Suborganelle sensing of mitochondrial cAMP-dependent protein kinase activity

Article abstract of DOI:10.1021/ja909652q

A fluorescent sensor of protein kinase activity has been developed and used to characterize the compartmentalized location of cAMP-dependent protein kinase activity in mitochondria. The sensor functions via a phosphorylation-induced release of a quencher from a peptide-based substrate, producing a 150-fold enhancement in fluorescence. The quenching phenomenon transpires via interaction of the quencher with Arg residues positioned on the peptide substrate. Although the cAMP-dependent protein kinase is known to be present in mitochondria, the relative amount of enzyme positioned in the major compartments (outer membrane, intermembrane space, and the matrix) of the organelle is unclear. The fluorescent sensor developed in this study was used to reveal the relative matrix/intermembrane space/outer membrane (85:6:9) distribution of PKA in bovine heart mitochondria.

Full text of DOI:10.1021/ja909652q

Published on Web 04/09/2010
Suborganelle Sensing of Mitochondrial cAMP-Dependent
Protein Kinase Activity
Richard S. Agnes,† Finith Jernigan,‡ Jennifer R. Shell,‡ Vyas Sharma,*,‡ and
David S. Lawrence*,‡
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 and
the Department of Chemistry, DiVision of Medicinal Chemistry and Natural Products,
Department of Pharmacology, UniVersity of North Carolina, Chapel Hill, North Carolina 27599
Received November 13, 2009; E-mail: lawrencd@email.unc.edu
Abstract: A fluorescent sensor of protein kinase activity has been developed and used to characterize the
compartmentalized location of cAMP-dependent protein kinase activity in mitochondria. The sensor functions
via a phosphorylation-induced release of a quencher from a peptide-based substrate, producing a 150-
fold enhancement in fluorescence. The quenching phenomenon transpires via interaction of the quencher
with Arg residues positioned on the peptide substrate. Although the cAMP-dependent protein kinase is
known to be present in mitochondria, the relative amount of enzyme positioned in the major compartments
(outer membrane, intermembrane space, and the matrix) of the organelle is unclear. The fluorescent sensor
developed in this study was used to reveal the relative matrix/intermembrane space/outer membrane (85:
6:9) distribution of PKA in bovine heart mitochondria.
Protein kinases are a large enzyme family that have been
implicated in nearly every cell-based behavior, from ATP
generation to unrestrained growth and division.1 These enzymes
are linked by their ability to catalyze phosphoryl transfer from
ATP to the hydroxyl moieties of serine, threonine, and/or
tyrosine residues in proteins. A variety of factors limit protein
kinase-catalyzed phosphorylation to intended protein targets: (a)
the ability to phosphorylate serine/threonine or tyrosine, but only
rarely both aliphatic and aromatic residues, (b) differential
expression as a function of cell type, (c) recognition of specific
amino acid sequences encompassing the hydroxyl phosphoryl
acceptor moiety, and (d) localization to specific intracellular
sites. The cAMP-dependent protein kinase (PKA) exhibits many
of these attributes as a serine/threonine-specific protein kinase
with a special preference for sequences of the general form Arg-
Arg-Xaa-Ser/Thr-Xaa in protein substrates.2 In addition, PKA
is anchored to a variety of intracellular sites via coordination
to A-kinase anchoring proteins (AKAPs), and thus the biological
consequences of its action are location-dependent.3 For example,
mitochondrial PKA is implicated in the regulation of apoptosis
and ATP synthesis.4 However, as is true for protein kinases in
general, presumed intracellular PKA activity is commonly
assessed in an indirect fashion: either by the mere presence of
the enzyme (immunofluorescence or Western blots) or by the
effect of small molecule modulators, such as inhibitors, on the
phosphorylation of presumed PKA protein substrates. Unfor-
tunately, these commonly employed methods do not furnish a
direct measure of kinase activity.
