Bio-orthogonal affinity purification of direct kinase substrates

Article abstract of DOI:10.1021/ja050727t

Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signaling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Herein, we describe a chemical strate

Full text of DOI:10.1021/ja050727t

Published on Web 03/24/2005
Bio-orthogonal Affinity Purification of Direct Kinase Substrates
Jasmina J. Allen,‡ Scott E. Lazerwith,† and Kevan M. Shokat*
Departments of Cellular and Molecular Pharmacology, UniVersity of California, San Francisco, California 94143,
and Department of Chemistry, UniVersity of California, Berkeley, California 94720
Received February 3, 2005; E-mail: shokat@cmp.ucsf.edu
Scheme 1. Tandem Approach for Creating Bio-orthogonal Affinity
Tagged Kinase Substrates (Ser R ) H, Thr R ) Me)
Protein kinases mediate signal transduction through phosphory-
lation of their protein substrates.1 Up to one-third of proteins in a
cell are phosphorylated,2 and a major goal of phosphoproteomics
is to characterize phosphorylation mediated signaling cascades by
identifying phosphorylated proteins. This feat is analytically chal-
lenging because most phosphoproteins are of low abundance and
substoichiometrically phosphorylated. Further, once a phospho-
protein is identified, it is difficult to integrate the role of the
phosphorylation event into signal transduction networks without
knowledge of the upstream kinase. Affinity purification techniques,
such as strong cation exchange (SCX),3 immobilized metal ion
affinity chromatography (IMAC),4 chemical tagging of phospho-
rylated residues with biotin,5 and phospho-motif specific antibodies6
can enrich phosphopeptides or proteins, but the information
regarding the kinase responsible for phosphate transfer is uncoupled
from the phosphorylation event. We previously have described
methodology that allows selective labeling of direct kinase sub-
strates, using analogue specific (as) kinases and orthogonal un-
natural nucleotides (A*TP and A*TPγS);7 however, it remains
challenging to biochemically isolate the labeled substrates, impeding
their identification.
Here, we report a technique that combines direct substrate
labeling with immunoaffinity purification (Schemes 1 and 2). To
label the substrates of a given kinase, an as allele is used to
enzymatically label substrates with A*TPγS. The selectively
introduced thiophosphate is then chemically derivatized to construct
a bio-orthogonal affinity tag. This approach is similar to other bio-
orthogonal tagging strategies using ketones8 or azides,9 except
thiophosphate cannot be selectively tagged in a single chemical
step. For example, an alkylating agent will label both thiophosphate
and other cellular nucleophiles, but we envisioned that an antibody
could discriminate the thiophosphate alkylation products from other
undesired alkylation products. The alkylating agent p-nitroben-
zylmesylate (PNBM) was selected to construct the epitope because
we predicted antibodies could recognize the product of thiophos-
phate alkylation over other nitrobenzyl alkylated amino acid
residues, based on unique size and charge. Also, several high affinity
antibodies have been raised against haptens containing p-nitrophenyl
moieties,10 increasing the chances of eliciting an antibody capable
of immunoprecipitation. Antibodies raised against hapten 4 are
likely to be sequence independent because the binding determinants
are relatively distant from the peptide backbone.
Scheme 2. Immunoaffinity Purification
ments, cyclin dependent kinase 1 (Cdk1)12 substrates, Histone H1
(H1) and Swe1, were derivatized and tested in immunoassays.
The R-3-IgY antibodies only successfully recognized H1 that
had been thiophosphorylated and PNBM alkylated. In control
experiments, untreated H1, PNBM alkylated H1, or thiophospho-
rylated H1 were not detected by ELISA (Supporting Information
Figure S2). Similarly, R-3-IgY recognition of the substrate Swe113
also required both thiophosphorylation and alkylation (Figure 1,
lanes 1-4). Cell lysates contain abundant free thiols and other
