A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity

Article abstract of DOI:10.1021/ja108258d

We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV i

Full text of DOI:10.1021/ja108258d

Published on Web 12/13/2010
A Photocleavable Rapamycin Conjugate for Spatiotemporal Control of Small
GTPase Activity
Nobuhiro Umeda,†,‡,¶,§ Tasuku Ueno,†,§ Christopher Pohlmeyer,† Tetsuo Nagano,*,‡,¶ and
Takanari Inoue*,†
Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins UniVersity, Baltimore,
Maryland 21205, United States, Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan, and CREST, JST, 3-5, Chiyoda-ku, Tokyo, Japan
Received September 13, 2010; E-mail: tlong@mol.f.u-tokyo.ac.jp; jctinoue@jhmi.edu
completely inert until UV illumination. It has been reported that
Abstract: We developed a novel method to spatiotemporally
the modification of a C40 hydroxyl group of rapamycin with a
control the activity of signaling molecules. A newly synthesized
relatively large functional group such as fluorescein (molecular
photocaged rapamycin derivative induced rapid dimerization of
weight: 332) reduces affinity against FKBP by more than 5000-
FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin
fold,14 making this position an ideal link to a caging group.
binding protein) upon UV irradiation. With this system and the
However, even 5000-fold reduction may not be sufficient to
spatially confined UV irradiation, we achieved subcellularly local-
completely prevent the dimerization event in cells given the very
ized activation of Rac, a member of small GTPases. Our
high concentration of caged molecules (10-5 to 10-3 M) commonly
technique offers a powerful approach to studies of dynamic
used in the cellular environment to complement the generally poor
intracellular signaling events.
uncaging efficiency.11
To circumvent this pitfall, we designed a caged rapamycin that
is tethered to a macromolecule such that the resulting large complex
Dynamic regulation of the Rho family of small guanosine
does not cross the plasma membrane, leading to virtually no
triphosphatases (GTPases) with great spatiotemporal precision is
background activity as a chemical dimerizer inside cells. However,
essential for various cellular functions such as cell migration,
UV irradiation releases rapamycin that can now permeate cells to
phagocytosis, adhesion, and secretion.1 In order to drive these
induce dimerization events (Figure 1). To connect rapamycin to a
events, Rho GTPases become activated at specific intracellular
locations to interact with distinctive downstream effector molecules.2-5
Their spatiotemporally dynamic nature has been revealed by
visualization of the activity and localization in real time.6-8 In order
to gain a deeper understanding of their roles in diverse cellular
functions at the molecular level, the next step should be perturbation
of protein activities at a precise subcellular location and timing.
Unfortunately, few techniques are available for rapidly controlling
the location and activity of Rho GTPases in living cells. We have
previously developed a rapamycin-triggered heterodimerization
strategy for activation or inactivation of Rho GTPases,9 in which
rapamycin induces translocation of FKBP-fused GTPases or its
activator to the plasma membrane where FRB is anchored.9 We
then assumed that such inducible activation of Rho GTPases can
be spatially confined to a subcellular level, if administration of
rapamycin is localized to the small region of the cells.
The photocaging method has been frequently used to release
small molecules in a spatially and temporally controlled manner
by means of light.10,11 A photocaged compound is a small molecule
whose activity is suppressed with a photocleavable protecting group
known as a caging group. Light irradiation at an appropriate
wavelength initiates a photochemical reaction that results in removal
of the caging group from the mother compound, leading to
restoration of the original activity. While there have been two
Figure 1. Schematic of spatially confined protein dimerization using caged
rapamycin-avidin conjugate (cRb-A) and UV light. UV irradiation cleaves
reports on the synthesis of caged rapamycin,12,13 neither of these
the linker between rapamycin and biotin, resulting in the release of chemical
dimerizer (HE-Rapa) and its byproduct. The released dimerizer then diffuses
into cells and induces dimerization between FKBP-POI (protein of interest)
and plasma membrane anchored FRB only in the proximity of the irradiated
region (shown as a blue circle).
has shown that the caged rapamycin is capable of inducing FKBP-
FRB dimerization at the subcellular level. A general principle of
the photocaging technique requires a caged molecule to be
† Johns Hopkins University.
‡ The University of Tokyo.