Fluorescent sensors have been used to directly and continu-
ously assess kinase action.5 However, these either display a
limited dynamic range or employ fluorophores with photophysi-
cal properties (short λex/λem,6 small ε,7 low Φ) that are
incompatible (due to interference from autofluorescence) with
cells, cell lysates, or organelles. With the latter limitation in
mind, we report herein the application of a quenched fluores-
cence strategy6 to create a kinase sensor of unprecedented
dynamic range. Sensors with a large dynamic range can be used
in relatively small quantities, thereby diminishing the likelihood
of interference with ongoing biochemical processes (i.e.,
Observer Effect).8 The favorable properties associated with the
sensor have allowed us to assess the relative abundance of PKA
in the major mitochondrial microenvironments (outer membrane,
intermembrane space, and matrix).9-13
Results and Discussion
The fluororophores described in this report are coumarin
derivatives, which possess photophysical properties (Table 1)
that are readily amenable to biological applications. Three
coumarin-derivatized peptides (1-3, Figure 1) of the general
form coumarin-Aoc-GRTGRRFSYP-amide were prepared (where
(5) Lawrence, D. S.; Wang, Q. ChemBioChem 2007, 8, 373–278.
(6) Sharma, V.; Agnes, R. S.; Lawrence, D. S. J. Am. Chem. Soc. 2007,
129, 2742–2743.
† Albert Einstein College of Medicine.
‡ University of North Carolina.
(7) Shults, M. D.; Imperiali, B. J. Am. Chem. Soc. 2003, 125, 14248–
14249.
(1) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,
S. Science 2002, 298, 1912–1934.
(8) Sharma, V.; Lawrence, D. S. Angew. Chem., Int. Ed. 2009, 48, 7290–
7292.
(2) Shabb, J. B. Chem. ReV. 2001, 101, 2381–2411.
(3) Smith, F. D.; Langeberg, L. K.; Scott, J. D. Trends Biochem. Sci. 2006,
31, 316–323.
(9) Ma, Y.; Taylor, S. S. J. Biol. Chem. 2008, 283, 11743–11751.
(10) Sardanelli, A. M.; Signorile, A.; Nuzzi, R.; Rasmo, D. D.; Technikova-
Dobrova, Z.; Drahota, Z.; Occhiello, A.; Pica, A.; Papa, S. FEBS Lett.
2006, 580, 5690–5696.
(4) Feliciello, A.; Gottesman, M. E.; Avvedimento, E. V. Cell Signal.
2005, 17, 279–87.
9
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J. AM. CHEM. SOC. 2010, 132, 6075–6080 6075

A R T I C L E S
Agnes et al.
Table 1. Photophysical properties, fluorescent-fold increase, Km,
and Vmax for the PKA-catalyzed phosphorylation of sensors 1-3
(where Sensor ) Fluorophore-Aoc-GRTGRRFSYP-amide)a
sensor (λex/λem
)
fluorescent fold-increase
Km (µM)
Vmax (µmol/min·mg)
1 (420/475 nm)
2 (437/477 nm)
3 (450/490 nm)
152
150
28
2.2 ( 0.1
1.9 ( 0.1
6.2 ( 0.1
0.53 ( 0.03
0.34 ( 0.04
0.20 ( 0.09
a Kinetic properties were acquired in the presence of quencher 4 and
the 14-3-3τ domain. See “Acquisition of apparent Km and Vmax values”
in the Material and Methods section for experimental details.
Figure 2. Fluorescence change as a function of incubation time of the
PKA-catalyzed phosphorylation of sensors (A) 1, (B) 2, (C) 3, and (D) a
nonphosphorylatable Ala-for-Ser control peptide 5 (coumarin-Aoc-GRT-
GRRFAYP-amide). Experiments were conducted in the presence of
fluorescent quencher (4) and 14-3-3τ domain. See “Optimization of Enzyme-
Dependent Fold-Change” in the Material and Methods section for experi-
mental details.
Scheme 1. Protein Kinase-Catalyzed Phosphorylation of a
Fluorescently Quenched Peptide Generates a Fluorescent
Response in the Presence of the Phospho-Ser 14-3-3τ Binding
Domain
Figure 1. Structures of the coumarin derivatives 1-3 of the general form
fluorophore-Aoc-GRTGRRFSYP-amide. The fluorescent quencher Acid
Green 27 (4) was identified from a library of 47 dyes (Table S1, Supporting
Information).
Aoc ) aminooctanoic acid). These positively charged peptides
were exposed to 47 different negatively charged dyes (Sup-
porting Information, Table S1) with the expectation that complex
formation would result in the quenching of coumarin fluores-
cence. In addition, we anticipated that PKA-catalyzed phos-
phorylation of coumarin-Aoc-GRTGRRFSYP-amide would
promote association of the phosphorylated peptide with the 14-
3-3τ domain,14 thereby releasing the quenching dye and
restoring fluorescence (Scheme 1). The lead quencher dye for
all three peptides proved to be Acid Green 27 (4) and the
observed PKA-catalyzed changes in fluorescence in the presence
of 4 are: peptide 1 (152-fold), peptide 2 (150-fold), and peptide
3 (28-fold) (Table 1 and Figure 2). Given its large dynamic
range and relatively high V max (Table 1), we decided to employ
peptide 1 in all subsequent studies.
