Molecular mechanisms in cell polarity and asymmetric cell division

During development, cell divides and differentiates into various cell types with differentfunctions through asymmetric cell division. The processes of cell polarity establishment andmaintenance, cell fate determinants asymmetric localization and cell spindle alignment are allprecisely regulated. Regulation through phosphorylation plays a significant role throughoutthese processes. Substrate recognitions by specific kinases and the downstream signalingpathways of phosphorylated substrates are critical for understanding the functional roles ofkey kinases regulating asymmetric cell division. Elucidation of the three dimensionalstructures is one of the best way to reveal the molecular mechanism governing the roles ofkinases and their substrates in asymmetric cell division. In this thesis, I use X-raycrystallography as the major method to determine the atomic structures of one of such kinases(PKC&igr;) and several of its downstream signaling complexes.;In multicellular organisms, phosphorylation is involved in almost all known cell polarityevents. Phosphorylations of Par3 in the Par complex, Crumbs in the Crumbs complex, andLgl in the Scribble complex are all indispensable regulation processes for proper apical-basalpolarity formation in epithelial cells. Phosphorylation of Numb and Miranda is also one of themain regulatory steps to allocation cell fate determinants into different daughter cells. Andphosphorylation of Lgn/Pins is one critic process to align mitotic spindle with polarity axisduring cell division. Atypical Protein Kinase C (aPKC) is reported to play major roles in allthese 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 carefulmapping of the minimal PKC&igr; (one of aPKC isozymes) binding region of Par3, wedetermined the crystal structure of PKC&igr; in complex with a peptide from Par3 at 2.4 A. PKC&igr;in the complex adopts catalytically competent, closed conformation without phosphorylationof Thr402 in the activation loop. The Par3 peptide binds to an elongated groove formed by theN- and C-lobes of the kinase domain. The PKC&igr;/Par3 complex structure, together withextensive biochemical studies, reveals a set of substrate recognition sites common to all PKCisozymes as well as a hydrophobic pocket unique to aPKC. A consensus aPKC's substraterecognition sequence pattern can be readily identified based on the complex structure. Theseadditional 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 PKC&igr;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 duringasymmetric cell division. The C-terminal Goloco motifs of LGN bind to G? proteins on cellcortex, and the N-terminal TPR repeats of LGN bind to NuMA, which is in turn associatedwith dynein on astral microtubules and thus generate a pulling force to align the spindle withcell cortex. In this process, Discs Large (Dlg) guanylate kinase domain (GK) is reported toassociate with Pins (drosophila homolog of LGN) linker region to facilitate a robust spindlealignment 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 tissuedevelopments, cell-cell communications, cell polarity control, and cellular signaltransductions. Despite extensive studies over the past two decades, the functions of thesignature GK of MAGUKs are poorly understood. Through biochemistry experiments, weshow that the GK of Dlg1 binds to LGN in a phosphorylation-dependent manner. The atomicstructure of the Dlg1 SH3-GK tandem in complex with a phospho-LGN peptide reveals thatthe GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residuesboth N- and C-terminal to the pSer are also critical for the specific binding of thephospho-LGN peptide to Dlg1 GK. We further demonstrate that the previously reported GKdomain-mediated interactions of Dlgs with other targets, such as GKAP/DLGAP1/SAPAP1and SPAR are also strictly phosphorylation dependent. Finally, we provide evidence that otherMAGUK GKs may also function as phospho-peptide binding modules. The discoveries of thephosphorylation-dependent MAGUK GK/target interactions indicate that MAGUKscaffold-mediated signaling complex organizations are dynamically regulated.;The discovery of GK domain as a phosphor-peptide binding module shed lights on thefunctions 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 inmetazoan. Dlg, Lgl and Scribble are known to interact strongly with each other geneticallyand form the evolutionary conserved Scribble complex. Despite of more than a decade ofextensive research in the past, it has not been demonstrated whether Dlg, Lgl and Scribblephysically interact with each other. We show that Dlg directly interacts with Lgl in aphosphorylation-dependent manner. Phosphorylation of any one of the three conserved Serresidues 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 themolecular mechanism underlying the specific and phosphorylation-dependent Dlg/Lglcomplex formation. In addition to providing a mechanistic basis underlying the regulatedformation of the Scribble complex, the structure of the Dlg/Lgl complex may also serve as astarting point for designing specific Dlg inhibitors for targeting the Scribble complex formation.