Caged phosphoproteins

Article abstract of DOI:10.1021/ja043875c

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the α-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported. Copyright

Full text of DOI:10.1021/ja043875c

Published on Web 12/30/2004
Caged Phosphoproteins
Deborah M. Rothman,† E. James Petersson,‡ M. Eugenio Va´zquez,† Gabriel S. Brandt,‡
Dennis A. Dougherty,*,‡ and Barbara Imperiali*,†,§
Departments of Chemistry and Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, and DiVision of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, California 91125
Received October 7, 2004; E-mail: dadoc@caltech.edu (D.A.D); imper@mit.edu (B.I.)
The phosphorylation of serine, threonine, and tyrosine residues
in proteins is a central mechanism of cellular regulation.1 Phos-
phorylation can profoundly modulate the role of the protein in a
biological system, either by altering the activity of the isolated
protein or by influencing interactions with other proteins. The
phosphorylation state of a protein is dynamic; it is determined by
the interplay of kinases, which append the phosphoryl group, and
phosphatases (PPases), which remove it.2 Currently available
biological experiments, such as chemical inhibition, gene knockout,
or point mutation studies, do not allow real-time studies of the
effects of protein phosphorylation in complex biochemical signaling
pathways.
Caged amino acids afford researchers spatial and temporal control
over the effective phosphorylation state at a specific site in a target
protein (Scheme 1).3 In general, the “cage” comprises a photo-
cleavable protecting group that masks the essential functionality.
Photolysis of the protecting group generates a bioactive protein.
Caged amino acids have been available for incorporation into full-
length proteins for some time.4 Caged phosphopeptides have
recently become accessible as tools for studying cell signaling
pathways both in vitro and in vivo.5 These peptides have already
proven useful for elucidating the role of a specific protein family
in real-time studies of cell cycle regulation.6 Due to the centrality
of protein phosphorylation in cell biology, a general method for
preparing caged phosphoproteins is highly desirable.
poor expression of bulkier, bis-caged analogues. For this purpose,
a new phosphitylation reagent (3; Scheme 2) was developed to
enable the synthesis of the singly caged phosphate. Transient, acid-
labile t-butyl protection enables manipulation of the serine and
threonine amino acid derivatives and avoids â-elimination reactions
that would result during the basic deprotection of groups such as
the common cyanoethyl moiety. The phosphoramidite (3) was
synthesized from hexaethyl phosphorus triamide in a one-pot
procedure. Each of the target amino acids (Ser, Thr, and Tyr) was
synthesized as the corresponding NR-4-pentenoylphospho(1-nitro-
phenylethyl)cyanomethyl ester (5a, 5b, and 6; Scheme 2) for coup-
ling to the dinucleotide, dCA.7,8 The tyrosine derivative 6 was
synthesized using a slightly modified version of the CpSer/CpThr
route.7
When coupling the CpAAs to dCA, tetrabutylammonium acetate
(NBu4OAc) salt beyond the typical 2.4 equiv was required; we
believe that the additional salt aided in solubilizing the negatively
charged amino acids in the presence of the negatively charged dCA.
It was found that 250 mM NBu4OAc optimally increased coupling
efficiency with minimal â-elimination side reactions. Next, the
aminoacylated dCAs were ligated to truncated tRNAs11 using a
slightly modified literature procedure.7,8 This gave full-length,
aminoacylated tRNAs (tRNACUA-CpAAs), designed to deliver the
CpAA to a position specified by an amber (UAG) codon.
Scheme 2. Syntheses of Amino Acid Derivativesa
Scheme 1. Control of Phosphorylation Dynamics with Caged
Residues
Herein is presented the chemical and biological synthesis of
caged phosphoproteins using the in vitro nonsense suppression
methodology.7,8 Although Hecht has used this method for the
incorporation of phosphonoserine and phosphotyrosine into firefly
luciferase,9,10 this is the first report of caged phosphoamino acids
(CpAAs) in proteins via in vitro translation. Caged phosphoproteins
will allow the real-time study of kinase targets with phosphorylated
residues in any position of a native protein sequence.
In these studies, phosphoamino acid analogues with a single
o-nitrophenylethyl-caging moiety were strategically targeted (Scheme
1). The cage renders the phosphoprotein stable to phosphatases in
biological systems and affords temporal control over the release
of the target phosphoprotein. Use of a single cage avoids anticipated
a (a) 4-Pentenoic anhydride, THF/water; (b) chloroacetonitrile, DBU; (c)
TFA; (d) 4,5-dicyanoimidazole, 3, THF; (e) tBuOOH, CH2Cl2; (f) TFA/
TIS, CH2Cl2. 1a, 2a, 4a, 5a: R ) H, 1b, 2b, 4b, 5b, R ) Me.