We confirmed that phosphoryl transfer from ATP to the serine
hydroxyl moiety of the peptide sensor is required for the
observed fluorescent enhancement by examining analogues of
the peptide substrate 1 and ATP. First, substitution of peptide
1 with its nonphosphorylatable Ala-for-Ser counterpart 5 fails
to elicit a change in fluorescence (Figure 2D). Second, substitu-
tion of ATP with the corresponding thio-derivative, ATP(γ)S,
dramatically reduces the rate of fluorescence change (Supporting
Information, Figure S1). ATP(γ)S is known to serve as a weak
thiophosphoryl donor in protein kinase-catalyzed reactions.15,16
Both 14-3-3τ and the fluorescent quencher, Acid Green 27
(4), are required for the large phosphorylation-induced fluores-
cence yield. In the absence of 14-3-3τ (but with quencher 4
present), no change in fluorescence is observed (Supporting
Information, Figure S2). Furthermore, there exists a direct
correlation between the relative amount of 14-3-3τ present and
the observed phosphorylation-dependent fluorescent enhance-
ment (Supporting Information, Figure S2). Surprisingly, in the
absence of quencher 4 (but with 14-3-3τ present), there is a
50% decrease in observed fluorescence (Supporting Information,
Figure S3). One possible explanation for the unexpected
fluorescence decrease is the likely orientation of the 14-3-3τ-
bound phosphopeptide 1 based on a previously described crystal
(11) Chen, Q.; Lin, R. Y.; Rubin, C. S. J. Biol. Chem. 1997, 272, 15247–
15257.
(12) Schwoch, G.; Trinczek, B.; Bode, C. Biochem. J. 1990, 270, 181–
188.
(13) Ryu, H.; Lee, J.; Impey, S.; Ratan, R. R.; Ferrante, R. J. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 13915–13920.
(14) Yaffe, M. B.; Rittinger, K.; Volinia, S.; Caron, P. R.; Aitken, A.;
Leffers, H.; Gamblin, S. J.; Smerdon, S. J.; Cantley, L. C. Cell 1997,
91, 961–971.
(15) Zou, K.; Cheley, S.; Givens, R. S.; Bayley, H. J. Am. Chem. Soc.
2002, 124, 8220–8229.
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A. E.; Welsh, M. J. Cell 1991, 67, 775–784.
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A R T I C L E S
Table 2. KD Values of Sensor 1 and Various Ala-for-Arg
Analogues (6-12) of Sensor 1, with the Fluorescent Quencher 4a
peptide
peptide sequence
KD (µM)
1
6
7
8
Cou-Aoc-GRTGRRFSYP-amide
Cou-Aoc-GATGRRFSYP-amide
Cou-Aoc-GRTGARFSYP-amide
Cou-Aoc-GRTGRAFSYP-amide
Cou-Aoc-GATGRAFSYP-amide
Cou-Aoc-GATGARFSYP-amide
Cou-Aoc-GRTGAAFSYP-amide
Cou-Aoc-GATGAAFSYP-amide
0.04 ( 0.07
1.6 ( 0.3
2.7 ( 0.6
1.8 ( 0.2
130 ( 80
130 ( 65
130 ( 70
>200
9
10
11
12
a See “Acquisition of Apparent KD Values for 4 with Peptides 1 and
6-12” in the Material and Methods section for experimental details.
Figure 4. Reaction rate (nM of phosphopeptide formation/min) as a
function of PKA concentration (pM).
Figure 5. Strategy for assessing PKA activity on the outer membrane (blue),
in the intermembrane space (red), and in the matrix (yellow). PKA activity
of intact mitochondria (A) is due to enzyme present on the outer membrane
and in the intermembrane space. Trypsinized (i) mitochondria (B) lack outer
membrane proteins and thus only intermembrane space PKA is present.
Sonicated (ii) mitochondria (C) furnishes enzyme from all three compart-
ments and thus represents total mitochondrial PKA.
