Synthetic phosphorylation of p38α recapitulates protein kinase activity

Article abstract of DOI:10.1021/ja4095318

Through a "tag-and-modify" protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38α. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38α bearing only a single phosphocysteine (pCys) as a mimic of pThr at 180 was sufficient to switch the kinase to an active state, capable of processing natural protein substrate ATF2; 172 site phosphorylation did not. In this way, we chemically recapitulated triggering of a relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric phosphorylation events catalyzed by p38α and revealed that site 180 is a sufficient activator alone and engenders dominant mono-phosphorylation activity. Moreover, a survey of kinase inhibition using inhibitors with different (Type I/II) modes (including therapeutically relevant) revealed unambiguously that Type II inhibitors inhibit phosphorylated p38α and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II.

Full text of DOI:10.1021/ja4095318

Communication
pubs.acs.org/JACS
Synthetic Phosphorylation of p38α Recapitulates Protein Kinase
Activity
K. Phin Chooi,† Sebastien R. G. Galan,† Ritu Raj,† James McCullagh,† Shabaz Mohammed,†
́
Lyn H. Jones,‡ and Benjamin G. Davis*,†
†Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
‡Chemical Biology Group, BioTherapeutics Chemistry, WorldWide Medicinal Chemistry, Pfizer, 200 Cambridge Park Drive,
Cambridge Massachusetts 01890, United States
S
* Supporting Information
ing Information (SI) Figure S1) are typically heterogeneous14 (as
ABSTRACT: Through a “tag-and-modify” protein chem-
ical modification strategy, we site-selectively phosphoryl-
ated the activation loop of protein kinase p38α.
Phosphorylation at natural (180) and unnatural (172)
sites created two pure phospho-forms. p38α bearing only a
single phosphocysteine (pCys) as a mimic of pThr at 180
was sufficient to switch the kinase to an active state,
capable of processing natural protein substrate ATF2; 172
site phosphorylation did not. In this way, we chemically
recapitulated triggering of a relevant segment of the
MAPK-signaling pathway in vitro. This allowed detailed
kinetic analysis of global and stoichiometric phosphoryl-
ation events catalyzed by p38α and revealed that site 180 is
a sufficient activator alone and engenders dominant mono-
phosphorylation activity. Moreover, a survey of kinase
inhibition using inhibitors with different (Type I/II)
modes (including therapeutically relevant) revealed
unambiguously that Type II inhibitors inhibit phosphoryl-
ated p38α and allowed discovery of a predictive kinetic
analysis based on cooperativity to distinguish Type I vs II.
for many post-translationally modified proteins), alternative
strategies for their precise study are required.15 For detailed in
vitro studies, such as activity/signaling triggering, a direct non-
enzymatic method would be useful. Here we use such a strategy
in the chemical recapitulation of an activation−protein
phosphorylation sequence from a relevant section of MAPK-
mediated signaling (Figure 1a, box).
p38α is difficult to activate non-enzymatically.16 A common
technique for kinases mutates phosphorylation sites to Asp/Glu.
However, at physiological pH, the sp2 carboxylate side chains are
poor phospho-mimetics, having not only different pKa values
(pKa ≈ 4 C(O)OH, cf. pKa1 ∼ 2, pKa2 ∼ 6 OP(O)(OH)2)17 but
also insufficient charge (−1 cf. −2) or structural (trigonal planar
cf. tetrahedral) mimicry. Indeed, in p38α, successful constitutive
activation has only been achieved indirectly by mutating off-
target residues to favor the stabilization of an active
conformation.16,18 To our knowledge, recapitulation of effective
charge mimicry in p38α has not been possible until now, and this
has precluded key hypotheses regarding the effect of point charge
localization in such critical signaling cascades.
In vitro, site-selective chemical modification gives an
alternative potential strategy. Although an elegant approach for
chemical attachment of a single negative charge19 has shown
some modulation of kinase activity in MEK1, the groups used
were not phospho-derived or charge-matched and were redox-
sensitive, bringing disadvantages associated with non-represen-
tative instability, charge, and pKa. We previously demonstrated
the first examples of site-selective, chemical protein phosphoryl-
ation,20 although not in naturally phosphorylated enzymes. This
employs a “tag-and-modify” approach, which involves introduc-
ing an orthogonally reactive functional groupthe tagthat can
be used as a selective chemical handle for further modification
and introduction of the desired group.21 Incorporating
dehydroalanine (Dha)20a,22,23 as a tag allows diverse modifica-
tion,20,23,24 including phosphorylation.
