Abstract
Scribble is a crucial adaptor protein that plays a pivotal role during establishment and control of cell polarity, impacting many physiological processes ranging from cell migration to immunity and organization of tissue architecture. Scribble harbours a leucine-rich repeat domain and four PDZ domains that mediate most of Scribble's interactions with other proteins. It has become increasingly clear that post-translational modifications substantially impact Scribble-ligand interactions, with phosphorylation being a major modulator of binding to Scribble. To better understand how Scribble PDZ domains direct cell polarity signalling and how phosphorylation impacts this process, we investigated human Scribble interactions with MCC (Mutated in Colorectal Cancer). We systematically evaluated the ability of all four individual Scribble PDZ domains to bind the PDZ-binding motif (PBM) of MCC as well as MCC phosphorylated at the -1 Ser position. We show that Scribble PDZ1 and PDZ3 are the major interactors with MCC, and that modifications to Ser at the -1 position in the MCC PBM only has a minor effect on binding to Scribble PDZ domains. We then examined the structural basis for these observations by determining the crystal structures of Scribble PDZ1 domain bound to both the unphosphorylated MCC PBM as well as phosphorylated MCC. Our structures indicated that phospho-Ser at the -1 position in MCC is not involved in major contacts with Scribble PDZ1, and in conjunction with our affinity measurements suggest that the impact of phosphorylation at the -1 position of MCC must extend beyond a simple modulation of the affinity for Scribble PDZ domains.
Highlights
Scribble is a large multi-domain scaffold protein comprising 16 Leucine Rich Repeats and 4 PSD-95/Disc-large/ZO-1 (PDZ) domains, and belongs to the LAP family of proteins
To understand the impact of phosphorylation on the ability of the Mutated in Colorectal Cancer (MCC) PDZ-binding motifs (PBMs) to bind to Scribble PDZ domains, we systematically examined the affinities of recombinant human Scribble PDZ domains for peptides spanning the MCC PBM with either phosphorylated or unphosphorylated Ser at the -1 position, as well as peptides harboring a phosphomimic Ser to Glu mutation
To understand the impact of phosphorylation on the binding of MCC to Scribble we examined the affinity of recombinant human Scribble PDZ1, 2, 3 and 4 domains for a panel of 8-mer peptide corresponding to the MCC PBM (Figure 1, Table 1)
Summary
Scribble is a large multi-domain scaffold protein comprising 16 Leucine Rich Repeats and 4 PSD-95/Disc-large/ZO-1 (PDZ) domains, and belongs to the LAP family of proteins. It was shown that MCC harbors a C-terminal PBM that is able to engage human Scribble PDZ1 and 3 domains to impact epithelial cell polarity [16]. To understand the impact of phosphorylation on the ability of the MCC PBM to bind to Scribble PDZ domains, we systematically examined the affinities of recombinant human Scribble PDZ domains for peptides spanning the MCC PBM with either phosphorylated or unphosphorylated Ser at the -1 position, as well as peptides harboring a phosphomimic Ser to Glu mutation. We determined crystal structures of human Scribble PDZ1 bound to a MCC PBM peptide as well as the MCC PBM with phosphorylated Ser at the -1 position to examine the structural basis for this interaction and the impact of phosphorylation on binding to Scribble PDZ1. Our findings suggest that phosphorylation of the MCC PBM at the -1 position is not a major determinant of interactions with human Scribble PDZ1 and 3 domains
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