Figure 3. Fluorescence change as a function of mole fraction of sensor 1.
See “Job Plot for Determination of Stoichiometry” in the Material and
Methods section for experimental details.
of initial rate versus PKA concentration (Figure 4). Figure 4
provides a standard curve for assessing “PKA activity equiva-
lents” in biological systems.
structure.17 The N-terminus of the peptide, when complexed
with the 14-3-3τ domain, is positioned adjacent to two tryp-
tophan residues (Supporting Information, Figure S4). We,18 as
well as others,19 have shown that tryptophan can serve as a
fluorescent quencher. 14-3-3τ domain-mediated quenching of
coumarin-phosphopeptide fluorescence is intriguing since it
suggests that removal of the tryptophan residues in the 14-3-3τ
domain could furnish fluorescence fold changes even larger than
those observed in this study.
We examined the underlying assumption that the negatively
charged quencher 4 engages the positively charged peptide
substrate via electrostatic interactions. A small library of Arg-
to-Ala substituted peptide analogues were prepared and KD
values were acquired for each of these with Acid Green 27,
using fluorescence quenching as an indicator of complex
formation. As is clear from Table 2, the quenching phenomenon
is Arg residue-dependent, with the apparent KD displaying an
approximate 2 orders of magnitude loss for every Arg replaced
by an Ala. The effect is independent of the site of the Ala-for-
Arg substitution (cf. 6 vs 7 vs 8), implying a diffuse electrostatic
interaction between the negatively charged dye and the peptide
substrate. Job plot analysis revealed the formation of a 1:1
complex between 1 and 4 (Figure 3).
Although the indispensable nature of the PKA signaling
pathway in mitochondrial physiology is beyond dispute,4 its
suborganelle location within the mitochondria (i.e., matrix,
intermembrane space, and/or on the outer membrane oriented
toward the cytoplasm; Figure 5) is enigmatic. Orr and colleagues
demonstrated (via electron microscopy) that type-II PKA is
primarily associated with the outer membrane of mitochondria
in male germ cells.20 Indeed, a major mitochondrial A-kinase
anchoring protein (AKAP121 in mice and AKAP149 in
humans), and its splice variants, position PKA on the cytoplas-
mic face of the outer membrane.11 Proteolysis of this AKAP21,22
appears to promote apoptosis by releasing PKA from the outer
membrane, which is known to promote antiapoptotic behavior.23,24
By contrast, Schwoch et al showed (via electron microscopy)
that both types-I and -II PKA are primarily associated with the
mitochondrial matrix/inner membrane in mitochondria isolated
from a wide variety of organs.12 Matrix localization of PKA
has also been observed in neuronal mitochondria.13 Papa and
his colleagues found that PKA is associated with the inner
membrane of bovine heart mitochondria.25 Furthermore, these
(20) Lieberman, S. J.; Wasco, W.; MacLeod, J.; Satir, P.; Orr, G. A. J. Cell
Biol. 1988, 107, 1809–1816.
(21) Yoo, H.; Cha, H. J.; Lee, J.; Yu, E. O.; Bae, S.; Jung, J. H.; Sohn, I.;
Lee, S. J.; Yang, K. H.; Woo, S. H.; Seo, S. K.; Park, I. C.; Kim,
C. S.; Jin, Y. W.; Ahn, S. K. Oncol. Rep. 2008, 19, 1577–1582.
(22) Carlucci, A.; Adornetto, A.; Scorziello, A.; Viggiano, D.; Foca, M.;
Cuomo, O.; Annunziato, L.; Gottesman, M.; Feliciello, A. EMBO J.
2008, 27, 1073–1084.
The large fluorescent dynamic range of the PKA-catalyzed
phosphorylation of peptide 1 furnishes a sensitive measure of
kinase activity. The catalytic activity of PKA at a concentration
as low as 160 pM can be detected as demonstrated by the plot
(23) Affaitati, A.; Cardone, L.; de Cristofaro, T.; Carlucci, A.; Ginsberg,
M. D.; Varrone, S.; Gottesman, M. E.; Avvedimento, E. V.; Feliciello,
A. J. Biol. Chem. 2003, 278, 4286–4294.
(17) Rittinger, K.; Budman, J.; Xu, J.; Volinia, S.; Cantley, L. C.; Smerdon,
S. J.; Gamblin, S. J.; Yaffe, M. B. Mol. Cell 1999, 4, 153–166.
(18) Lee, H. M.; Priestman, M. A.; Lawrence, D. S. J. Am. Chem. Soc.