rotein kinases transfer phosphoryl onto side chains of
P
protein residues (typically Ser, Thr, and Tyr).1 Their activity
is often triggered or modulated by phosphorylation on a so-called
activation loop (Figure 1),2 which causes a change in
conformation.3 In this way, kinases in a specific sequence
generate consequential phosphorylation events that lead to
amplifying cascades in one of the dominant modes of
intracellular signaling and regulation (Figure 1a).4 p38α
mitogen-activated protein kinase (MAPK) is implicated in
critical widespread events such as inflammation,5 somatic,6
cardiac,7 and neural8 cellular regulation. It is the most studied
protein kinase9 and the most targeted therapeutically.10 Once the
MAPK cascade is triggered by external receptor engagement
(Figure 1a), “upstream” kinases are activated that in turn activate
p38α. This occurs naturally by phosphorylation at Thr180 and
Tyr182 in the activation loop (Figure 1b).11 p38α, in turn,
phosphorylates other protein substrates, including activating
transcription factor 2 (ATF2), which is implicated in many roles,
including pathways that, when disregulated, lead to melanoma.12
Such cascades are finely tuned, so small changes can be
amplified, leading to ready disregulation and hence pathoge-
nicity. Given that representative biological samples13 (Support-
To test the effect of site-selective chemical phosphorylation of
the activation loop in p38α, two sites were selected: 172 and
180only 180 is naturally phosphorylated (as pThr180). First, a
Dha “tag” was site-selectively installed in two different p38α
variants (SI sections 4 and 5) at 172 and 180 by treating p38α-
Received: September 14, 2013
Published: January 6, 2014
© 2014 American Chemical Society
1698
dx.doi.org/10.1021/ja4095318 | J. Am. Chem. Soc. 2014, 136, 1698−1701

Journal of the American Chemical Society
Communication
Figure 2. (a) Enzymatic phosphorylation by variants of p38α of ATF2
generated by both chemical and biological methods. (b) 1, Electro-
phoretic radioassay (ERA); 2, Coomassie stain; and 3, intact protein
ESI-MS were used to assay phosphorylation of ATF2. (c) Left:
Comparison of a representative time course of global phosphorylation
using p38α-pCys180 as enzyme, measured by ERA and MS. Right: MS-
allowed assay of precise ATF2 phospho state (representative time
course). (d) Quantitative kinetic parameters for global, mono- (P), and
di- (PP) phosphorylation of ATF2 derived from these assays.
Figure 1. (a) p38α and the MAP kinase cascade (shown here for ATF2
activation). (b) Structure, design, and synthesis of synthetically
phosphorylated p38α; box shows sites of natural (180, 182) and
unnatural (172) phosphorylation in the activation loop.
Cys172 and p38α-Cys180, respectively, with selective reagent
2,5-dibromohexane diacetamide (DBHDA, SI section 3; Figure
1b, step i).23 Next, reaction of Dha with sodium thiophosphate
(Figure 1b, step ii) proceeded cleanly and completely (SI section
5) to create phospho-amino acid phosphocysteine (pCys), to
yield p38α-pCys172 and p38α-pCys180, respectively. Protein
characterization confirmed generation of the intended folded
p38α pure phospho-forms: peptide mapping using proteolytic
digestion and LC-MS/MS (SI section 7 for Cys→Dha→pCys
mapping) confirmed chemistry at only intended sites, despite the
presence of other native Cys (Cys39, Cys211) in the designed
constructs (p38α-C119S:C162S:A172C and -C119S:C162S:
T180C), highlighting selectivity based on predicted accessibility
(Figure S3). Circular dichroism (CD) spectroscopy revealed that
these synthetically phosphorylated p38αs retained correct α-
helical/β-sheet structure (SI section 7 and Figure S2) and that
their temperature profiles were essentially identical to native.