¶ CREST JST.
macromolecule, we employed avidin and biotin, whose interaction
is extremely stable with a dissociation constant on the order of
§ These authors contributed equally.
9
12 J. AM. CHEM. SOC. 2011, 133, 12–14
10.1021/ja108258d  2011 American Chemical Society

C O M M U N I C A T I O N S
femtomolar. A similar strategy should be achieved by modifying
caged rapamycin with hydrophilic groups instead of macromolecules.
We first synthesized caged rapamycin-biotin conjugate cRb by
linking rapamycin to biotin through a caging group (see Supporting
Information for the detailed synthetic scheme). As a caging group
we used a 4,5-dimethoxy R-methyl nitrobenzyl group.15 An
additional linker was inserted between the nitrobenzyl group and
the C40 hydroxyl group of rapamycin, as direct caging of the C40
hydroxyl group was not successful in our hands despite previous
reports.12,16 The following high-performance liquid chromatography
(HPLC) analysis of cRb assured that the molecule releases C40-
O-(2-hydroxyethyl) rapamycin (HE-Rapa, also known as Everoli-
mus or Rad001) upon irradiation with UV light (Figure S1), which
was confirmed with high-resolution mass spectrometry analysis.
Independently, we also confirmed that HE-Rapa is fully functional
as a chemical dimerizer (Figure S2a-c). cRb itself functioned as
a weak chemical dimerizer (Figure S2d), suggesting that the biotin
and nitrobenzyl group provide insufficient steric hindrance. cRb
and the avidin conjugate (cRb-A) were then prepared by incubating
cRb in the presence of an excess amount of avidin in phosphate
buffered saline at room temperature, followed by purification with
a size-exclusion column. To test if rapamycin can be released to
function as a chemical dimerizer in cells, we transfected HeLa
epithelial cells with DNA constructs encoding YFP-FKBP (YF)
and membrane-targeted FRB (LDR).9 With this system we have
previously shown that addition of rapamycin or its analog to the
bulk media induces rapid dimerization of these two proteins,
promoting translocation of YF from cytoplasm to the plasma
membrane. Addition of the purified cRb-A to the bulk media did
not induce any protein translocation (Figure 2a and 2b). However,
of Rac, a member of Rho family GTPases, induces membrane ruffle
formation and plays an important role in cell migration especially
at the leading edge of the cells.3 We have previously shown that
recruitment of Tiam1, a specific guanine exchange factor for Rac,
to the plasma membrane using a rapamycin-triggered protein
dimerization technique is sufficient to induce Rac activation and
subsequent ruffle formation.9 In order to achieve localized Rac
activation, we transfected NIH3T3 fibroblasts with YFP-FKBP-
Tiam1 (YF-Tiam1) and LDR. As a light source for localized UV
irradiation, we employed a light-emitting diode (LED) coupled to
a microscope through an optical fiber. After adding cRb-A to the
bulk media, LED UV light was applied to a small region near a
cellular edge. This operation rapidly induced formation of localized
ruffles only in the irradiated area (Figure 3a, Supplementary movie
Figure 3. Spatially confined ruffle formation in a transfected NIH3T3 cell
induced by localized UV irradiation with cRb-A conjugate. (a) Confocal
fluorescence images of YF-Tiam1 before and after local UV irradiation at
365 nm (yellow circle). (b-c) Frequency of ruffle formation in each quadrant
with local or global UV irradiation in the presence of cRb-A. Cells were
divided into quadrants with the irradiated spot in the area i (b) and ruffle
formation was counted in each quadrant (c). Scale bar: 10 µm.
