets are associated with raw data deposition at MassIVE; access through ftp://massive.ucsd.edu/MSV000080570 (interactions with NDR, LATS and MOB proteins), ftp://massive.ucsd.edu/MSV000080331 (interactions with MOB mutants), ftp://massive.ucsd.edu/MSV000080566 (GST-pull down with MOB4) and ftp://massive.ucsd.edu/MSV000080315 (phosphorylation).


Copyright © 2012 Gingras labs, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital.