Download Hitting the Target: Emerging Technologies in the Search for Kinase

PERSPECTIVE
Hitting the Target: Emerging Technologies in
the Search for Kinase Substrates
Brendan D. Manning and Lewis C. Cantley*
(Published 10 December 2002)
If gene expression and protein translation represent the musicians and instruments of a cellular symphony, then posttranslational modification of proteins should be considered the
conductor. Aside from containing sequences necessary for
three-dimensional folding into domains, many proteins have
evolved motifs that allow posttranslational modifications that
modulate function. By far the largest group of enzymes that catalyze regulatory posttranslational modifications is the family of
protein kinases. These enzymes transfer phosphates from ATP
(adenosine triphosphate) to specific hydroxyl groups on substrate proteins. About 2% of the genes encoded by the human
genome are predicted to encode protein kinases. The reversible
phosphorylation of specif ic tyrosine, serine, or threonine
residues on a target protein can dramatically alter its function in
several ways, including activating or inhibiting enzymatic activity, creating or blocking binding sites for other proteins, altering
subcellular localization, or controlling protein stability. Therefore, our understanding of the molecular control of cell physiology requires the identification of substrates targeted by specific
protein kinases.
Despite the widespread interest in this area, the discovery of
physiological substrates for protein kinases has been slow and
often unreliable. The catalytic domains of protein kinases share
a high degree of sequence and structural similarity, and many
subfamilies of kinases exist whose members are likely to have
overlapping substrate specificities. The use of genetic or pharmacological disruption of specific kinases also presents inherent problems due to functional compensation by related kinases
and lack of specificity in currently available kinase inhibitors.
However, several recent in vitro and in vivo technologies have
emerged as promising new avenues toward identification of
new substrates of protein kinases.
“Classical” Methods for Identifying Kinase Substrates
Approaches to identifying candidate substrates of protein kinases
have historically included genetic screens, the isolation of kinasebinding proteins, and biochemical purification. Genetic screens and
subsequent epistasis analyses in model organisms such as yeast,
worms, and flies have been very successful in placing substrates
downstream of specific protein kinases within signaling pathways.
For example, in the budding yeast Saccharomyces cerevisiae, screens
for mating-defective mutants (sterile or ste mutants) led to the elucidation of a protein kinase cascade leading from cell surface
pheromone receptors to the downstream transcription factor Ste12p
(1). In Caenorhabditis elegans, epistasis analyses placed the forkhead-family transcription factor DAF-16 downstream of the PI3K
(phosphoinositide 3-kinase)-Akt pathway (2), leading to the subseDepartment of Cell Biology, Harvard Medical School, Division of Signal Transduction, Beth Israel Deaconess Medical Center, 4 Blackfan
Circle, Boston, MA 02115, USA.
*Corresponding author. E-mail: [email protected]
quent demonstration of direct phosphorylation and regulation of
mammalian forkhead-family members by the kinase Akt (3). Genetic screens in model organisms have often paralleled and complemented efforts in Xenopus and mammalian cell systems, which have
used primarily biochemical approaches to identify kinase substrates.
Many protein kinase substrates have been identified through
interaction screens, most commonly yeast two-hybrid screens,
with the kinase of interest as “bait.” The first demonstration that
this was a viable technique for detecting novel kinase substrates
came with the identification of Sip1p as a two-hybrid-interacting protein with the yeast Snf1p kinase (4). Sip1p was subsequently shown to be directly phosphorylated by Snf1p. In most
cases the interaction between a protein kinase and its substrate
is transient, and upon substrate phosphorylation the association
is disrupted, allowing a single kinase to catalyze the
phosphorylation of multiple substrates. Such interactions are
difficult to trap and identify. However, in some cases, kinases
bind their substrates by interactions outside of the catalytic
pocket, and such associations can often be detected in two-hybrid screens. For instance, filamin was found in a two-hybrid
screen for proteins that associate with the NH2-terminal regulatory domain of Pak1 (p21-activated kinase 1) and is a Pak1 substrate (5). However, in cases where a third protein acts as a scaffold to stabilize the interaction between a kinase and its substrate, direct-interacting screens such as two-hybrid screens will
likely fail. Although two-hybrid screens to identify kinase substrates have been used with some success, problems with a high
rate of false-positives and with distinguishing between kinasebinding partners and bona fide in vivo substrates limit the usefulness of this method. As with all the approaches discussed
here, confirmation of candidate substrates by secondary assays
is critical.