With pure, synthetic phospho-forms in hand, we characterized
catalytic activity (Figure 2). While small peptide substrates allow
qualitative assay, useful for high-throughput, our intention in
creating homogeneous constructs was to develop precise
mechanistic understanding, aided by prior analyses with mixed
systems.13 Use of relevant protein substrate ATF225 therefore
allowed us to recapitulate a segment of MAPK signaling (Figure
1a) and to consider both global and individual phosphorylation
events. This was assessed by both mass spectrometry (MS,
Figure 2b,c and SI section 8) and electrophoretic radioassay
(ERA, Figure 2b and SI section 9): ERA using [32P]-labeled ATP
provides a measure of total phosphorylation, while direct intact
protein ESI-MS kinetics allows mono-/di-phosphorylation states
of ATF2 to be determined (Figure 2b). Consistent with an
anticipated need for activation loop phosphorylation, non-
phosphorylated synthetic p38 precursors (p38α-Cys172, p38α-
Dha172, p38α-Cys180, p38α-Dha180) showed no phosphoryl-
ating activity. Synthetic phosphorylation at natural (180) and
unnatural (172) phosphorylation sites, both within the loop, to
create p38α-pCys180 and p38α-pCys172, respectively, had
dramatically contrasting effects. Although correctly formed,
unnaturally phosphorylated p38α-pCys172 was inactive. How-
ever, excitingly, phosphorylation at the single natural site 180 in
p38α-pCys180 was sufficient to generate clear activity (Figure
2c).
Kinetic characterization of this active, pure, synthetic
phospho-form p38α-pCys180 provided key mechanistic insights.
1699
dx.doi.org/10.1021/ja4095318 | J. Am. Chem. Soc. 2014, 136, 1698−1701

Journal of the American Chemical Society
Communication
Quantitative analysis revealed that pCys is a sufficient mimic of
pThr; it triggers activity (kcat/KM(global) = 0.65 0.03 μM−1
h−1) at a level equal to that created through site Thr180
phosphorylation in biologically derived, mixed samples.13d This
is ∼10% of the activity (Figure 2d and SI sections 8 and 9) of
doubly phosphorylated p38α-pThr180-pTyr182 (generated
enzymatically14), consistent with results from other mixed,
biologically derived samples.13d This mimicry is successful
despite βO→βS, likely α-epimers, and lack of Thr Cβ-Me
substituent. Lack of activity for p38α-pCys172 shows that correct
charge and mimicry in the activation loop are not enoughsite is
also important. We note this generation of 172-phosphorylated
variants (applicable, in principle, to any site) would not be
possible with current biological methods.
The MS method used here is a rare example of on-protein
modification kinetic determination.26 Comparison (Figure 2c)
revealed good correlation between global ATF2 phosphorylation
determined by MS and, more typical, ERA. However, MS also
revealed key subtleties in phospho-state: ATF2 is phosphoryl-
ated at Thr69 and Thr71. Compared to initial mono- (ATF2-P)
and di-phosphorylation (ATF2-PP) rates with p38α-pThr180-
pTyr182, those for pure phospho-form p38α-pCys180 show a
bias toward ATF2-P. This switch (ATF2-P:ATF2-PP = 1.9→
8.9) suggests that phosphate at site 180 engenders critical
activation of mono-P kinase activity and that at site 182 then
enables di-P. MSn analysis suggested that ATF2-P is phosphoryl-
ated only at Thr69 (SI section 8.6). Moreover, this difference in
selectivity highlights that current analyses of protein phosphoryl-
ation at a global level may ignore key elements of selectivity,
resulting in mechanistic implications in kinase signaling. The
design of synthetic kinase (in activated phosphorylated form)
used here also maintains an essentially intact, unaltered activation
loop. While alterations can be achieved through disruptive
mutational analyses13c,16,27 (e.g., Tyr→Phe182) to “mutate out”
phosphorylation sites, it is known that loop residues act in
concert,3 and losses of key functional groups (e.g., Tyr-OH) may
create an unclear mechanistic picture.
To test the scope of mechanistic analysis possible with the
synthetic system described here, we also evaluated the effect of
protein kinase inhibitors as powerful probes of function.9 These
have been characterized on the basis of observations made in
inhibitory co-crystal structures, according to induction of so-
called DFG-loop (Figure 1) “in” (Type I) or “out” (Type II)
states.28 A representative range of Type I (VX745,29 TAK71530
SB202190, and 131), Type II (BIRB-79610b and 231), and
unknown (JX401) inhibitors was tested. The pure phospho-form
p38α-Cys180 generated high-quality inhibitory profiles (Figure
3c, SI section 10), consistent with known potencies for Type I.
