Biology Reference
In-Depth Information
7 Profiling PDE Activity in Strains Lacking Adenylyl Cyclase
The expression of PDEs in strains lacking adenylyl cyclase provides a favorable
context for observing the properties of PDEs in live cells (although to be sure,
S. pombe
lacks many of the PDE-interacting proteins found in mammalian cells).
By comparing the cyclic nucleotide-mediated growth response of a strain that
lacks both adenylyl cyclase and PDE activity to that of a PDE-expressing strain,
one can assess the relative activity of the PDE against cAMP and cGMP (Fig.
6
).
We see, for example, that a strain expressing PDE4B requires a final concentra-
tion of ~4 mM cAMP in the medium to stimulate cell growth to OD
600
¼
0.6,
while the same strain grown in the presence of 40
m
M rolipram (Fig.
6a
)ora
strain lacking PDE activity (Fig.
6b, c
) requires
50
m
M cAMP in the medium to
reach this OD
600
. In contrast, expression of PDE4B shifts the concentration of
cGMP needed to stimulate growth by only ~60
m
M (Fig.
6a
), although this may
represent enough cGMP hydrolyzing activity by PDE4B to play a role in
controlling cGMP levels in certain mammalian cells. A strain expressing the
cGMP-specific PDE9A requires
<
3 mM more cGMP to stimulate growth to
>
OD
600
¼
0.6 than does a strain lacking PDE activity, while these strains show
identical responses to cAMP (Fig.
6b
). A strain expressing the dual cAMP/cGMP
hydrolyzing PDE1B enzyme (Repaske et al.
1992
) requires 10
m
M more cAMP or
70
m
M more cGMP in the medium to stimulate growth to OD
600
¼
0.6 than does
a strain lacking PDE activity (Fig.
6c
). Low activity of mammalian calcium/
calmodulin-activated PDE1B expressed in
S. pombe
could be because
S. pombe
calmodulin is only 74% identical to mammalian calmodulin and may be unable to
activate PDE1B.
8 Additional Uses of this System
The reciprocal growth behavior of strains expressing the
fbp1-ura4
reporter in
medium containing 5FOA (in which high cAMP levels are required for growth)
versus medium lacking uracil (in which low cAMP levels are required for growth),
allows for screens of PDE activators as well as PDE inhibitors. While such
compounds should be detectable by their ability to promote growth of strains
with high cAMP levels in medium lacking uracil, two pilot screens failed to identify
compounds that activated either PDE7A or PDE8A. This may be due to the fact that
PDE activators will be much less common than PDE inhibitors, just as dominant
gain-of-function mutations are less common than recessive loss-of-function muta-
tions. The reciprocal growth behavior in medium lacking uracil versus 5FOA
medium conferred by the
fbp1-ura4
reporter could also be used to facilitate growth
enrichment and genetic screens for strains expressing mutant alleles of PDE genes
that encode inhibitor-resistant enzymes. The ability of
S. pombe
to maintain
autonomous plasmids allows for the screening of cDNA libraries, cloned in