With the tRNACUA-CpAAs in hand, the suppression efficiency
of these bulky and negatively charged residues was tested in a
system known to accept natural and non-natural amino acids using
nonsense suppression: position 122 of the nicotinic acetylcholine
receptor (nAChR) R-subunit.12 The mRNA for nAChR R with an
amber stop codon in position 122 was introduced into a rabbit
reticulocyte lysate translation system, and the results were monitored
by Western blot analysis (Figure 1). Uncaged, phosphorylated amino
acids (pAA) appear to incorporate, but with relatively low efficiency
(Figure 1, lanes 4, 7, and 10), consistent with earlier findings from
Hecht.9,10 However, adding a single caging group (CpAA) does
† Department of Chemistry, MIT.
‡ California Institute of Technology.
§ Department of Biology, MIT.
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10.1021/ja043875c CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
as the phosphorylated species (lane B3). Next, the suppression of
VASP-S153TAG mRNA with caged phosphoserine was evaluated
(Figure 2C). The full-length protein with the caged phosphoserine
migrated as the unphosphorylated species (lane C1). The uncaged
protein migrated with a shift characteristic of that for the phos-
phorylated species (lane C2). Finally, upon uncaging and subsequent
reaction with λ-PPase, the protein migrated as the unphosphorylated
species (lane C3). As a negative control, wild-type VASP (λ-PPase-
treated) was irradiated under the uncaging conditions to show that
this did not alter the gel mobility of the wild-type protein (lane
A4).
In conclusion, we present a synthesis of tRNA aminoacylated
with caged phosphoserine, threonine, and tyrosine. Several sup-
pression reactions were performed in order to evaluate the ability
of the native ribosomal machinery to incorporate these large and
charged residues into full-length proteins. Additionally, the protein
products were tested for nativelike behavior in biochemical studies.
Access to such caged phosphoproteins will enable studies of a large
number of kinase targets in real-time using both in vitro and in
vivo4 translation systems.
Figure 1. Test suppression in nAChR R subunit: A122TAG mRNA with
tRNACUA charged with amino acids listed above lanes. “No aa” refers to
full-length, but uncharged, tRNA.
not further diminish suppression efficiency (Figure 1, lanes 3, 6,
and 9). The inefficient suppression may be attributable to low
affinity binding of the aminoacyl tRNAs by elongation factors (i.e.,
EF-1A). Uhlenbeck has shown that elongation factor Tu (EF-Tu)
binds tRNAs bearing negatively charged amino acids poorly,13 and
that glutaminyl tRNAs (from which our tRNACUA is derived) have
a low inherent EF-Tu affinity.14 It should be noted that masking
this charge by doubly caging the phosphate moiety resulted in far
lower incorporation efficiency for tyrosine derivatives,7 and use of
bis-caged phosphoserine and threonine was intractable due to
â-elimination side reactions.
Next, the suppression ability of the tRNACUA-CpAAs was tested
in a biologically significant system relating to phosphoregulated
signaling with a centrally targeted residue. The vasodilator-stimu-
lated phosphoprotein, VASP, is involved in cell migration processes.
Specifically, phosphorylation of serine 153 has been associated with
cell leading edge protrusion and forward cell movement.15 VASP
with CpSer153 would thus be a valuable tool for determining the
precise role of this phosphoserine in the complex process of cell
migration. Figure 2A depicts the translation of the wild-type (WT)
VASP in rabbit reticulocyte lysate (lane A1). The VASP appears
as two bands due to a gel shift caused by phosphorylation at serine
153; the shift is not caused by the increase in molecular weight,
but rather by a conformational change and altered SDS binding
capacity of VASP that results in decreased mobility in the SDS-
PAGE analysis.16
Acknowledgment. The work has been supported by the Cell
Migration Consortium Grant GM64346 to B.I., NS-34407 to D.A.D,
and the HFSP Foundation for M.E.V. The authors thank Professor
F. Gertler for the VASP gene and antibody.
Supporting Information Available: Full experimental procedures.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S.
Science 2002, 298, 1912-1934.
(2) Woodgett, J. R. Protein Kinase Functions, 2nd ed; Oxford University
Press, New York, 2000.
(3) Marriott, G. Methods Enzymol. 1998, 291, 925-1116.