Our data also directly addressed whether Type II inhibitors bind
active phosphorylated protein kinase states.28 It has been
suggested that the Type II mode of action may depend on
sequestering inactive kinase in DFG-out non-phosphorylated
form.28 However, to be consistent with the Curtin−Hammett
principle, this would depend critically on suitable equilibration
kinetics. With impure, “phosphorylated” p38α-pThr180-
pTyr182 generated by enzymatic activation14 (>10% non-
phospho, only ∼30% di-phospho), such a possibility cannot be
discounted. Now, through formation of pure active form, we can
demonstrate direct inhibition (Figure 3). Moreover, this
inhibition was characterized by a marker that distinguished
inhibitor types. Under the conditions employed,28 Type I
inhibitors are favored, as reflected in more potent inhibition by
Type I inhibitors (Figure 3d) over Type II. However, Type II
Figure 3. (a) Inhibition of p38α-mediated phosphorylation of ATF2.
(b) Type I and II inhibitors used. (c) Inhibition−dose curves (dotted for
chemically generated p38α-Cys180 and solid for biologically
generated14 p38α-pThr180-pTyr182; see SI section 10 for individual
curves). (d) Inhibition data.
showed a clear cooperativity (shown by Hill coefficients) for
their inhibition. Large coefficients are typically ignored32 but may
be consistent33 with conformation-alteration mechanisms (e.g.,
“DFG-out” of phosphorylated loop triggered by site-directed,
localized unfolding from the Type II urea motif). This readily
applied measurement might complement current structural
distinction of inhibitor type during development.28 Interestingly,
our method also highlights an ambiguity of TAK-715, which
displays intermediate characteristics; this has also recently been
noted structurally.34 We also predict JX401 to be a Type I
inhibitor.
Finally, to explore the generality of synthetic chemical
phosphorylation in other key kinases, we also tested MEK1.
Site-selective reaction at activation site 222 successfully gave
phospho-form MEK1-pCys222 (SI sections 11 and 12).
In summary, we have rationally designed and constructed
synthetic variants of p38α that allow precise recapitulation of
activation through chemical phosphorylation. Choice of a natural
phosphorylation site seems critical in this kinase; modification
causes activation, as would be the case with enzymatic
phosphorylation at the same position. Through site-selective
phosphorylation at a non-native site, we show that location is key
to activity, not simply the addition of charge alone. The
methodology for kinase activation described here opens further
possibilities for in vitro functional studies of other kinases for
which it has been difficult to obtain intrinsically active
mutants.24b Interestingly, differences in tolerance were also
noted during our synthetic protein phosphorylation chemistry:
p38α-pCys172 was formed from p38α-Dha172 more rapidly
than p38α-pCys180 from p38α-Dha180 (SI section 5.5). This is
consistent with potential electrostatic interactions35 (SI Figure
S3) and suggests such chemistry might also be used to probe the
inherent tolerance of phospho-sites within proteins. The
mechanistic origins of the intriguing p38α mono- vs di-
1700
dx.doi.org/10.1021/ja4095318 | J. Am. Chem. Soc. 2014, 136, 1698−1701

Journal of the American Chemical Society
Communication
Graham, A.; Grob, P.; Hickey, E.; Moss, N.; Pav, S.; Regan, J. Nat. Struct.
Biol. 2002, 9, 268.
phosphorylation selectivity we observed here are unknown but
may also relate to electrostatic modulation in concert with
Tyr182.
(11) Pearson, G.; Robinson, F.; Beers Gibson, T.; Xu, B.-e.;
Karandikar, M.; Berman, K.; Cobb, M. Endocrinol. Rev. 2001, 22, 153.
(12) Papassava, P.; Gorgoulis, V. G.; Papaevangeliou, D.; Vlahopoulos,
S.; van Dam, H.; Zoumpourlis, V. Cancer Res. 2004, 64, 8573.
(13) (a) LoGrasso, P.; Frantz, B.; Rolando, A.; O’Keefe, S.; Hermes, J.;
O’Neill, E. Biochemistry 1997, 36, 10422. (b) Szafranska, A.; Dalby, K.
FEBS J. 2005, 272, 4631. (c) Humphreys, J. M.; Piala, A. T.; Akella, R.;
He, H.; Goldsmith, E. J. J. Biol. Chem. 2013, 288, 23322. (d) Zhang, Y.-
Y.; Mei, Z.-Q.; Wu, J.-W.; Wang, Z.-X. J. Biol. Chem. 2008, 283, 26591.
(14) Ready biological access to pure phospho-p38 is difficult. For
enzymatically “activated and phosphorylated” p38-pT180-pY182 used
in many studies, we determined >6 species including non-:mono-:di-
phosphorylated as ∼10:60:30% mixture (see Figure S1).
