Structural insights into the aPKC regulatory switch mechanism of the human cell polarity protein lethal giant larvae 2 Lior Almagora,b, Ivan S. Ufimtseva, Aruna Ayera,b, Jingzhi Lia,b, and William I. Weisa,b,1 aDepartment of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305; and bDepartment of Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305 Edited by Wesley I. Sundquist, University of Utah Medical Center, Salt Lake City, UT, and approved March 5, 2019 (received for review December 28, 2018) Metazoan cell polarity is controlled by a set of highly conserved down-regulation of Lgl occurs in various human cancers (18). Lgl proteins. Lethal giant larvae (Lgl) functions in apical-basal polarity has important roles in all aspects of cell polarity, including in the through phosphorylation-dependent interactions with several development of epithelial apical-basal polarity, asymmetrical cell other proteins as well as the plasma membrane. Phosphorylation division, and cell migration. of Lgl by atypical protein kinase C (aPKC), a component of the The molecular basis of Lgl function in cell polarity is poorly partitioning-defective (Par) complex in epithelial cells, excludes Lgl understood, but it is clear that it depends upon aPKC phosphorylation- from the apical membrane, a crucial step in the establishment of dependent cellular localization. In early stages of Drosophila epithelial cell polarity. We present the crystal structures of human epithelial development, when polarity is being established, Lgl is Lgl2 in both its unphosphorylated and aPKC-phosphorylated states. both cytoplasmic and uniformly localized at the cell cortex. At Lgl2 adopts a double β-propeller structure that is unchanged by later stages, aPKC phosphorylation excludes Lgl from the apical aPKC phosphorylation of an unstructured loop in its second β-pro- domain, where Par-6 and aPKC concentrate. This apical exclu- peller, ruling out models of phosphorylation-dependent conforma- sion does not occur in a nonphosphorylatable mutant Lgl, which tional change. We demonstrate that phosphorylation controls the results in aberrant polarity (19). In fully polarized cells, Lgl is direct binding of purified Lgl2 to negative phospholipids in vitro. We located mostly at the lateral membrane, where it actively excludes also show that a coil–helix transition of this region that is promoted Par-6 from the cell cortex (19). A similar spatial and temporal by phosphatidylinositol 4,5-bisphosphate (PIP2) is also phosphorylation- localization of Lgl in relation to the aPKC/Par-6 complex is also dependent, implying a highly effective phosphorylative switch for observed in mammalian epithelial cultures (20). membrane association. In a polarized epithelial cell, Lgl colocalizes with the polarity proteins scribble (Scrib) and discs large (Dlg) at the apical margin Lgl | cell polarity | aPKC of the lateral membrane. Experiments in Drosophila have dem- onstrated a strong genetic interaction among these three genes, he development of structurally and functionally distinct cell indicating that they act together in a common pathway in the Tsurfaces is essential for the proper function of most animal regulation of cell polarity (21). Lgl and Dlg mutations have been cells. The establishment and maintenance of such cell polarity shown to produce similar effects on fly development (21), and a require a set of so-called polarity proteins, whose core compo- nents are conserved throughout metazoa. In epithelial cells, Significance apicobasal polarity is controlled by the spatial and temporal cross-talk between polarity complexes located on the apical or – Epithelial cells have a spatially polarized organization. For ex- basolateral membranes, as well as the cell cell junctions that ample, one surface of an intestinal epithelial cell, called the delineate these membrane domains (1, 2). A central modulator apical side, faces the lumen of the gut and has a membrane of this process is atypical protein kinase C (aPKC) (3). aPKC composition distinct from those of the basolateral sides. robustly interacts with partitioning-defective 6 (Par-6), and they Several proteins that control the development and mainte- are found together in a subapical epithelial region in complex nance of apical-basolateral polarity have been identified, but with the aPKC substrate Par-3 (bazooka in Drosophila) (4, 5). their molecular mechanisms are poorly understood. Lethal The aPKC/Par-6/Par-3 complex is important for the establish- giant larvae (Lgl) is a basolateral polarity protein that is lost ment of apical-basal polarity, as well as for the maturation of selectively from the apical membrane during development, epithelial junctions in Drosophila and mammals. It also