Historically, the positions of phosphorylation sites on important
cellular proteins were often determined prior to identification of the
responsible kinase. To identify kinases, proteins in cell lysates were
separated by column chromatography and protein fractions were assayed for kinase activity toward a substrate of interest. For example,
the 70-kD ribosomal protein S6 kinase (S6K1) was identified with
this approach (6, 7). Similar techniques have been used with limited
success for the identification of proteins within lysate fractions that
can be phosphorylated in vitro by specific protein kinases, as scored
by incorporation of 32P. Obvious problems arise from such an approach, including the presence of kinases within the lysates phosphorylating themselves and other proteins, leading to high levels of
background phosphorylation and false-positives. Some of these
problems have been circumvented in a recent study by the Cohen
laboratory searching for novel substrates of SAPK/p38-kinase family members (8). These authors reduced the background phosphorylation problem by making several small adjustments to standard
protocols. Together, these modifications allowed the detection of
specific bands in lysate fractions that can be phosphorylated by distinct SAPK/p38 family members. However, after the initial detec-
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tion, this approach still depends on a rather laborious biochemical
purification of the candidate substrate. Furthermore, there are intrinsic caveats to all in vitro kinase substrate identification techniques,
and these are discussed below.
ground phosphorylation of phage and bacterial proteins is a problem with the kinase of interest, it will be detected early on during pilot studies because each plaque will contain the same background
proteins. The frequent improper folding of cDNA-encoded proteins
within E. coli presents one potential problem. In general, the utility
of this approach for identifying genuine substrates of a given kinase
will depend greatly on the in vitro specificity of the kinase (11). Kinases that utilize secondary (noncatalytic domain) contacts with
their substrates will likely give a higher degree of success in most in
vitro kinase screens against full-length native proteins.
Oriented peptide library screens for determining optimal
substrate motifs of specific kinases have been useful in distinguishing the substrate specificities of related kinases as well as
for identifying candidate substrates through bioinformatics (1215). This approach uses a degenerate library of peptides (that is,
a large mixture of peptides of random sequences) oriented
around an invariant serine, threonine, or tyrosine residue to be
phosphorylated by a purified kinase in vitro (Fig. 1A). The ki-
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ATP
ATP
Inhibitor
ATP
ATP
ATP
In Vitro Identification of Kinase Substrate Candidates
In recent years, several novel approaches for the identification of in
vitro protein kinase substrates have been developed. Although useful for the identification of candidate substrates for a given kinase,
in vitro kinase assays can often be misleading and provide too many
candidate substrates to be further validated by more rigorous in vivo
analyses. Indeed, protein kinases often display a high degree of
promiscuity in vitro. The use of high concentrations of purified kinase in in vitro assays is partially responsible for this loss of specificity. In addition, removal of the kinase from the cell often results
in a loss of its physiological regulatory mechanisms. A better understanding of the regulation of a kinase of interest and subsequent adjustments to the kinase purification scheme and assay can circumvent some of these complications. However, all candidate
substrates identified with in vitro
A
B
approaches require validation in
−5 −4 −3 −2 −1 0 +1 +2 +3 +4 +5
vivo. Nevertheless, some of the
WT kinase +
NH2−X−X−X−X−X−S−X−X−X−X−X−COOH
ATP
recently developed in vitro techKinase
P
+ ATP
niques allow a rapid identificaSubstrate
Substrate
tion of candidate substrates for
XXXXX XXXXXSXXXXX
SXXXX P XXXX
both known and novel kinases
XXXXXS
XSXXXXX
X
XXXXX
and are, therefore, valuable tools
X
XXXXXSXXXXX
X
X
X
SXXXXX
X
XXXXXSXXXXXXXXXX
XXS
WT kinase +
in the search for new kinase tarXXX XXXXX
XXX
X
SXXXXX P
XSX
bumped ATP
XXXX
gets.