Given the mixtures produced by biological methods,14 this
represents a rare, pure phospho-form of a kinase. Other powerful
methods, such as protein semi-synthesis or assembly of
phosphorylated peptide fragments, could also be considered.36
Such precise methods should allow further insight into enzyme
reaction mechanisms, excluding other confounding factors (e.g.,
dominant catalyst impurities). For example, the chemically
controlled switch-like nature of our method employs substrates
(e.g., p38α-Dha180) that can only be chemically phosphorylated
and so unambiguously remove other possible mechanisms (auto-
phosphorylation) of kinase activation.37 We now have chemical
control of the site 180 “on switch” for p38α not found through
traditional biology. We also note that, despite extensive structural
work, some mechanistically informative kinase structures, such as
Type II inhibitors bound to active form, still do not exist, and our
strategy for generating pure forms may also facilitate their
formation. Cys phosphorylation has also recently been noted in
bacterial signaling.38 In this way, we aim to unlock further details
of the key chemical mechanisms behind signaling.
(15) (a) Davis, B. G. Science 2004, 303, 480. (b) Gamblin, D. P.; van
Kasteren, S. I.; Chalker, J. M.; Davis, B. G. FEBS J. 2008, 275, 1949.
(16) Diskin, R.; Askari, N.; Capone, R.; Engelberg, D.; Livnah, O. J.
Biol. Chem. 2004, 279, 47040.
́
(17) Smiechowski, M. Chem. Phys. Lett. 2010, 501, 123.
(18) Tzarum, N.; Diskin, R.; Engelberg, D.; Livnah, O. J. Mol. Biol.
2011, 405, 1154.
(19) Lamoureaux, T.; Lee, D. Chem. Commun. 2011, 47, 8623.
(20) (a) Bernardes, G.; Chalker, J.; Errey, J.; Davis, B. G. J. Am. Chem.
Soc. 2008, 130, 5052. (b) Chalker, J. M.; Lercher, L.; Rose, N. R.;
Schofield, C. J.; Davis, B. G. Angew. Chem., Int. Ed. 2012, 51, 1835.
(21) Chalker, J. M.; Bernardes, G. J. L.; Davis, B. G. Acc. Chem. Res.
2011, 44, 730.
(22) (a) Levengood, M.; van der Donk, W. A. Nat. Protoc. 2007, 1,
3001. (b) Wang, J.; Schiller, S. M.; Schultz, P. G. Angew. Chem., Int. Ed.
2007, 46, 6849.
ASSOCIATED CONTENT
* Supporting Information
Experimental details and supplementary figures. This material is
■
S
available free of charge via the Internet at http://pubs.acs.org.
(23) Chalker, J. M.; Gunnoo, S.; Boutureira, O.; Gerstberger, S.;
Fernandez-Gonzalez, M.; Bernardes, G.; Griffin, L.; Hailu, H.; Schofield,
C. J.; Davis, B. G. Chem. Sci. 2011, 2, 1666.
(24) (a) Timms, N.; Windle, C. L.; Polyakova, A.; Ault, J. R.; Trinh, C.
H.; Pearson, A. R.; Nelson, A.; Berry, A. ChemBioChem 2013, 14, 474.
(b) Rowan, F.; Richards, M.; Bibby, R.; Thompson, A.; Bayliss, R.; Blagg,
J. ACS Chem. Biol. 2013, 8, 2184.
AUTHOR INFORMATION
Corresponding Author
ben.davis@chem.ox.ac.uk
Notes
The authors declare the following competing financial
interest(s): L.H.J. is an employee and shareholder of Pfizer.
■
(25) Cuenda, A.; Rousseau, S. Biochim. Biophys. Acta 2007, 1773, 1358.
(26) For an example applied to a chemical protein reaction and
extended discussion, see: Lin, Y. A.; Boutureira, O.; Lercher, L.;
Bhushan, B.; Paton, R. S.; Davis, B. G. J. Am. Chem. Soc. 2013, 135,
12156.
ACKNOWLEDGMENTS
■
We thank EPSRC/Pfizer (K.P.C.), BBSRC/AstraZeneca
(S.R.G.G.), Felix Scholarship (R.R.), and Welcome Trust
(B.G.D., J.M., S.M.) for funding; K. Scott and L. Gong for
assistance; A. Breeze and S. Royer for discussion; R. Bazin and E.
Hett for p38αC119SC162SA172C plasmid and reagents. B.G.D.
is a Royal Society Wolfson Merit Award Recipient.