has roles due to its phosphorylation by atypical protein kinase C. Here, – in asymmetric cell division (6 8). In Caenorhabditis elegans zy- we describe the 3D structure of Lgl in both its unmodified and gotes, a homologous PKC3/Par-6/Par-3 complex, which localizes phosphorylated states, and show that phosphorylation of Lgl to the anterior half following the entry of the sperm cell, is es- mediates a structural switch that controls its association with sential for anterior/posterior polarity (6). The aPKC/Par-6 com- the plasma membrane. plex also colocalizes with the crumbs/PALS1/PATJ (crumbs/ stardust/discs-lost in Drosophila) polarity complex located at the Author contributions: L.A. and W.I.W. designed research; L.A., I.S.U., and A.A. performed apical domain. This complex is also crucial for the establishment research; I.S.U., A.A., and J.L. contributed new reagents/analytic tools; L.A., I.S.U., and of epithelial apicobasal polarity, and it is likewise regulated by W.I.W. analyzed data; and L.A. and W.I.W. wrote the paper. aPKC phosphorylation (9). The authors declare no conflict of interest. In addition to its roles in the apical polarity complexes, aPKC This article is a PNAS Direct Submission. is responsible for the phosphorylation of other polarity proteins, Published under the PNAS license. including lethal giant larvae (Lgl) (3). First identified in Dro- Data deposition: The atomic coordinates and structure factors have been deposited in the sophila genetic screens, lgl mutant flies develop cancer-like de- Protein Data Bank, www.wwpdb.org (PDB ID codes 6N8P, 6N8Q, 6N8R, and 6N8S). fects, leading to uncontrolled growth of larval brain neuroblasts 1To whom correspondence should be addressed. Email: [email protected]. and imaginal discs (10–13). Lgl is conserved in eukaryotes and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. can be found in two isoforms in mammals (14). Its mutations 1073/pnas.1821514116/-/DCSupplemental. result in polarity defects in mice and other animals (15–17), and Published online May 14, 2019. 10804–10812 | PNAS | May 28, 2019 | vol. 116 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1821514116 Downloaded by guest on September 23, 2021 direct low-affinity interaction between the guanylate kinase do- by multiple serine phosphorylations in the aPKC target site could main of human Dlg4 and a Lgl2 peptide containing phosphory- be the major determinant of membrane targeting and/or protein– lated aPKC target sites has been characterized biochemically and protein interaction. structurally (22). Interactions between Scrib and Lgl have also Here, we report crystal structures of human Lgl2 in both its been demonstrated (23), but strong biochemical evidence for the phosphorylated and unphosphorylated forms. Our structural existence of a ternary Lgl/Dlg/Scrib complex is lacking. Interaction results confirm the double β-propeller core structure for Lgl2. of Lgl, controlled by aPKC phosphorylation, has been reported There are no apparent structural variations between the with additional targets, including nonmuscle myosin II (NMII) unphosphorylated and phosphorylated states of the protein. We (15, 24, 25), syntaxin4 (26, 27), and others (28–32). In addition to demonstrate the preferential interaction of Lgl2 protein with aPKC, cytoplasmic Lgl is phosphorylated by the aurora A and B phosphatidylinositol 4,5-bisphosphate (PIP2)-containing mem- kinases at mitosis in epithelial cells, which promotes its mitotic branes that is abolished by aPKC phosphorylation, as previously relocalization (33–35). demonstrated with a polybasic peptide fragment of this loop (41, Budding yeast express the Lgl homolog Sro7, a 1,033-residue 42). We also show that a coil–helix transformation of this region, protein that is essential for polarized exocytosis in bud growth promoted by PIP2, is phosphorylation-dependent, which implies (36). The Sro7 structure (37) comprises 14 WD40 repeats arranged an effective membrane switch mechanism. These results set a in two seven bladed β-propeller barrels. The distant Lgl/Sro7 se- firm basis for understanding the mechanistic roles of Lgl in quence homology (∼10% identity) suggests them to be structurally cell polarity. and functionally related. Mouse Lgl1 was shown to partially rescue low salt tolerance and temperature sensitivity associated with the Results loss of Sro7 and its homolog, Sro77 (38, 39). The role of Lgl in Structure of Lgl2. To explore the structure of Lgl in its different exocytosis in vertebrates has yet to be solidly established, however. functional forms, we crystallized human Lgl2 both in its The target serine-rich sequence for aPKC phosphorylation unphosphorylated state and in its in vitro aPKCι-phosphorylated