Biology Reference
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(kinase activity assay for kinome profiling) method
[261]
can be used for the multiplexed measurement of phos-
phorylation events on 90 different peptides directly from
cell lysates (see
Box 5.4
for details on the KAYAK tech-
nology). Phosphorylated peptides are enriched using
immobilized metal-ion affinity chromatography and
analyzed by LC-MS techniques.
In addition to providing direct measurements on a pro-
teome-wide scale, quantitative MS approaches facilitate the
comparison of phosphoproteomes between wild-type cells
and cells that have undergone manipulations of their
signaling network components. In a recent study using
mutant strains of S. cerevisiae, a label-free, quantitative
phosphoproteomics approach was employed to determine
the relationships between 97 kinases and 27 phosphatases
and more than 1000 phosphoproteins
[262]
. Strikingly,
inactivation of most (77%) kinases and phosphatases
affects their immediate downstream targets as well as
a large proportion of the overall signaling network. Owing
to the inherent variation that exists between LC-MS
experiments from run to run, label-free quantitation
provides a relatively imprecise measurement of the differ-
ences in the phosphoproteome between wild-type and
mutant yeast cells. Recent advances in techniques such as
SILAC and chemical labeling, as well as improved sensi-
tivity and dynamic range of peptide identification by
current MS-based technologies, is enabling comprehensive
and reproducible assessment of differences in phospho-
proteomes
[263]
. Many groups have begun to apply this
promising global approach to identify the effects of
network perturbations on changes in the phosphoproteome
[262,264
266]
. iTRAQ labeling and phosphatase treat-
ment was used to identify phosphorylation sites on the
purified, auto-activated tyrosine kinase domain of fibroblast
growth factor receptor 3 (FGFR3-KD) and to analyze
complexes formed around the insulin receptor substrate
homologue (chico) immunopurified from Drosophila mel-
anogaster cells that were either stimulated with insulin or
left untreated
[267]
. In two recent studies of the insulin
signaling network in mammalian cells, Grb10 was identi-
fied as a mTORC1 substrate and was shown to be involved
in feedback inhibition of the phosphatidylinositol 3-kinase
(PI3K) and extracellular signal-regulated, mitogen-acti-
vated protein kinase (ERK-MAPK) pathways
[268,269]
.
iTRAQ has also been used to compare the phosphopro-
teomes of cells treated with insulin to activate the pathway
and cells that were pretreated with Torin 1 before insulin
activation
[268]
. Torin 1 is a novel adenosine 5
0
-triphos-
phate (ATP)-competitive mTOR kinase domain inhibitor
that blocks all known activities of both mTORC1 and
mTORC2 complexes
[270]
. Yu and colleagues
[269]
used
SILAC to quantify differences in the phosphoproteome of
TSC2
/
MEFs in the presence and absence of rapamycin,
as well as
e
in the absence or presence of a drug
Box 5.4 KAYAK (Kinase Activity Assay for Kinome Profiling) Method for Multiplex Analysis of Kinase Activities
KAYAK is a multiplexed, MS-based kinase assay developed to
measure the activity of multiple kinases from the same sample
lysate
[261,274]
. A single MS run directly measures the phos-
phorylation of 90 synthetic peptides, thus providing a multi-
plexed assay to simultaneously monitor kinase activities from
multiple signaling pathways. Key to the success of the KAYAK
method is the design and synthesis of substrate peptides that
can represent activity through the different core cellular
signaling pathways. This set of substrate peptides also includes
synthetic peptides containing phosphorylation sites with no
associated kinase. Such peptides are identified from large-scale
phosphoproteomics studies of cellular signaling networks and
can be used to identify the responsible kinase via perturbation
assays. The peptides are composed of 10
identical sequence (as internal standards), at a known
concentration. Immobilized metal-ion affinity chromatography
is used to enrich the phosphorylated substrate peptides and
internal standard phosphopeptides, which are then analyzed by
LC-MS techniques. The light (product) and heavy (internal
standard) peptide pairs differ in mass by 6 daltons (Da) and
although they co-elute, they can be quantified by the ratio of
light-to-heavy areas under the curve from the raw spectra. Since
the amount of each heavy phosphopeptide added is known, the
ratio of the light to the heavy phosphopeptide provides the
absolute amount of each product formed during the kinase
reaction. The in vitro kinase reactions are carried out in
a reaction volume of 50 mL and require only nanogram to
microgram amounts of cell lysate. 5 mM of each substrate
peptide is used in the reaction to reduce cross-phosphorylation
of peptides by different kinases.
The KAYAK method was first applied to profile the activity of
kinases in different cellular contexts, including mitogen-
induced cell proliferation, inhibition of signaling pathways by
known small molecule inhibitors, and a number of breast
cancer cell lines
[261]
. This study also identified that a peptide
derived from a PI3K regulatory subunit was a novel Src family
kinase site in vivo.
15 amino acid resi-
dues, with five residues upstream and four residues downstream
of the phospho-acceptor site, and a C-terminal tripeptide of
proline
e
arginine to facilitate the incorporation
and quantification of the stable isotope. The set of peptide
substrates whose phosphorylation reflects activity through
multiple signaling pathways is incubated together with a cell
lysate to allow for phosphorylation by active kinases in the
lysate. The in vitro kinase reactions are quenched, followed by
the addition of stable isotope-labeled phosphopeptides of
phenylalanine
e
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