APKC/LGN^Linker/DLG Pathway In Asymmetric Cell Division Regulation

Asymmetric cell division(ACD) is crucial for generating diversity during development and for the function of self-renewing stem cells. Conserved cellular principles and molecular players that govern the process have been identified in Drosophila melanogaster and Caenorhabditis elegans,mostly appliable to vertebrate.In brief there are two main types of mechanism governing asymmetric cell divisions. The first relies on the asymmetric partition of cell components that determine cell fate,called intrinsic mechanism. The second involves the asymmetric placement of daughter cells relative to external cues,called extrinsic mechanism.A sequence of four steps are needed for intrinsic asymmetric cell divisions:symmetry breaking,cell polarization, cell fete determinants asymmetrically segregation and mitotic spindle orientation.The Par3/Par6/aPKC complex plays an important role in symmetry breaking, cell polarization and cell fate determinants asymmetric segregation while the G a i/LGN/NuMA/Dynein compex mainly functions in spindle orientation regulation. The two complex can be linked by Insc which bind Par3and Pins TPR. Newly identified Pins/DLG/Khc73pathway is also thought to be playing an important role in microtubules induced spindle orientation.Whether this pathway is conserved in vertebrate and the structure information for Pins/DLG/Khc73interaction need further exploring. What does the role DLG play in this pathway?DLG belongs to a large family of scaffold proteins Membrane-associated guanylate kinases (MAGUKs) family which play essential roles in tissure developments,cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood and how does the GK domain act in Pins/DLG/Khc73pathway is also a mystery.Here we show that the GK domain of DLG1/SAP97binds to asymmetric cell division regulatory protein LGN(homologue of Pins) in a phosphorylation-dependent manner. aPKC directly phosphorylate LGNlinker Ser401thus mediate the interaction between LGN and DLG Through sequence alignment of LGNLinker,we find a conserved Linker region(aa.395-414) with Ser401included.By fluorescence polarization assay and other detection methods,we assure that the p-LGN18(395-414) fragment is the binding site for DLG GK domain and the affinity is0.22uM.After that we obtained the complex crystals of SAP97/p-LGN18and PSD95/p-LGN18and solved the crystal structure. According to the three dimension structure, we have mutated some of the critical residues for p-LGN18/GK binding and find that residues both N-and C-terminal to the pSer are also critical for the specific binding.Through comparing the crystal structure of SAP97GK/p-LGN18,PSD95GK/GMP and yeast GK/GMP we find that the GMP-binding site of yeast GK has evolved to be a specific pSer/pThr-binding pocket. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1and SPAR, are also phosphorylation-dependent.Through sequence analysis of the MAGUK family members,we predict that some MAGUK GKs such as CASK and MPPs, also function as phospho-peptide-binding modules while the others like Zo-1,DLG5may not. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold mediated signalling complex organizations are dynamically regulated.The binding between phosphorylated LGN and SAP97paves the way for further exploring of aPKC/LGNLinker/DLG pathway.