1). To confirm that this was indeed local activity, we then globally
irradiated with UV, or added rapamycin to the bulk media, both of
which induced global ruffle formation throughout the membrane
(Figure S6, Supplementary movie 2). Global UV irradiation after
local irradiation induced relatively smaller global ruffle formation,
at least partly because the molecular machinery for ruffle formation,
such as Rac and WAVE, may have been exploited for the preceding
local ruffling. Consistent with this, we observed more striking,
global ruffles with global stimulation without any prior operation
(Supplementary movie 3). For a quantitative analysis, we divided
target cells into four quadrants and counted the frequency of ruffle
formation upon local as well as following global irradiation in each
quadrant (Figure 3b and c). While all the cells tested showed ruffle
formation in an irradiated quadrant (n ) 13, Table S1), only a small
number of cells showed ruffling in nonirradiated quadrants (p <
0.0002). Ruffle formation in nontargeted quadrants may result from
rapamycin overspill or lateral diffusion of dimerization complex
and/or activated endogenous Rac along the plasma membrane. With
global UV irradiation, on the other hand, more than 90% of the
quadrants showed ruffle formation upon irradiation (n ) 12, Table
S2). We confirmed that local UV irradiation with or without avidin
did not induce any ruffle formation in these cells (Figure S7). These
Figure 2. Translocation of YF in HeLa cells induced by global UV
irradiation with cRb-A conjugate. (a) Confocal fluorescence images of YF
before and after photoirradiation. (b) Time course of fluorescence intensity
in cytosol of the irradiated or nonirradiated cell. Arrows indicate the time
of acquiring images in (a). UV was irradiated for 0.5 s at the period shown
as a gray bar. Scale bar: 20 µm.
subsequent UV irradiation to a broad region of the transfected cells
resulted in a rapid translocation of YF proteins to the plasma
membrane (Figure 2a and 2b). We confirmed that neither UV
irradiation with avidin (without cRb) nor UV irradiation with an
avidin and rapamycin mixture that had been purified with a size-
exclusion column (which should exclude any free rapamycin)
induced translocation of YF (Figure S3, S4). In another control
experiment we observed no effect of UV irradiation on the
translocation of YF that had been induced by bulk rapamycin
(Figure S5). These results indicate that cRb-A can release a
functional chemical dimerizer upon UV irradiation.
We then tested if spatially restricted UV irradiation could trigger
a dimerization event at the subcellular level. Toward this end, we
aimed to achieve localized activation of Rho GTPases. Activation
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C O M M U N I C A T I O N S
results indicated that spatially restricted uncaging of cRb-A can
achieve Rac activation at the subcellular level.
Acknowledgment. We thank Prof. T. Furuta and Dr. T.
Kobayashi for helpful discussions. This study was supported in part
by the NIH (DK090868 and GM092930 to T.I.). N.U. and T.U.
are recipients of a fellowship from the Japan Society for the Pro-
motion of Science.
In conclusion, we demonstrated the development of a technique
that employs a novel caged compound together with a FKBP-FRB
heterodimerization system in order to manipulate Rho GTPase
activity at a precise subcellular location on a time scale of seconds.
Although it was predicted to be difficult to “cage” the effectiveness
of rapamycin using only small functional groups, a large protein
such as avidin could achieve this goal by precluding rapamycin’s
access into cytosol. This strategy greatly helped to silence the
compound activity as a chemical dimerizer until UV irradiation.
Upon cleavage of the linker between rapamycin and biotin-avidin
with UV irradiation, local release of a chemical dimerizer was
achieved, leading to subcellularly localized Rho GTPase activation.
Supporting Information Available: Synthesis, cell culturing,
transfection, confocal microscopy, and other experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Takai, Y.; Sasaki, T.; Matozaki, T. Physiol. ReV. 2001, 81, 153–208.
(2) Etienne-Manneville, S.; Hall, A. Nature 2002, 420, 629–635.
(3) Ridley, A. J.; Schwartz, M. A.; Burridge, K.; Firtel, R. A.; Ginsberg, M. H.;
Borisy, G.; Parsons, J. T.; Horwitz, A. R. Science 2003, 302, 1704–1709.
(4) Van Keymeulen, A.; Wong, K.; Knight, Z. A.; Govaerts, C.; Hahn, K. M.;
Shokat, K. M.; Bourne, H. R. J. Cell Biol. 2006, 174, 437–445.
(5) Sander, E. E.; ten Klooster, J. P.; van Delft, S.; van der Kammen, R. A.;
Collard, J. G. J. Cell Biol. 1999, 147, 1009–1021.
