Exploring the regulation and function of human Lats1 and Aurora A-kinases in mitosis [Elektronische Ressource] / vorgelegt von Eunice Ho Yee Chan

Hoyee Chan

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Exploring the regulation and function of human Lats1
and Aurora A kinases in mitosis


Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München



Vorgelegt von
Eunice Ho Yee Chan

Martinsried / München 2007
















Dissertation eingereicht am: 26.06.2007
Datum der mündlichen Prüfung: 30.08.2007

Erstgutachter: Prof. Dr. Erich A. Nigg
Zweitgutachter: Prof. Dr. Heinrich Leonhardt
Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne
unerlaubte Hilfe angefertigt habe. Sämtliche Experimente sind von mir selbst
durchgeführt worden, falls nicht explizit auf dritte verwiesen wird. Ich versichere, daß ich
weder versucht habe, eine Dissertation oder Teile einer Dissertation an einer anderen
Stelle einzureichen, noch eine Doktorprüfung durchzuführen.

Eunice H.Y. Chan
München, den 31-05-2007



Table of contents
Table of contents

Table of contents…………………………………………………………………………..I-IV

Acknowledgements

Summary........................................................................................................................ 1

Introduction ................................................................................................................... 3
An overview of the cell cycle........................................................................................ 3
An overview of mitosis................................................................................................. 3
Regulation of mitotic progression by kinases............................................................... 5
Cyclin-dependent kinase 1....................................................................................... 5
Polo-like kinase 1 (Plk1)........................................................................................... 6
Aurora kinase family 8
MEN/SIN kinases?................................................................................................. 11

Aim of this thesis ........................................................................................................ 12

Part I: Basic characterization of human Lats1/2 kinases and their regulation by
Ste20-like kinases Mst1/2 ........................................................................................... 13
Introduction I ................................................................................................................. 14
LATS: a tumor suppressor gene ............................................................................ 14
Proposed mitotic function of human Lats ............................................................... 14
Drosophila Lats is required for cell cycle exit and apoptosis .................................. 15
Results I ........................................................................................................................ 17
LATS1 is ubiquitously expressed in contrast to LATS2.......................................... 17
Lats1 is phosphorylated during mitosis .................................................................. 19
Lats1 and Cdk1 do not interact in either coimmunoprecipitation or yeast two-hybrid
............................................................................................................................... 21
Lats1 shows a diffuse cytoplasmic staining throughout the cell cycle .................... 22
I Table of contents

Lats1 is absent from a spindle preparation ............................................................ 24
Lats1 is active in okadaic acid (OA) treated cells, but not in mitotic cells............... 25
Mst2 interacts with hWW45.................................................................................... 27
Lats1 is phosphorylated by Mst2............................................................................ 28
Lats1 is activated by Mst2 -mediated phosphorylation........................................... 30
Specific activation of Lats1 and Lats2 by Mst2/1 kinases ...................................... 32
The Lats1 activation segment resides in the C-terminal (catalytic) domain............ 34
Phosphorylation of S909 and T1079 is essential for Lats1 kinase activity ............. 36
Discussion I................................................................................................................... 40
Ste20 family members as upstream regulators of Lats/Dbf2-related kinases ........ 40
What is the role of hWW45 in the regulation of Lats kinases? ............................... 42
Emerging evidence for an evolutionarily conserved signaling pathway ................. 44
Summary I..................................................................................................................... 45

Part II Exploring the function and regulation of Aurora A
kinase………………………… ....................................................................................... 47
Introduction II ................................................................................................................ 48
Centrosome maturation in mitotic spindle assembly.................................................. 48
Plk1 and Aurora A are required for centrosome maturation and spindle assembly ... 48
Regulation of Plk1 and Aurora A ............................................................................... 49
Bora is a novel Aurora A interactor and activator ...................................................... 50
Results II ....................................................................................................................... 51
1. hBora, a novel Aurora A binding partner links Plk1 functions with Aurora A.......... 51
1.1 hBora interacts with Aurora A........................................................................... 51
1.2 Cell cycle expression of hBora......................................................................... 53
1.3 Identification of multiple phosphorylation sites in hBora................................... 55
1.4 Depletion of hBora causes aberrant spindle formation..................................... 57
1.5 Excess hBora causes Aurora A mislocalization and monoastral spindle
formation ................................................................................................................ 60
1.6 hBora interacts with Plk1 during mitosis........................................................... 62
β-TrCP1.7 Plk1 triggers the SCF mediated degradation of hBora ............................. 65
II Table of contents
1.8 Plk1 regulates Aurora A by controlling hBora levels......................................... 68
2. Functional studies of Aurora A............................................................................... 71
2.1 Aurora A depletion leads to long/multipolar spindle formation and abnormal
centriole splitting .................................................................................................... 71
2.2 Aurora A activity is required for centrosome separation................................... 74
2.3 Aurora A localization is required for centriole cohesion.................................... 74
Discussion II.................................................................................................................. 76
1. Plk1 controls the function of Aurora A kinase by regulating the protein levels of
hBora......................................................................................................................... 76
hBora levels are critical for proper spindle assembly ............................................. 76
hBora interacts not only with Aurora A but also with Plk1 ...................................... 77
Plk1 regulates hBora stability................................................................................. 78
Through hBora Plk1 acts as an upstream regulator of Aurora A............................ 78
2. Functions of human Aurora A kinase..................................................................... 79
Summary II.................................................................................................................... 81

Materials and Methods................................................................................................ 82
Plasmid constructions and site directed mutagenesis ............................................... 82
Cell culture, synchronization, and transfection .......................................................... 82
Generation of stable cell lines.................................................................................... 83
Cell extracts and Western blot analysis..................................................................... 83
Spindle preparation.................................................................................................... 84
Preparation of Baculoviruses, Sf9 cell culture, and purification of reco