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Cross-talk between non receptor tyrosine kinases and caspases in cancer and in apoptosis
present on the Procaspase-8 molecule and it is therefore lost during autoprocessing
and in the tetrameric complex.
The lack of structural data for the procaspase, containing Tyr380, does not
allow structural and modeling approaches to address the effect of phosphorylation
on the structure and on the function of Caspase-8. Tyrosine phosphorylation
may modulate one of the steps of Caspase-8 activation, such as DISC
recruitment, dimerization and activation, processing and release of the active
tetramer. Alternatively, phosphorylation on Tyr380 may drive new proteinprotein
interactions that either change the conformation of Caspase-8 preventing
its activation, or sequester it far from its substrates, impinging on its apoptotic
function. At present all these models are merely speculative only further
experiments will address this point.
Milestone A1: Analysis of the effect of Caspase-8 tyrosine phosphorylation on
the Death Inducing Signaling Complex – To address the possibility that tyrosine
phosphorylation might somehow affect DISC formation or Caspase-8 activation
and release at the DISC, Fas-sensitive cell lines that stably overexpress a
constitutive active form of Src will be generated. These cell lines, along with
the parental cell lines as a control, will be stimulated to undergo apoptosis by
anti-Fas antibodies. The recruitment of Caspase-8 to the DISC will be revealed
upon DISC immunoprecipitation and immunoblot with specific anti-Caspase-8
antibodies (24) . The use of the phospho-Tyr380-Caspase-8 antibody will help
to clarify whether phosphorylation interferes or not with the recruitment of
Caspase-8 to the DISC.
Milestone A2: Studies to address whether tyrosine phosphorylation might
interfere with Caspase-8 dimerization – Recently it has been proposed that two
dimers of procaspase in which the large and small subunits have not been yet
separated by cleavage, are already active. Receptor trimerization would allow the
formation of dimers of proCaspase-8 at the DISC level. Dimerization would then
allow proCaspase-8 activation, resulting in the first cleavage that separates the
large and the small subunit, the so-called “ interdimer processing ”. The produced
tetramers would then be able to perform the second cleavage event
between the prodomain and the large subunit, resulting in the release of the
active Caspase-8 from the DISC (37) . To test whether tyrosine phosphorylation
impairs proCaspase-8 dimerization we will take advantage of an in vitro translation
approach based on FK506 binding protein (FKBP). This system allows the
production of Caspase-8 in vitro which can be further induced to dimerize by the
addition of a compound that recognizes the FKBP sequence allowing dimerization.
Upon dimerization the procaspase will be activated and the autoprocessing
will occur (37) . In vitro translated proCaspase-8 might be phosphorylated in vitro
by Src kinase before the induction of its dimerization. The dimerization-induced
processing of in vitro translated phosphorylated proCaspase-8 will be compared
to the one of unphosphorylated proCaspase-8. The Caspase-8-Y380F mutant will
be used as a negative control.
An alternative approach will be based on experiments where proCaspase-8
dimerization will be induced by incubation of protein extracts from cells transfected
with the constitutively active SrcY527F or with the empty vector as con-
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