Molecular mechanisms in cell polarity and asymmetric cell division

Abstract

During development, cell divides and differentiates into various cell types with different functions through asymmetric cell division. The processes of cell polarity establishment and maintenance, cell fate determinants asymmetric localization and cell spindle alignment are all precisely regulated. Regulation through phosphorylation plays a significant role throughout these processes. Substrate recognitions by specific kinases and the downstream signaling pathways of phosphorylated substrates are critical for understanding the functional roles of key kinases regulating asymmetric cell division. Elucidation of the three dimensional structures is one of the best way to reveal the molecular mechanism governing the roles of kinases and their substrates in asymmetric cell division. In this thesis, I use X-ray crystallography as the major method to determine the atomic structures of one of such kinases (PKCι) and several of its downstream signaling complexes. In multicellular organisms, phosphorylation is involved in almost all known cell polarity events. Phosphorylations of Par3 in the Par complex, Crumbs in the Crumbs complex, and Lgl in the Scribble complex are all indispensable regulation processes for proper apical-basal polarity formation in epithelial cells. Phosphorylation of Numb and Miranda is also one of the main regulatory steps to allocation cell fate determinants into different daughter cells. And phosphorylation of Lgn/Pins is one critic process to align mitotic spindle with polarity axis during cell division. Atypical Protein Kinase C (aPKC) is reported to play major roles in all these regulations. Despite the above important functions of aPKC and its various substrates, the binding mechanisms of aPKC to its substrates are still largely unknown. After careful mapping of the minimal PKCι (one of aPKC isozymes) binding region of Par3, we determined the crystal structure of PKCι in complex with a peptide from Par3 at 2.4 Å. PKCι in the complex adopts catalytically competent, closed conformation without phosphorylation of Thr402 in the activation loop. The Par3 peptide binds to an elongated groove formed by the N- and C-lobes of the kinase domain. The PKCι/Par3 complex structure, together with extensive biochemical studies, reveals a set of substrate recognition sites common to all PKC isozymes as well as a hydrophobic pocket unique to aPKC. A consensus aPKC’s substrate recognition sequence pattern can be readily identified based on the complex structure. These additional aPKC’s substrates include LGN and Lgl, which are the two proteins studied in detail in this thesis work. Finally, we demonstrate that the pseudo-substrate sequence of PKCι resembles its substrate sequence, directly binds to and inhibits the activity of the kinase. Phosphorylation of LGN by aPKC is crucial for proper spindle orientation during asymmetric cell division. The C-terminal Goloco motifs of LGN bind to Gα proteins on cell cortex, and the N-terminal TPR repeats of LGN bind to NuMA, which is in turn associated with dynein on astral microtubules and thus generate a pulling force to align the spindle with cell cortex. In this process, Discs Large (Dlg) guanylate kinase domain (GK) is reported to associate with Pins (drosophila homolog of LGN) linker region to facilitate a robust spindle alignment with cell polarity axis. Dlg belongs to membrane associated guanylate kinases (MAGUKs), which are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature GK of MAGUKs are poorly understood. Through biochemistry experiments, we show that the GK of Dlg1 binds to LGN in a phosphorylation-dependent manner. The atomic structure of the Dlg1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to Dlg1 GK. We further demonstrate that the previously reported GK domain-mediated interactions of Dlgs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR are also strictly phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs may also function as phospho-peptide binding modules. The discoveries of the phosphorylation-dependent MAGUK GK/target interactions indicate that MAGUK scaffold-mediated signaling complex organizations are dynamically regulated. The discovery of GK domain as a phosphor-peptide binding module shed lights on the functions of Dlg GK in other processes. The tumor suppressors Dlg, Lethal giant larvae (Lgl) and Scribble are essential for the establishment and maintenance of epithelial cell polarity in metazoan. Dlg, Lgl and Scribble are known to interact strongly with each other genetically and form the evolutionary conserved Scribble complex. Despite of more than a decade of extensive research in the past, it has not been demonstrated whether Dlg, Lgl and Scribble physically interact with each other. We show that Dlg directly interacts with Lgl in a phosphorylation-dependent manner. Phosphorylation of any one of the three conserved Ser residues situated in the central linker region of Lgl is sufficient for its binding to the Dlg GK. The crystal structures of the Dlg4 GK in complex with two phosphor-Lgl2 peptides reveal the molecular mechanism underlying the specific and phosphorylation-dependent Dlg/Lgl complex formation. In addition to providing a mechanistic basis underlying the regulated formation of the Scribble complex, the structure of the Dlg/Lgl complex may also serve as a starting point for designing specific Dlg inhibitors for targeting the Scribble complex formation.

Date of Award	2014
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology