Biological Regulation by Protein Phosphorylation
669
in an attempt to modify the phosphoryla-
tion state of a particular protein in intact
cells and to test for changes in its bio-
logical activity. By incubating cells with a
molecule that selectively activates or in-
hibits a particular kinase or phosphatase,
one can study the downstream events that
develop as a consequence of the pharma-
cological manipulation. The number of
pharmacological agents that target protein
kinases and phosphatases have increased
signiFcantly in recent years, and have been
used to provide a wealth of information
linking a particular enzyme with a particu-
lar biological response. Agents that modify
protein phosphorylation can be classiFed
into a number of different categories
including activators or inhibitors, and
natural or synthetic molecules. The syn-
thetic molecules can be further separated
into small molecules, peptides/proteins,
and nucleic acids (e.g. antisense oligonu-
cleotides and small interfering RNAs to
regulate expression of an enzyme). Re-
gardless of the classiFcation scheme, these
agents have great value when trying to
identify the physiological substrates for a
particular kinase or phosphatase, and to
dissect the role of a particular phospho-
proteinincellfunction.
Several synthetic analogs of cAMP have
been developed and shown to activate
cAMP-dependent
protein
kinase
in
a
manner analogous to cAMP. Some of these
compounds are membrane-permeable and
therefore can activate the enzyme when
applied to intact cells. Because these
compounds effectively mimic the actions
of extracellular signals on the cAMP
signal-transduction pathway, these cAMP
analogs are useful to determine if a cellular
response to a particular hormone, growth
factor or neurotransmitter is mediated
through the cAMP pathway.
Phorbol esters are a class of small or-
ganic molecules Frst identiFed in croton
oil. These compounds mimic the actions
of diacylglycerol and activate members of
the protein kinase C family. Both dia-
cylglycerol and inositol trisphosphate are
generated by the hydrolysis of inositol
phospholipids, which occurs in response
to a number of different extracellular sig-
nals. Diacylglycerol activates the protein ki-
nase C signal-transduction pathway while
inositol trisphosphate stimulates the re-
lease of calcium from intracellular stores.
Because the phorbol esters are potent and
selective activators of protein kinase C in
intact cells, they have become invaluable
tools for investigating the involvement of
protein kinase C isoenzymes in various
biochemical pathways and to dissociate
responses mediated via the diacylglycerol
signal-transduction pathway from the in-
ositol trisphosphate pathway. However,
caution must be used when interpreting
the results from such experiments, for
recent studies have revealed additional tar-
gets for diacylglycerol and phorbol esters
that can regulate cell function independent
of protein kinase C.
Several natural toxins have been iden-
tiFed that exert their biological effects
through direct and potent inhibition of
the phosphatases PP1 and PP2A. These
toxins are found in biologically diverse or-
ganisms. Okadaic acid, the Frst inhibitor
identiFed within this class, is produced by
several species of marine plankton. This
plankton can accumulate in shellFsh and,
when ingested by humans, cause an ill-
ness termed diarrhetic seafood poisoning.
Cantharidin is another interesting exam-
ple, for it has a rich history of medicinal
and recreational use prior to understand-
ing its mechanism of action. Cantharidin
is produced by the blister beetle as a de-
fense mechanism against predators. The
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