2010, 132, 1446–1447.
(24) Harada, H.; Becknell, B.; Wilm, M.; Mann, M.; Huang, L. J.; Taylor,
S. S.; Scott, J. D.; Korsmeyer, S. J. Mol. Cell 1999, 3, 413–422.
(25) Papa, S.; Sardanelli, A. M.; Scacco, S.; Technikova-Dobrova, Z. FEBS
Lett. 1999, 444, 245–249.
(19) Doose, S.; Neuweiler, H.; Sauer, M. ChemPhysChem 2005, 6, 2277–
2285.
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Agnes et al.
investigators reported a detailed electron microscopic analysis
of rat heart mitochondria and concluded that more than 90% of
all mitochondrial-bound PKA is present in the inner mitochon-
drial compartment.10 However, an additional complication, not
described by these studies, is the enzymatic reach of PKA. In
particular, the majority of mitochondrial proteins are nuclear
encoded and thus must be imported from the cytoplasm.
Consequently, although a variety of mitochondrial proteins are
phosphorylated in a cAMP-dependent fashion, they may suffer
PKA-catalyzed phosphorylation during import (e.g., at the outer
membrane) and thus prior to being embedded within their final
destination (e.g., in the matrix).25 In short, the mitochondrial
location of known PKA substrates is not necessarily a valid
indicator of microenvironmental PKA activity. Finally, the active
form of PKA (C subunit), which is released from the inactive
holoenzyme (R2C2) upon exposure to cAMP, can diffuse through
membranes.26 Therefore, the suborganelle location of the
holoenzyme (as determined by electron microscopy) does not
necessarily recapitulate the location of the active form of the
enzyme. With these factors in mind, we turned our attention to
the suborganelle analysis of cAMP-activated PKA in isolated
bovine heart mitochondria.
Perhaps the most obvious approach for assessing PKA activity
as a function of mitochondrial microenvironment is purification
of the enzyme from the outer membrane, the intermembrane
space, and the matrix. However, such a strategy is not practical
for both structural and technological reasons. The holoenzyme
resides as an AKAP-bound membrane-associated species on the
outer membrane and likely, in an analogous AKAP-dependent
fashion, at the inner membrane. Unfortunately, even if it were
feasible to isolate and purify the outer and inner membrane
components, it would not be clear which direction the enzyme
is facing in the intact mitochondrion (i.e., an outer membrane-
bound species could be oriented outward toward the cytoplasm
or inward toward the intermembrane space). An alternative
approach might involve the cAMP-induced release of the C
subunit and its subsequent isolation from the extra-mitochon-
drial, intermembrane space, and matrix regions. However, as
demonstrated with bovine heart mitochondria, the standard
technique used to expose the contents of the intermembrane
space (digitonin treatment) results in some disruption of the
matrix as well, leading to contamination of the intermembrane
space contents with components from the matrix.27 Therefore,
we resorted to the strategy depicted in Figure 5.
Figure 6. Assessment of mitochondrial purity and extent of trypsinolysis
by Western blot analysis. (A) The mitochondrial preparation was examined
for the presence of ER (calnexin), plasma membrane (Na+/K+-ATPase),
and cytoplasmic (glyceraldehyde phosphate dehydrogenase) proteins. A
minimal amount of cytoplasmic contamination is observed. (B) Trypsin
digestion of intact mitochondria. Untreated mitochondria (lane 1) and
mitochondria treated with trypsin for 1 h at 37 °C (lane 2) were analyzed
by Western blot for Hsp 60 and Tom 20, matrix and outer membrane
markers, respectively, and the PKA catalytic subunit. Complete loss of Tom
20 upon trypsin exposure implies extensive trypsinolysis of the outer
membrane surface. Complete retention of Hsp 60 implies that the mito-
chondrial matrix is preserved upon exposure to trypsin.
of externally added cAMP29). This approach avoids the use of
digitonin and thus should prevent contamination by the unin-
tended release of matrix contents. In short, cAMP-treated
mitochondria should furnish outer membrane and inter mem-
brane space PKA activity. Furthermore, since trypsin digests
only outward-facing outer membrane mitochondrial proteins,9
we expected that cAMP-exposed trypsin-treated mitochondria
should provide only intermembrane space PKA activity (Figure
5). Finally, mitochondrial structural integrity is completely
disrupted by sonication, and thus mitochondria treated in this
fashion should yield, upon cAMP exposure, total mitochondrial
PKA activity. These three activities can then be used to assign
relative PKA activity to the three separate mitochondrial
microenvironments.
