The role of MEK1 in progression through G2/M phase of the cell cycle

Tanya Pike (2010). The role of MEK1 in progression through G2/M phase of the cell cycle PhD Thesis, UQ Diamantina Institute, The University of Queensland.

       
Attached Files (Some files may be inaccessible until you login with your UQ eSpace credentials)
Name Description MIMEType Size Downloads
S33554097_phd_finalthesis.pdf S33554097_phd_finalthesis.pdf Click to show the corresponding preview/stream application/pdf 5.42MB 14
Author Tanya Pike
Thesis Title The role of MEK1 in progression through G2/M phase of the cell cycle
School, Centre or Institute UQ Diamantina Institute
Institution The University of Queensland
Publication date 2010-12
Thesis type PhD Thesis
Supervisor Brian Gabrielli
Angus Harding
Total pages 185
Total colour pages 21
Total black and white pages 164
Subjects 11 Medical and Health Sciences
Abstract/Summary The primary endpoint for signalling through the canonical Ras-Raf-MEK-MAP kinase cascade is ERK activation. The work undertaken in this thesis describes an alternative outcome for signalling through this pathway which utilises a proteolytically truncated form of MEK1to inhibit activation of the pathways' recognised downstream effector, ERK. Activation of the mitogen activated protein kinase (MAPK) pathway by growth factors during G1 phase promotes cell cycle progression but activation of the MAPK pathway during G2 phase by growth factors (EGF) or phorbol esters (TPA) results in delayed G2/M phase progression. We have previously identified a novel proteolytically truncated form of MEK1 (tMEK - truncated MEK1) (Harding, Giles et al. 2003), which is cleaved to remove its N-terminal ERK binding domain. We show that induction of tMEK with activation of MAPK signalling during G2 phase, delays entry into mitosis. RNAi depletion in somatic cells or knockout in mouse embryonic fibroblasts of MEK1 but not MEK2, results in loss of the G2 delay. The G2 delay can be recapitulated with addition of recombinant tMEK protein. Investigation of G2 checkpoint proteins demonstrated a lack of involvement of classical G2 phase DNA damage/stress response molecules such as p38MAPK, Chk1/2 and ATM/ATR kinases. Examination of the proximal mediators of mitotic entry, the Cdc25 family of dual specificity phosphatases, revealed the rapid degradation of Cdc25B in response to exogenous growth factor or phorbol ester addition. This degradation could be inhibited by the addition of the proteasome subunit inhibitor MG132 or interestingly, the addition of the MEK1/2 inhibitor U0126. A potential ERK dependent phosphorylation site proximal to a β-TRCPSCF binding motif which targets Cdc25B to the proteasome for degradation was identified. Phosphorylation of Cdc25B at Ser249 occurs with TPA treatment and could be inhibited by the MEK1/2 inhibitor, U0126, strongly suggesting that this site is phosphorylated by ERK in response to TPA treatment. Addition of recombinant tMEK protein also resulted in reduced levels of the Cdc25B isoform indicating that tMEK mediates its effect of delaying G2/M progression through regulating the protein level of the key cell cycle regulator, Cdc25B. Cdc25B is, by nature, an unstable protein, which begs the question: why further destabilise an already unstable protein? What we observed was, after the initial activation of ERK following mitogenic stimulation; ERK activity was rapidly down-regulated. This correlated with the maintenance of decreased Cdc25B protein levels and concomitant G2 phase delay. The data presented herein led us to hypothesise that the role of tMEK is to inhibit de novo synthesis of critical cell cycle proteins required for progression from G2 phase to mitosis. Further investigation into the role of protein synthesis during G2 phase revealed a marked reduction in the phosphorylation and corresponding inhibition of the activity of key components of the translational initiation machinery and mRNA cap binding proteins in response to TPA treatment. The existence of a novel form of MEK1 which functions to inhibit its normal effector ERK is of great biological interest. In many cancers, signalling through the MAPK pathway involving Ras-Raf-MEK-ERK is enhanced, usually via oncogenic mutations in Ras or Raf, providing proliferative and pro-survival advantages to cells. The existence of another function for components of this pathway provides an entirely novel spectrum of possible outcomes for aberrant activation of this pathway.
Keyword Cell cycle
G2 phase checkpoint
mitogen activated protein kinse
Ras/Raf/MEK/ERK signalling cascade
Translation inititation
Cdc25B
Additional Notes Please print the following pages in colour: 17, 20, 31, 33, 48, 52, 67, 82-83, 88, 90, 93, 95, 97, 128, 131, 133-135, 148, 151

 
Citation counts: Google Scholar Search Google Scholar
Access Statistics: 173 Abstract Views, 14 File Downloads  -  Detailed Statistics
Created: Wed, 15 Jun 2011, 23:31:20 EST by Ms Tanya Pike on behalf of Library - Information Access Service