(27) Askari, N.; Beenstock, J.; Livnah, O.; Engelberg, D. Biochemistry
2009, 48, 2497.
(28) Liu, Y.; Gray, N. Nat. Chem. Biol. 2006, 2, 358.
(29) Duffy, J. P.; Harrington, E. M.; Salituro, F. G.; Cochran, J. E.;
Green, J.; Gao, H.; Bemis, G. W.; Evindar, G.; Galullo, V. P.; Ford, P. J.;
Germann, U. A.; Wilson, K. P.; Bellon, S. F.; Chen, G.; Taslimi, P.; Jones,
P.; Huang, C.; Pazhanisamy, S.; Wang, Y.-M.; Murcko, M. A.; Su, M. S. S.
ACS Med. Chem. Lett. 2011, 2, 758.
(30) Miwatashi, S.; Arikawa, Y.; Kotani, E.; Miyamoto, M.; Naruo, K.;
Kimura, H.; Tanaka, T.; Asahi, S.; Ohkawa, S. J. Med. Chem. 2005, 48,
5966.
REFERENCES
(1) Adams, J. Chem. Rev. 2001, 101, 2271.
■
(2) Johnson, L.; Noble, M.; Owen, D. Cell 1996, 85, 149.
(3) (a) Johnson, L.; Lewis, R. Chem. Rev. 2001, 101, 2209. (b) Huse,
M.; Kuriyan, J. Cell 2002, 109, 275.
(4) (a) Nolen, B.; Taylor, S.; Ghosh, G. Mol. Cell 2004, 15, 661.
(b) Ono, K.; Han, J. Cell. Signall. 2000, 12, 1. (c) Zhang, Y.; Dong, C.
Cell. Mol. Life Sci. 2007, 64, 2771. (d) Junttila, M.; Li, S.-P.;
Westermarck, J. FASEB J. 2008, 22, 954.
(5) Schieven, G. Curr. Top. Med. Chem. 2009, 9, 1038.
(6) Ventura, J.; Tenbaum, S.; Perdiguero, E.; Huth, M.; Guerra, C.;
Barbacid, M.; Pasparakis, M.; Nebreda, A. Nat. Genet. 2007, 39, 750.
(7) Ren, J.; Zhang, S.; Kovacs, A.; Wang, Y.; Muslin, A. J. Mol. Cell.
Cardiol. 2005, 38, 617.
(8) Xing, B.; Bachstetter, A.; van Eldik, L. Mol. Neurodegen. 2011, 6, 84.
(9) Knapp, S.; Arruda, P.; Blagg, J.; Burley, S.; Drewry, D.; Edwards, A.;
Fabbro, D.; Gillespie, P.; Gray, N.; Kuster, B.; Lackey, K.; Mazzafera, P.;
Tomkinson, N.; Willson, T.; Workman, P.; Zuercher, W. Nat. Chem.
Biol. 2013, 9, 3.
(31) Braganza, J.; Letavic, M.; McClure, K. Patent WO2004072072.
(32) Hafenbradl, D.; Baumann, M.; Neumann, L. In Protein Kinases as
Drug Targets; Klebl, B., Muller, G., Hamacher, M., Eds.; Wiley-VCH
̈
Verlag & Co. KGaA: Weinheim, 2011; Vol. 49, pp 3−43.
(33) Prinz, H. J. Chem. Biol. 2010, 3, 37.
(34) Azevedo, R.; van, Z. M.; Raaijmakers, H.; Kazemier, B.; de, V. J.;
Oubrie, A. Acta Crystallogr. D: Biol.Crystallogr. 2012, 68, 1041.
(35) Baker, N.; Sept, D.; Joseph, S.; Holst, M.; McCammon, A. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 10037.
(36) Siman, P.; Brik, A. Org. Biomol. Chem. 2012, 10, 5684.
(37) Lochhead, P. Sci. Signal. 2009, 2, pe4 DOI: 10.1126/
scisignal.254pe4.
(38) Sun, F.; Ding, Y.; Ji, Q.; Liang, Z.; Deng, X.; Wong, C.; Yi, C.;
Zhang, L.; Xie, S.; Alvarez, S.; Hicks, L.; Luo, C.; Jiang, H.; Lan, L.; He,
C. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 15461.
(10) (a) Lee, M.; Dominguez, C. Curr. Med. Chem. 2005, 12, 2979.
(b) Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P.; Gilmore, T.;
1701
dx.doi.org/10.1021/ja4095318 | J. Am. Chem. Soc. 2014, 136, 1698−1701