The purity of mitochondria isolated from bovine heart was
assessed via Western blot analysis using antibodies against
proteins that are localized to mitochondrial (cytochrome C) and
nonmitochondrial sites, including the endoplasmic reticulum
(calnexin), the plasma membrane (Na+/K+ ATPase), and the
cytosol (GAPDH) (Figure 6A). The results demonstrate that the
mitochondrial preparation is not contaminated with proteins from
other membranes or the ER and displays only minimal
contamination from the cytoplasm. Mitochondrial structural
integrity (intactness) was assessed as previously described30,31
and found to be greater than 90% (data not shown).
Intact mitochondria, upon exposure to cAMP, exhibit an
initial rate of PKA activity equivalent to 29 ( 4 pg PKA/µg
mitochondria. By contrast, in the absence of cAMP, phospho-
rylation activity is minimal (Supporting Information, Figure S5);
demonstrating that sensor 1 is phosphorylated in a cAMP-
dependent fashion. Indeed, previous studies have shown that
active-site directed sequences, similar to the sequence employed
in sensor 1, are highly selective for PKA.32-34 Finally, we have
found that H-89, a PKA inhibitor, blocks the phosphorylation
of sensor 1 by mitochondrial preparations (Supporting Informa-
We reasoned that exposure of intact mitochondria to cAMP
should activate PKA located on the outer membrane as well as
any PKA present in the intermembrane space (i.e., inner leaflet
of the outer membrane and/or outer leaflet of the inner
membrane). The mitochondrial outer membrane contains a
channel-forming protein-based complex (porin; also known as
the voltage-dependent anion channel) that allows small mol-
ecules (<5000 molecular weight) to passively diffuse between
the extra-mitochondrial environment and the intermembrane
space.28 We expected that, in addition to cAMP, our PKA sensor
1 and quencher 4 should be able to freely migrate into and out
of the intermembrane space and thus report any PKA activity
(by contrast, the inner membrane blocks access to the matrix
(29) Acin-Perez, R.; Salazar, E.; Kamenetsky, M.; Buck, J.; Levin, L. R.;
Manfredi, G. Cell Metab. 2009, 9, 265–276.
(30) Wharton, D. C.; Tzagoloff, A. Methods Enzymol. 1967, 10, 245–250.
(31) Rice, J. E.; Lindsay, J. G. In Subcellular Fractionation; Graham, J. M.,
Rickwood, D., Eds.; IRL Press: Oxford, England, 1997; pp 107-142.
(26) Harootunian, A. T.; Adams, S. R.; Wen, W.; Meinkoth, J. L.; Taylor,
S. S.; Tsien, R. Y. Mol. Biol. Cell 1993, 4, 993–1002.
(27) Burnette, B.; Batra, P. P. Anal. Biochem. 1985, 145, 80–86.
(28) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P.
Molecular Biology of the Cell, Reference ed.; Garland Science: New
York, 2007; pp 818.
(32) Su, J.; Bringer, M. R.; Ismagilov, R. F.; Mrksich, M. J. Am. Chem.
Soc. 2005, 127, 7280–7281.
(33) Min, D. H.; Su, J.; Mrksich, M. Angew. Chem., Int. Ed. 2004, 43,
5973–5977.
(34) He, Y.; Yeung, E. S. Electrophoresis 2003, 24, 101–108.
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A R T I C L E S
10-carboxylic acid], and Atto 425-NHS ester were purchased from
Sigma-Aldrich. Fmoc-aminooctanoic acid (Fmoc-Aoc-OH) was
purchased from Advanced ChemTech (Louisville, KY, U.S.A.).
Bovine heart mitochondria was purchased from MitoSciences, and
trypsin (sequencing grade) and PKA were purchased from Promega.