nucleophiles, which can react with PNBM, producing close
structural variants of 3. Therefore, one of the most demanding
requirements of R-3-IgY is the ability to discriminate phospho-
rothioate 3 from thioether 2 and other undesired alkylation products.
To address this point, whole HeLa cell lysate (WCL) was treated
with PNBM and analyzed by western blot. WCL was not recognized
by R-3-IgY irrespective of treatment with PNBM (lanes 5 and 6,
Figure 1). However, if Swe1 thiophosphate (Swe1-PS) was added
to the WCL in the presence of PNBM, Swe1-PS+PNBM was
readily detected (Figure 1, lane 7), indicating R-3-IgY exhibits the
desired specificity for 3 in the context of whole cell lysate. Similar
results were obtained with another Cdk1 substrate, Mob1 (Sup-
porting Information Figure S3). These results demonstrate that
alkylation of thiophosphate with PNBM produces an epitope that
can be specifically recognized in a complex protein mixture,
validating this approach.
Utilizing hapten 4, polyclonal antibodies (IgY and IgG) were
raised in chickens and rabbits, respectively.11 To enrich for specific
binders, immune antibodies were purified on an affinity column
containing immobilized hapten 4. Chicken IgY antibodies (R-3-
IgY) performed best in immunoprecipitations and were used in
subsequent experiments. To investigate R-3-IgY binding require-
Because many antibodies do not recognize their targets with
sufficient affinity for immunoprecipitation,14 it was important to
determine if R-3-IgY could immunopurify an epitope-tagged
substrate from a complex protein mixture. To enable rapid analysis
and quantitation of immunoprecipitation experiments, we prepared
rhodamine-labeled versions of the kinase substrate Histone H1
(Rh-H1-PS and Rh-H1-PS+PNBM). R-3-IgY or preimmune IgY,
‡ Program in Chemistry and Chemical Biology, UC San Francisco.
† Current Address: Pfizer Inc. Ann Arbor, MI 48105.
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10.1021/ja050727t CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
strates from whole cell lysates will likely require production of
anti-3 specific monoclonal antibodies, which is currently underway.
The method we report here requires a kinase to utilize ATPγS as
a phosphodonor, and although it is unclear what percentage of the
kinome can utilize ATPγS, several kinases have been shown to
thiophosphorylate their substrates (see Supporting Information Table
S5 for a partial list). Combining this purification method with as
kinase substrate labeling should provide a general route to the
identification of direct kinase substrates. Other biological questions
may be approached with tandem chemical/immunological strate-
gies,16 which are providing new routes to interrogate the proteome.
Acknowledgment. The authors are thankful to Mart Loog for
providing Mob1 and Swe1, and Justin Blethrow for Cdk1F80G/
cyclinB and assistance with early histone labeling experiments.
HeLa cells were generously provided by the NCCC. This work
was supported by a grant from the NIH (RO1 EB001987); S.L.
was supported by a NIH grant (1 F32 GM63312-01A1). We
acknowledge Dustin Maly, Matthew Simon, and Zachary Knight
for helpful comments on this manuscript. Mass spectra were
provided by the Center for Mass Spectrometry at UC-San Francisco,
supported by the NIH Division of Research Resources.
Figure 1. Recognition determinants for R-3-IgY immunoreactivity mea-
sured by western blotting; 25 ng of Swe1 or Swe1-PS was treated with
DMSO (lanes 1 and 3) or 2.5 mM PNBM in DMSO (lanes 2 and 4); 15 µg
of WCL was treated with DMSO (lane 5), 2.5 mM PNBM in DMSO (lane
6), and 25 ng of Swe1-PS plus 2.5 mM PNBM in DMSO (lane 7). Lanes
8 and 9 show coomassie staining of samples identical to 6 and 7,
respectively.
Supporting Information Available: Details for experimental
procedures, synthesis of hapten 4, ELISA data, Mob1 Western Blot,
immunoprecipitation controls, and a table of ATPγS utilizing kinases.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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