XXXXXSXXXXX
XXXXXS
XXXXX
P
S
XXXXX
XXXXX
Solid-phase phosphorylation
No phosphorylation
Substrate
screening of phage expression libraries has been used successfulFe3+
ly to identify bona fide substrates
Holed kinase +
of some kinases [for example,
XXXXXSXXXXX
bumped ATP
extracellular signal-regulated kiXXXXXSXXXXX
P
P
Substrate
Substrate
nase 1 (9) and cyclin-dependent
kinases (10)]. This approach uses
or
cDNA libraries cloned into a
Quantitative Edman sequencing
phage expression vector. Followto determine substrate specificity
Holed kinase +
ing plaque formation on lawns of
bumped inhibitor
Escherichia coli, proteins within
Scansite
Phospho-motif
Substrate
No phosphorylation
selectivity matrix
antibodies
the plaques, including those ex+
pressed from an individual
cDNA clone as well as phage and
Identification of
candidate substrates
bacterial proteins, are then immobilized onto nitrocellulose filters. Proteins on these filters are
then subjected to phosphoryla- Fig. 1. Schematic of two approaches for identifying substrates of protein kinases. (A) The use of an orienttion by the purified kinase of in- ed peptide library to determine substrate specificity of a kinase and subsequent identification of candidate
terest in the presence of [γ- substrates. A degenerate mixture of peptides (x, any amino acid) oriented around a Ser, Thr, or Tyr to be
phosphorylated is subjected to an in vitro kinase assay with the kinase of interest. The small fraction, typi32 P]ATP (adenosine triphoscally 1%, of resulting phosphopeptides is then isolated on a ferric column. After elution from the column,
phate), and plaques containing the phosphopeptides are quantitatively sequenced by Edman degradation to determine the peptide-subpositive phosphorylation signals strate specificity of the kinase. This information can then be used both to search protein databases with
are identified by autoradiogra- the Scansite program and to generate phospho-motif-specific antibodies, the combination of which identiphy. One of the main benefits of fies candidate substrates. (B) A method for the in vivo study of kinase function and for kinase substrate
this approach comes from the identification developed by Shokat and colleagues (22) (see text for details). This approach uses ATP
ease of identifying the candidate analogs (bumped ATP) that can distinguish between wild-type and mutant (holed) kinases. The in vitro or
substrate by isolating the clone in vivo use of a bumped-ATP/holed-kinase pair allows selective labeling of substrates specific for the kifrom the phosphorylation-posi- nase of interest. The same technology has been modified for use of allele-specific inhibitors (bumped intive plaque. Furthermore, if back- hibitors) to study kinase function in vivo. WT, wild type.
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nase phosphorylates a small percentage of the billions of peptides within the mixture, and these phosphopeptides are then
isolated on a ferric column. This mixture of phosphopeptides is
then sequenced by Edman degradation, and the quantity of each
amino acid at each position relative to that of the phosphorylated residue is determined. The data obtained from this technique
provide a list of residues preferred, tolerated, and selected
against by the kinase at each site surrounding the phosphorylation site. These data can then be formatted into a selectivity matrix for protein database searches for candidate kinase substrates with the Web-based program Scansite [http://scansite.
mit.edu (15)]. Thus far, the amino acid selectivities of kinases
analyzed with this peptide library approach match those for
known substrates of the tested kinases (11), and Scansite
searches with the corresponding selectivity matrices correctly
predict previously identified substrates (15, 16). Therefore, this
approach will identify those proteins that may be phosphorylated by the given kinase. However, selecting which candidate substrates predicted by Scansite to test in more conventional substrate characterization assays is the real limitation of this approach. The technique is better suited for use in combination
with other substrate identification approaches, such as immunoblotting with phospho-motif antibodies (discussed below),
and can be very useful in predicting which sites on an independently identified substrate are likely to be phosphorylated.
