668
Biological Regulation by Protein Phosphorylation
phosphorylates and inactivates phospho-
protein phosphatase-1. Because a single
protein kinase molecule can phosphory-
late many substrate molecules, each step in
this cascade amplifes the signaling path-
way. In addition, some protein kinases in
these cascades can phosphorylate many
diFFerent substrate proteins, thereby mod-
ulating multiple biochemical pathways,
which lead to an amplifcation oF a sig-
nal. This is most prominent in the MAPK
cascade. In response to an extracellular
stimulus, several stress-activated protein
kinases also converge onto key protein ki-
nases, resulting in the amplifcation oF
a cell stress signal. Thus, protein kinase
cascades contain sequential steps to am-
pliFy an extracellular signal as well as
divergent steps to coordinate diFFerent cel-
lular processes.
Many proteins appear to be phospho-
rylated at multiple sites with varying
consequences on their biological activ-
ity. Examples oF multisite phosphorylation
have revealed that (1) diFFerent protein ki-
nases can phosphorylate a common site on
a protein; (2) diFFerent protein kinases can
phosphorylate distinct sites on a protein
yet produce similar changes in biological
activity; (3) diFFerent protein kinases can
phosphorylate distinct sites on a protein
resulting in opposite changes in biolog-
ical activity; and (4) phosphorylation oF
one site by a protein kinase can gener-
ate a consensus sequence For a second
protein kinase. Proteins that are phospho-
rylated at multiple sites oFten Function at
critical points in a biochemical cascade.
Thus, multisite phosphorylation repre-
sents one approach by which diFFerent
biological signals, acting through diFFer-
ent transduction pathways, can converge
to regulate the Function oF a single pro-
tein. This convergence would allow For
coordinated regulation oF a single cellu-
lar process. Reversible phosphorylation
oF proteins represents a uniFying and
dynamic mechanism by which diFFer-
ent signaling systems can be integrated
to
produce
the
appropriate
biological
response.
5
Genetic and Pharmacological Modulation
of Protein Phosphorylation
In 1979, the Following Four criteria For
establishing that an enzyme undergoes
physiologically
signifcant
phosphoryla-
tion were defned:
1. The enzyme is phosphorylated at sto-
ichiometric levels at a signifcant rate
by a protein kinase and is dephospho-
rylated by a protein phosphatase.
2. There is a correlation between the
degree oF phosphorylation oF an enzyme
and changes in its Functional pro-
perties.
3.
In vivo
studies or studies with intact
cells show that the enzyme can be
phosphorylated and dephosphorylated
with subsequent changes in its Func-
tional properties.
4. The cellular levels oF protein kinase
and
phosphatase
eFFectors
correlate
with the extent oF phosphorylation oF
the enzyme.
These criteria can be extended to all
other classes oF phosphoproteins such as
receptors, ion channels, and proteins that
Form the cytoskeleton and proteins that
regulate transcription and translation. OF
these Four criteria, the third element is
oFten diFfcult to demonstrate. Selective
activators or inhibitors oF a specifc pro-
tein kinase or phosphatase are oFten used
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