Characterisation of the role of ING5 [Elektronische Ressource] / vorgelegt von Poornima Basavarajaiah

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 1 Mo

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Characterisation of the role of ING5




Von der Fakultät für Mathematik, Informatik and Naturwissenschaften
der Rheinisch Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften
genehmigte Dissertation
vorgelegt von
Master of Science
Poornima Basavarajaiah
aus
Bangalore, Indien

Berichter: Universitäts Professor Dr. rer. Nat. Bernhard Lüscher
Universitäts Professorin Dr. rer. Nat. Ursula Priefer

thTag der Mündlichen prüfung: 6 July 2005

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

ICONTENTS
1. INTRODUCTION......................................................................................................... 1
1.1 Cancer.................................................................................................................1
1.1.1 Self-sufficiency in growth signals.......................................................... 2
1.1.2 Insensitivity to growth-inhibitory (antigrowth) signals ......................... 3
1.1.3 Evasion of programmed cell death......................................................... 4
1.1.4 Limitless replicative potential ................................................................ 5
1.1.5 Sustained Angiogenesis..........................................................................6
1.1.6 Tissue invasion and Metastasis .............................................................. 6
1.2 The Cell cycle..................................................................................................... 7
1.3 Inhibitors of cyclin/CDK complexes 10
1.3.1 Checkpoint Signalling11
1.3.2 Other cell cycle related diseases........................................................... 11
1.3.3 Significance of Cyclin E/CDK2 12
1.4 The tumour suppressor p53 .............................................................................. 13
1.4.1 p53 protein............................................................................................13
1.4.2 p53 function..........................................................................................15
1.4.3 p53 as a tumour suppressor .................................................................. 16
1.4.4 p53 protein regulation..........................................................................17
1.4.5 The p53 Family .................................................................................... 18
1.5 ING Family......................................................................................................20
1.5.1 Structure of the ING proteins ............................................................... 20
1.5.2 Role of ING proteins ............................................................................ 21
1.6 Identification of ING5 in a screen for the substrates of cyclin E/CDK2 ......... 22
1.6.1 ING5 23
1.7 Objectives.........................................................................................................23
II2. MATERIALS AND METHODS ............................................................... 25
2.1 Materials...........................................................................................................25
2.1.1 Primers ................................................................................................25
2.1.2 Vectors and Plasmid constructs............................................................ 25
2.1.3 Antibodies............................................................................................29
2.1.4 Materials required for the modifications, resolution and recovery of DNA
30
2.1.5 Materials required for the bacterial work............................................. 30
2.1.6 Materials required for the Yeast-Two Hybrid work............................. 31
2.1.7 Materials required for the cell culture work......................................... 33
2.2 Methodology....................................................................................................34
2.2.1 RNA and DNA extraction and analysis ............................................... 34
2.2.2 Bacterial work......................................................................................36
2.2.3 GST fusion protein purification ........................................................... 37
2.2.4 Methods for Yeast-Two Hybrid screening........................................... 40
2.2.5 Methods for cell culture ....................................................................... 45
2.2.6 Methods for protein analysis ................................................................ 49
3. RESULTS .............................................................................................................. 55
3.1 Interaction of ING5 with p53 ........................................................................... 55
3.1.1 ING5 activates p53............................................................................... 55
3.1.2 ING5 binds to p53 both in vitro and in vivo......................................... 57
3.2 ING5 mediates apoptosis.................................................................................58
3.3 The function of ING5 is regulated by cyclin E/CDK2..................................... 60
3.4 Mechanism of ING5 action .............................................................................. 63
3.4.1 ING5 is not a transactivator ................................................................. 64
3.4.2 Screening for interaction partners ........................................................ 65
3.4.3 Mapping the interaction sites ............................................................... 79
3.5 p63 and p73 also interact with ING5 84
III3.5.1 ING5 modulates the transactivational capacity of p63 and p73........... 84
3.5.2 ING5 interacts with p63 and p73 physically........................................ 86
4. DISCUSSION ................................................................................................ 88
4.1 Interaction of ING5 with p53 and its physiological relevance......................... 88
4.1.1 ING5 enhances the transactivational capacity of p53 .......................... 89
4.1.2 ING5 interacts with p53 both in vitro and in vivo................................ 91
4.1.3 Phyisological relevance of p53 and ING5 interaction.......................... 91
4.1.4 Mechanism of ING5-p53 interaction ................................................... 92
4.2 Effect of phosphorylation on the function of p53 ............................................ 96
4.3 The interaction partners of ING5 ..................................................................... 98
4.3.1 Yeast Two Hybrid Screening ............................................................... 98
4.3.2 p63 and p73 are the other interactors of ING5................................... 101
5. References ................................................................................................................. 105
6. Appendix ................................................................................................................... 121
Abberivations 121
IVABSTRACT
The tumour suppressor protein p53 stops cell division by activating the expression of
WAF1/CIP1p21 , whenever it senses that a cells DNA is damaged. Thus, giving the cell a chance to
repair the damaged DNA before its errors are duplicated and passed onto daughter cells. When
p53 is mutated, it loses its protective function. This allows mutations to accumulate in other
WAF1/CIP1genes and leads to more than 50% of all human cancers. p21 inhibits cyclin E/CDK2
complexes to prevent G1-S progression of cell cycle. Though the cyclin/CDK complexes are
required for the orderly progression of the cell cycle, overexpression of cyclin E/CDK2 has been
reported to lead to poor prognosis in breast cancer. The molecular mechanism underlying this is
poorly understood.
The current investigation was undertaken to elucidate the role of ING5, in relation to cyclin
E/CDK2 activity and p53 function. ING5, which was identified in a novel approach for screening
the substrates of cyclin E/CDK2 is a member of the ING family of proteins that inhibit growth.
The in vitro and in vivo interaction experiments and the reporter gene assays demonstrate that
ING5 interacts with p53 to strongly activate p53-mediated transactivation of the promoters of the
p21, bax and mdm2 genes. The extensive mapping of their interaction domains showed that the
PH domain of ING5 interacts with the DNA binding domain of p53 indicating that ING5 does
not interfere with p53-MDM2 interaction. ING5 mediates apoptosis of breast cancer cells with
an intact p53 pathway as demonstrated in MCF-7 cells. It was observed that overexpression of
cyclin E/CDK2 negatively regulated ING5-mediated activation of p53 and apoptosis. This work
for the first time also illustrates a member of the ING family to be interacting with p63 and p73.
ING5 interacts with p63 and p73 both in vitro and in vivo to differentially modulate p63 and p73-
mediated transactivation of bax, p21 and pig3 genes.
While one important function of p53 is to prevent S phase entry in response to cellular stress by
activating the CKI p21, which interfer