In Vivo Identification of Bona Fide Kinase Substrates
Aside from genetic approaches, in vivo screens for kinase substrates
have been slow to develop. In vivo [γ-32P]ATP labeling of cells followed by separation of phosphoproteins by chromatography or twodimensional gel electrophoresis has limited value when used alone.
In general, in vivo detection of substrate candidates for individual
protein kinases requires an effective and highly specific means of
inactivating that kinase. This can be done by pharmacological inhibition [although inhibitors that can strictly distinguish between specific kinases are rare (17)], antibody injection, mouse knockout
technology, or, more recently, by small-interfering RNAs (siRNAs).
Knowledge of a specific stimulus or condition that maximally activates the kinase of interest is also crucial. Although less prevalent
than during in vitro assays, overexpression of kinases within cells
will often lead to nonspecific phosphorylation of some proteins. In
addition, unlike in vitro methods, it is difficult to determine if a
phosphorylation event detected in vivo is the direct action of the kinase of interest or occurs through a downstream kinase. With these
caveats in mind, some new technologies have recently been developed that seem quite promising for the identification of in vivo targets of protein kinases.
Knowledge of the minimal requirements for a kinase to phosphorylate a given protein sequence, determined by previously identified phosphorylation sites or peptide library methods, can be used
to raise phospho-motif-specific antibodies (18). These antibodies
are raised against a library of degenerate phosphopeptides with
residues required by the specific kinase and a phosphoserine, phosphothreonine (pT), or phosphotyrosine locked in at the appropriate
positions. In theory, these antibodies should recognize substrates of
the given kinase only once they are phosphorylated. Phospho-motif
antibodies can be used to identify specific kinase substrates. This
was demonstrated with an antibody to the minimal Akt substrate
motif (16, 19), which was raised against an xxxRxRxx(pT)xxxx
peptide mixture (where x represents any amino acid) (18). The activity of PI3K, an upstream activator of Akt, was required for im-
munoblotting of the majority of the proteins recognized by this antibody. Our laboratory used the apparent molecular size of protein
bands on phospho-Akt substrate immunoblots to narrow down
searches for candidate Akt substrates with Scansite (16). This approach successfully identified known and novel Akt substrates that
were confirmed by using conventional methods of substrate validation. One problem with this method is that in any given mass range
Scansite will predict several strong substrate candidates. However,
the search can be narrowed down even further by determining the
isoelectric point (pI) of a given protein recognized by the antibody,
because Scansite can search for proteins in restricted ranges of both
mass and pI. The existence of kinases related to Akt that phosphorylate the same motif and that are also regulated by PI3K (for example, serum- and glucocorticoid-regulated kinase and S6K1) presents
another potential problem with this approach. For example, the
phospho-Akt substrate antibody also recognizes the phosphorylated
form of S6 (a substrate of S6K1) (18). Use of Scansite with a matrix
based on the selectivity of the phospho-Akt substrate antibody,
rather than that of the Akt protein kinase, and with imposed restrictions of isoelectric point and molecular size range allowed prediction of S6 as a major spot visualized by the antibody on two-dimensional immunoblots (18). Novel phosphoproteins identified in this
manner can be further analyzed by Scansite to predict which related
kinase is likely to be responsible.
Phospho-motif and general phospho-specific antibodies have also been used to immunoprecipitate phosphorylated proteins in order
to identify candidate kinase substrates by mass spectrometry (MS)
methods. A recent study used the phospho-Akt substrate antibody to
immunoprecipitate and identify specific PI3K-dependent phosphoproteins from insulin-stimulated adipocytes (19). One problem with
this method is that phospho-motif antibodies are generated against
peptides and, hence, will often not be able to bind and immunoprecipitate proteins in their native folded state. In fact, efficient immunoprecipitation with the phospho-Akt substrate antibody requires prior denaturation of proteins within cell lysates (19). General phospho-tyrosine-specific antibodies and MS have also been
used to isolate and identify protein substrates of receptor-tyrosine
kinases (20, 21). As with other in vivo techniques, this approach requires specific means of activating and inhibiting the kinase whose
substrates are being analyzed.
