Clarification of Answer by
synarchy-ga
on
09 Dec 2002 08:56 PST
A) Cells transformed by SV40 Large T-Ag exhibit uncontrolled mitosis
and do not require mitogenic factors for proliferation. Regulation of
the cell cycle is normally achieved by halting mitosis at various
checkpoints. These checkpoints function to halt mitosis until the
proper signaling has occured to indicate that the cell should divide.
The SV40 T-Ag acts at the first checkpoint of mitosis, removing the
necessity of mitogenic factor stimulation for proliferation.
The Rb protein functions to enforce a mitosis checkpoint (at the
transition from Growth phase 1, G1, and synthesis phase, S) by binding
to and inhibiting the action of E2F, a transcription factor necessary
for induction of mitosis. Normally, growth factor receptors, upon
binding to their ligand, begin a signaling cascade that leads to the
phosphorylation of Rb. When Rb becomes phosphorylated it is
inactivated - when inactive, Rb releases E2F - free E2F is active and
is able to function as a transcription factor supporting mitosis.
SV40, by binding to Rb and interfering with its ability to bind to
E2F, inactivates the checkpoint function of Rb. This decouples
mitosis from the necessity of growth factor stimulation. Thus SV40
allows for uncontrolled cell growth by bypassing the checkpoint
function of Rb through it's inactivation.
In addition to the suggested links from the original answer - this
page provides a nice overview:
http://www.landesbioscience.com/journals/cellcycle/papersinpress/1.6/cc07250254.html
B) Normally, Rb protein must be phosphorylated in order for it to
release E2F (from it's inactive, phosphorylated form). This likely is
the result of a conformational change in Rb, such that when Rb is
phosphorylated, the binding to E2F is no longer favored.
Theoretically, T-Ag could lead to a dissociation of Rb:E2F either by
(1)increasing the phosphorylation of Rb or binding to the same
residues which are normally phosphorylated and producing a similar
conformational change in the protein, (2) by displacing E2F from it's
binding site on Rb, or (3) by binding to another region of Rb entirely
that leads to a conformational change which no longer favors Rb:E2F
association. The question mentions that the phosphorylation domain
can be deleted without affecting Rb:T-Ag binding, meaning that T-Ag
must not bind directly to the residues involved in phosphorylation or
lead to phosphorylation of the protein. The easiest answer to explain
how T-Ag then acheives it's effect would be through displacement of
E2F from it's binding site on Rb.
Either answer (2) or (3) could be investigated by expressing deletion
constructs of Rb and looking for binding to either T-Ag or E2F. By
doing these experiments separately, one could map the binding sites
for both T-Ag and E2F on Rb. If the binding sites are overlapping,
one could confirm the ability of T-Ag to displace E2F by first mixing
Rb and E2F, adding T-Ag and looking for binding to Rb - if T-Ag
displaces E2F, only T-Ag should be bound to Rb. If the binding sites
are non-overlapping one would expect to find a deletion construct
which only would bind to E2F and not to T-Ag - in such a case,
addition of T-Ag to a mixture of this Rb deletion construct and E2F
would not lead to a decrease in binding between the construct and E2F.
Here is the web-page of someone who has carried out numerous similar
type experiments (listed in the left-hand column):
http://www.hmc.psu.edu/depts/old%20pages-kms%20save/micro/faculty/mtevethia.htm
Let me know if you need further information.