There are two limitations of the present approach. First, a target
location needs to be at or near the plasma membrane. Second,
molecular diffusion can affect the precise confinement of intended
effects particularly at later time points. This effect depends on
parameters associated with UV irradiation (such as size, mode
(continuous vs pulsed), and energy), diffusion coefficients of
chemical dimerizers, the FRB-FKBP dimerization complex, and
activated endogenous signaling molecules such as Rac. Further
optimization of these parameters enables a more stringent confine-
ment of signal activities. For example, smaller sized, pulsed, and
higher energy UV irradiation should help minimize the effect of
diffusion without compromising the uncaging efficiency. It also
helps to reduce diffusion of chemical dimerizers by using a denser
extracellular medium. In order to achieve slower lateral diffusion,
a transmembrane motif, instead of lipid modification, could be used
to anchor FRB to the plasma membrane.
(6) Itoh, R. E.; Kurokawa, K.; Ohba, Y.; Yoshizaki, H.; Mochizuki, N.;
Matsuda, M. Mol. Cell. Biol. 2002, 22, 6582–6591.
(7) Wong, K.; Pertz, O.; Hahn, K.; Bourne, H. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 3639–3644.
(8) Pertz, O.; Hodgson, L.; Klemke, R. L.; Hahn, K. M. Nature 2006, 440,
1069–1072.
(9) Inoue, T.; Heo, W. D.; Grimley, J. S.; Wandless, T. J.; Meyer, T. Nat.
Methods 2005, 2, 415–418.
(10) Ellis-Davies, G. C. R. Nat. Methods 2007, 4, 619–628.
(11) Kao, J. P. Y. Current Protocols in Neuroscience; John Wiley & Sons: 2006,
Chapter 6, Unit 6.20.
(12) Borchardt, A.; Liberles, S. D.; Biggar, S. R.; Crabtree, G. R.; Schreiber,
S. L. Chem. Biol. 1997, 4, 961–968.
(13) Sadovski, O.; Jaikaran, A. S. I.; Samanta, S.; Fabian, M. R.; Dowling,
R. J. O.; Sonenberg, N.; Woolley, G. A. Bioorg. Med. Chem. 2010, in
press
(14) Banaszynski, L. A.; Liu, C. W.; Wandless, T. J. J. Am. Chem. Soc. 2005,
127, 4715–4721.
(15) Aujard, I.; Benbrahim, C.; Gouget, M.; Ruel, O.; Baudin, J. B.; Neveu, P.;
Jullien, L. Chem.sEur. J. 2006, 12, 6865–6879.
(16) Chen, Y.; Nakanishi, K.; Merrill, D.; Eng, C. P.; Molnar-Kimber, K. L.;
Failli, A.; Caggiano, T. J. Bioorg. Med. Chem. Lett. 1995, 5, 1355–1358.
(17) Wu, Y. I.; Frey, D.; Lungu, O. I.; Jaehrig, A.; Schlichting, I.; Kuhlman,
B.; Hahn, K. M. Nature 2009, 461, 104–108.
Recently, Wu et al.,17 Levskaya et al.,18 and Yazawa et al.19
reported an elegant approach to activate Rac at a subcellular location
using light-sensitive plant proteins. Compared to their strategies,
our current technique is more feasible to expand the number of
target proteins, since we and others have already developed a variety
of dimerization probes that can manipulate the activity of diverse
signaling molecules at the plasma membrane.9,20-23 Our caging
strategy is readily applicable to all these molecular probes to achieve
subcellularly targeted manipulation of cell signaling, thus making
it a powerful tool to probe spatiotemporally dynamic cellular events.
(18) Levskaya, A.; Weiner, O. D.; Lim, W. A.; Voigt, C. A. Nature 2009, 461,
997–1001.
(19) Yazawa, M.; Sadaghiani, A. M.; Hsueh, B.; Dolmetsch, R. E. Nat.
Biotechnol. 2009, 27, 941–945.
(20) Schreiber, S. L.; Kapoor, T. M.; Wess, G. Chemical biology; Wiley-VCH:
Weinheim, 2007.
(21) Suh, B. C.; Inoue, T.; Meyer, T.; Hille, B. Science 2006, 314, 1454–1457.
(22) Komatsu, T.; Kukelyansky, I.; McCaffery, J. M.; Ueno, T.; Varela, L. C.;
Inoue, T. Nat. Methods 2010, 7, 206–208.
(23) Balla, T.; Szentpetery, Z.; Kim, Y. J. Physiology (Bethesda) 2009, 24,
231–244.
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