A promising new chemical genetic technology for identifying
kinase substrates has been developed (Fig. 1B) (22). This approach
takes advantage of the structurally conserved ATP-binding pocket
within all kinases to generate mutant alleles that can utilize specific
ATP analogs, in addition to ATP. The mutations create a “hole” in
the nucleotide-binding domain in a region where the N6 amine of
ATP usually sits. This mutation allows the use of an ATP analog
with a large hydrophobic moiety, or “bump,” positioned off of the
N6 amine. Importantly, wild-type kinases cannot utilize these ATP
analogs, and these mutations appear to be silent with respect to kinase activity and substrate specificity (22). Assays with a “holed”kinase mutant in the presence of a “bumped”-[γ-32P]ATP derivative
allow the specific phosphorylation, and thus labeling, of substrates
targeted by this kinase. In principle, this approach should be applicable to both in vivo labeling of substrates when the mutant kinase
is expressed, as well as in vitro biochemical approaches discussed
above. Unlike other in vivo approaches, this method eliminates any
doubts as to whether the phosphorylation event is direct, because
only the kinase being assayed can transfer phosphate from the labeled-ATP analog. However, delivering sufficient quantities of radiolabeled “bumped”-ATP analog into the cell to allow visualization
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and purification of substrate proteins is likely to present a problem.
Although issues with in vitro specificity of kinases still arise, the
use of a “bumped”-ATP/”holed”-kinase pair eliminates all background problems when searching for substrates by in vitro kinase
assays on fractions from cell lysates. This latter approach has recently been used with a “holed”-JNK allele and has successfully identified a novel JNK substrate (23). The “bump and hole” technology
has also been adapted to create kinase-allele-specific inhibitors that
are powerful tools for further defining substrate specificities and the
overall cellular function of specific kinases (24). This method is particularly useful in model organisms such as yeast, where replacing
the wild-type allele with one encoding a “holed” kinase that is sensitized to the “bumped” inhibitor is trivial (25, 26). Overall, the use
of “bumped”-ATP analogs and inhibitors for specific “holed” kinases should be a valuable tool for kinase substrate identification.
However, the applicability of this approach to all families of protein
kinases has yet to be demonstrated.
The Future
The future progress of kinase substrate searches will involve refinement of the technologies discussed here, as well as the development
of novel in vitro and in vivo approaches. With the availability of
complete genome sequences, proteomic approaches are rapidly
emerging to study protein function and modification. Arrays of an
organism’s entire complement of kinases or its entire proteome ordered onto protein chips represent exciting new tools for the highthroughput identification of kinase substrates in vitro (27, 28). Technologies for the analysis of the “phosphoproteome” are also coming
to fruition. Methods for isolating phosphoproteins from in vivo
sources and rapidly identifying them by MS are under development
(29). This underscores the need for specific inhibitors of kinases to
identify candidate kinase substrates through these new approaches.
The use of siRNA to uncover the in vivo repertoire of substrates for
a specific kinase will also prove invaluable when studying wholeproteome phosphorylation. Finally, the best methods to identify
genuine substrates of specific kinases are likely to be combinatorial
approaches. For instance, protease cleavage of whole-cell lysates
and immunoprecipitation of specific phosphopeptides with phospho-motif antibodies followed by tandem MS (MS/MS) to rapidly
identify a specific subset of a cell’s phosphoproteins could be a
powerful technique in the near future. Regardless of the preferred
approach, the search for kinase substrates will go on. Our ability to
read the cellular sheet music depends on it.
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Citation: B. D. Manning, L. C. Cantley, Hitting the target: Emerging technologies in the search for kinase substrates. Science’s STKE (2002),
http://www.stke.org/cgi/content/full/sigtrans;2002/162/pe49.
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