The apical transmembrane protein Crumbs (Crb) is evolutionarily conserved in metazoan, and acts as a master cell polarity and growth regulator in polarized epithelia. Crb intra-cellular functions are mediated by its highly conserved 37-residue cytoplasmic tail (Crb-CT). However, the mechanistic basis governing Crb's role in cell polarity and growth remains unclear. Here, I discover that the PDZ-SH3-GK tandem of PALS1 directly binds to Crb-CT with a dissociation constant of 70 nM, which is -100-fold stronger than the PALSI PDZ/Crb-CT interaction. The crystal structure of the PALS1 PDZ-SH3-GK/Crb-CT complex reveals that the PDZ-SH3-GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of...[ Read more ]

The apical transmembrane protein Crumbs (Crb) is evolutionarily conserved in metazoan, and acts as a master cell polarity and growth regulator in polarized epithelia. Crb intra-cellular functions are mediated by its highly conserved 37-residue cytoplasmic tail (Crb-CT). However, the mechanistic basis governing Crb's role in cell polarity and growth remains unclear. Here, I discover that the PDZ-SH3-GK tandem of PALS1 directly binds to Crb-CT with a dissociation constant of 70 nM, which is -100-fold stronger than the PALSI PDZ/Crb-CT interaction. The crystal structure of the PALS1 PDZ-SH3-GK/Crb-CT complex reveals that the PDZ-SH3-GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of the PDZ-SH3-GK supramodule weaken the PALSl/Crb interaction and compromise PALSI-mediated polarity establishment in MDCK cysts. These findings not only elucidate the molecular basis regard to Crb/PALSl complex in determining the apical-basal cell polarity, but also indicate a common PDZ-SH3-GK mode for other members of MAGUKs in specific target recognition.

I also biochemically and structurally characterized the direct interaction between Crb-CT and Moesin FERM domain, which provides a direct linkage from the plasma membrane to the actin cytoskeleton. The 1.5 Å resolution crystal structure of the Moesin-FERM/Crb-CT complex reveals a typical FERM/FBM binding mode, in which the FBM of Crb-CT forms a short β-sheet and fits into the canonical F3-binding site. To our surprise, the PBM of Crb-CT also contributes to the binding to Moesin-FERM, by occupying the InsP₃ binding site at the Fl/F3 cleft, implying that Crb-CT may mimic the role of Ptdlns(4,5)P₂ in the activation of the ERM family proteins. Interestingly, phosphorylation of Crb-CT by aPKC disrupts the Crb/Moesin association, but has no impact on the Crb/PALSl interaction. The above findings suggest that establishment of cell polarity promotes aPKC-mediated Crb phosphorylation and subsequent Crb/Moesin complex dissociation. Thus, Crb becomes to be stably associated with tight junction-localized PALS1 in polarized epithelia. It is likely that aPKC-mediated phosphorylation of Crb functions to switch epithelial cells from proliferation to contact-mediated growth inhibition.

Actin cytoskeleton is a major regulator of the Hippo signaling pathway, and angiomotin (AMOT) can relay the signals initiated by F-actin structural changes to control YAP activity. I show in the fourth part of this dissertation and by high resolution crystal structures that the Lats1/2 binding site on the Merlin FERM domain is physically blocked by Merlin's auto-inhibitory tail. AMOT binding releases the auto-inhibition and promotes Merlin's binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin's auto-inhibitory tail prevents AMOT from binding, and thus inhibits Hippo kinase activation. These findings reveal that AMOT and Merlin interface the cortical actin filaments and the core kinases in the Hippo signaling, respectively; and thus allow construction of a complete Hippo signaling pathway initiated from cortical F-actin cytoskeleton and establish a connection between cell polarity and cell growth.