(4) (a) Petersson, E. J.; Brandt, G. S.; Zacharias, N. M.; Dougherty, D. A.;
Lester, H. A. Methods Enzymol 2003, 360, 258-273. (b) Cook, S. N.;
Jack, W. E.; Xiong, X.; Danley, L. E.; Ellman, J. A.; Schultz, P. G.;
Noren, C. J. Angew. Chem., Int. Ed. Engl. 1995, 34, 1629-1630. (c)
Miller, J. P.; Silverman, S. K.; England, P. M.; Dougherty, D. A.; Lester,
H. A. Neuron 1998, 20, 619-624. (d) Short, G. F., III; Lodder, M.;
Laikhter, A. L.; Arslan, T.; Hecht, S. M. J. Am. Chem. Soc. 1999, 121,
478-479.
(5) (a) Rothman, D. M.; Va´zquez, M. E.; Vogel, E. M.; Imperiali, B. Org.
Lett. 2002, 4, 2865-2868. (b) Rothman, D. M.; Va´zquez, M. E.; Vogel,
E. M.; Imperiali, B. J. Org. Chem. 2003, 68, 6795-6798.
(6) Nguyen, A.; Rothman, D. M.; Stehn, J.; Imperiali, B.; Yaffe, M. B. Nat.
Biotechnol. 2004, 22, 993-1000.
Figure 2. Suppression at VASP position 153 with CSer or CpSer. Lanes
(left to right): (A) 1, WT translation; 2, WT with λ-PPase; 3, WT with
PKA; 4, WT with λ-PPase, then UV-irradiated; (B) 1, CSer with PKA; 2,
CSer; 3, CSer subjected to UV, then PKA; (C) 1, CpSer; 2, CpSer subjected
to UV; 3, CpSer subjected to UV, then λ-PPase.
(7) See Supporting Information.
(8) Ellman, J.; Mendel, D.; Anthony-Cahill, S.; Noren, C. J.; Schultz, P. G.
Methods Enzymol. 1991, 202, 301-336.
(9) Arslan, T.; Mamaev, S. V.; Mamaeva, N. V.; Hecht, S. M. J. Am. Chem.
Soc. 1997, 119, 10877-10887.
To ascertain that the proteins produced by in vitro translation
behave as expected, several experiments were performed. Phos-
phorylation of the wild-type protein with protein kinase A (PKA)
caused all of the protein to migrate as the phospho-species (Figure
2, lane A3), and dephosphorylation by λ-phosphatase (λ-PPase)
caused all of the protein to migrate as the unphosphorylated species
(lane A2). Next, mRNA for VASP-S153TAG was suppressed with
caged serine (CSer, protected with the 6-nitroveratryl group), and
the behavior of the protein product was examined (Figure 2B). With
the caging group on the serine hydroxyl group, the full-length
protein migrated as the unphosphorylated species in the presence
or absence of PKA (lanes B1 and B2, respectively). Upon uncaging
with 350 nm light, the serine hydroxyl group was liberated, and
subsequent phosphorylation by PKA caused the protein to migrate
(10) Short, G. F.; Laikhter, A. L.; Lodder, M.; Shayo, Y.; Arslan, T.; Hecht,
S. M. Biochemistry 2000, 39, 8768-8781.
(11) Saks, M. E.; Sampson, J. R.; Nowak, M. W.; Kearney, P. C.; Du, F.;
Abelson, J. N.; Lester, H. A.; Dougherty, D. A. J. Biol. Chem. 1996,
271, 23169-23175.
(12) England, P. M.; Lester, H. A.; Dougherty, D. A. Biochemistry 1999, 38,
14409-14415.
(13) Dale, T.; Sanderson, L. E.; Uhlenbeck, O. C. Biochemistry 2004, 43,
6159-6166.
(14) (a) LaRiviere, F. J.; Wolfson, A. D.; Uhlenbeck, O. C. Science 2001,
294, 165-168. (b) Asahara, H.; Uhlenbeck, O. C. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 3499-3504.
(15) (a) Krause, M.; Bear, J. E.; Loureiro, J. J.; Gertler, F. B. J. Cell Sci. 2002,
115, 4721-4726. (b) Krause, M.; Dent, E. W.; Bear, J. E.; Loureiro, J.
J.; Gertler, F. B. Annu. ReV. Cell DeV. Biol. 2003, 19, 541-564.
(16) Butt, E.; Abel, K.; Krieger, M.; Palm, D.; Hoppe, V.; Hoppe, J.; Walter,
U. J. Biol. Chem. 1994, 269, 14509-14517.
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