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Isoform-specific roles of the adaptor protein ShcA in cell signaling

Kleiner, Sandra. Isoform-specific roles of the adaptor protein ShcA in cell signaling. 2005, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7331

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Abstract

ShcA is a bona fide adaptor protein without
any enzymatic activity. Upon activation of
receptor tyrosine kinases, ShcA associates
with the receptor and becomes tyrosine
phosphorylated. Phosphorylated ShcA recruits
the Grb2/SOS complex to the membrane,
where SOS stimulates the small GTPase Ras,
resulting in the activation of the Ras/MAPK
pathway. The fact that Grb2 binds directly to
most of the receptor tyrosine kinases raises
the question of how important is the role of Shc
in mediating MAPK activation? Moreover,
beside growth factor-induced MAPK activation,
are there other pathways in which ShcAmediated
MAPK activation is relevant?
ShcA is expressed in three different
isoforms: p46Shc, p52Shc, and p66Shc. These
isoforms are all derived from a single gene and
differ only in their N-terminal part. Although all
isoforms are phosphorylated by receptor
tyrosine kinases, and subsequently bind to
Grb2, the p66Shc isoform does not seem to
mediate MAPK activation. The individual
contribution of p46Shc and p52Shc in mediating
MAPK activation is also not clear. The fact that
all isoforms are ubiquitously expressed, with
some restrictions for p66Shc, complicates the
experimental investigation of each isoform.
Recently, p66Shc has been implicated in the
regulation of apoptosis in response to oxidative
stress.
Using siRNA, we established a system which
allows isoform-specific knockdown of ShcA
proteins in tissue culture. Further development
of this technique enabled us to express a
single isoform in the absence of endogenous
protein. This so-called “knockdown-in”
technique is applicable for most proteins which
are expressed in multiple isoforms, and allows
the investigation of specific mutations against
a clear background without overexpression.
We used this technique to investigate the
contribution of individual ShcA isoforms to
EGF-induced MAPK activation in epithelial
cells. Knockdown of all or single ShcA
isoforms had no effect on EGF-induced Erk
activation. Moreover, overexpression of p66Shc
in non p66Shc-expressing MCF7 cells did not
change EGF-induced proliferation or viability.
These data suggest that EGF-induced MAPK
activation in epithelial cells is ensured by a
redundant coupling of Grb2 to the receptor.
In a quest for growth factor-independent
pathways involving Shc-mediated Erk
activation, we investigated signaling
downstream of the cell-cell adhesion molecule
E-cadherin. We identified a previously
unknown signaling pathway which is induced
upon disruption of E-cadherin-dependent cellcell
adhesion This pathway involves Src- and
Shc-dependent Erk activation, which results
subsequently in the expression of the
urokinase plasminogen activator. Applying the
knockdown-in technique revealed that p46Shc
and p52Shc, but not p66Shc, were able to
mediate MAPK activation upon disruption of
cell-cell adhesion. This pathway directly links
disruption of cell-cell adhesion with the
expression of proteolytic enzymes, both
processes involved in metastasis and wound
healing.
To learn more about the role of p66Shc in
mediating oxidative stress-induced apoptosis
in epithelial cells, the effect of p66Shc on cell
viability was investigated. Although p66Shc has
been shown to enhance stress-induced
apoptosis in fibroblasts, endothelial cells, and
T-cells, no effect on p66Shc expression was
observed in two different epithelial cells,
suggesting that the apoptotic response in
epithelial cells is mediated in a p66Shc-
independent manner.
Advisors:Meins, Frederick
Committee Members:Nagamine, Yoshikuni and Christofori, Gerhard M.
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI
UniBasel Contributors:Christofori, Gerhard M.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7331
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:88
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 15:20

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