The antibodies against the PKA catalytic subunit, Tom 20, and
Hsp60 were purchased from BD Biosciences, and the goat
antimouse secondary antibody was purchased from Santa Cruz
Biotechnology. Total mitochondrial protein was quantified using
the BCA protein assay (Pierce). Immunoblots were performed using
Snap i.d. (Millipore), and visualized using an AlphaInnotech
FluorChem FC2 imager. The intactness of the isolated mitochondria
was assessed via a previously described protocol.30,31 PKA murine
catalytic subunit (cat.) plasmid and the GST-14-3-3τ plasmid were
generous gifts from Dr. Susan Taylor and Dr. Alistair Aitken,
respectively.
tion, Figure S6). These results are consistent with the notion
that peptide 1 serves as a selective PKA sensor.
As noted above (Figure 5), we expected that the PKA activity
associated with intact mitochondria would be a combination of
enzyme present on the outer membrane and enzyme oriented
into the intermembrane space. We addressed this possibility by
treating mitochondria with trypsin, which should selectively
hydrolyze the outer membrane proteins exposed to the external
milieu (Figure 6B), but not hydrolyze matrix- or intermembrane
space-embedded proteins. Indeed, an outer membrane marker
(TOM 20) is completely digested in trypsin-treated mitochon-
dria, whereas a matrix marker (Hsp 60) is unperturbed. Previous
studies with trypsin-exposed mitochondria demonstrated that an
intermembrane space marker is protected against proteolysis as
well.9 Subsequent analysis of PKA activity (cAMP exposure)
in trypsin-treated mitochondria revealed a drop in PKA activity
from 24 ( 4 pg PKA/µg mitochondria to 10 ( 1 pg PKA/µg
mitochondria, implying a 1:1.4 ratio of intermembrane space:
outer membrane (external) PKA. Finally, mitochondria were
sonicated to completely disrupt their structural integrity, thereby
exposing all mitochondria-associated PKA, including any
matrix-embedded enzyme. Total mitochondrial PKA activity
(cAMP treatment) is equivalent to 165 ( 24 pg PKA/µg
mitochondria, approximately 5.7 times greater than that observed
with intact mitochondria. These values suggest that the relative
distribution of PKA activity in the matrix (165 ( 24 pg PKA/
µg mitochondria - 24 ( 4 pg PKA/µg mitochondria ) 141 pg
PKA/µg mitochondria), the intermembrane space (10 ( 1 pg
PKA/µg mitochondria), and the outer membrane (24 ( 4 pg
PKA/µg mitochondria - 10 ( 1 pg PKA/µg mitochondria )
14 pg PKA/µg mitochondria) in bovine heart mitochondria is
85:6:9, respectively. The latter compares favorably with the
electron microscopy work of Papa and colleagues,10 who
reported that 90% of the PKA present in mitochondria is found
the matrix and the intermembrane space. However, our analysis
could be complicated by endogenous protein phosphatases if
they are present in different amounts in the three distinct
mitochondrial compartments. Consequently, an analogous series
of experiments were conducted in the presence of a phosphatase
inhibitor cocktail. The experimentally determined ratio (79:8:
13) of matrix/intermembrane space/outer membrane PKA activ-
ity corresponds to that acquired in the absence of phosphatase
inhibitors.
Synthesis of Fluorophore-Labeled PKA Substrates. Peptides
were synthesized using standard Fmoc solid-phase synthesis on a
Prelude peptide synthesizer (Protein Technologies Inc., Tucson, AZ,
U.S.A.). Novasyn TGR resin was swelled for 30 min in dichlo-
romethane (DCM) before synthesis. Amino acids were then
sequentially coupled using 5.0 equiv of amino acid, 4.9 equiv of
HCTU, 20 equiv of diisopropylethylamine (DIPEA) in N,N-
dimethylformamide (DMF) (2 × 5 min) followed by DMF wash
(6 × 30 s). The Fmoc-protecting group was removed using 20%
piperidine in DMF (2 × 2.5 min) followed by a DMF wash (6 ×
30 s). The free N-terminal amine was used for subsequent on-resin
fluorophore labeling. Fluorescent dyes diethylcoumarin and Cou-
marin 343 were coupled to the N-terminal amine using 5.0 equiv
of fluorophore, 4.9 equiv of HCTU, 20 equiv of DIPEA in DMF
(1× 60 min). Atto425-NHS ester was coupled using 1.0 equiv of
dye and 20 equiv of DIPEA in DMF (1 × 60 min). The resin was
washed (3× DMF, IPA, DCM) and then cleaved and deprotected
using a 95:2.5:2.5 trifluoroacetic acid (TFA)/H2O/triisopropylsilane
(TIPS). The peptides were isolated via filtration, precipitated with
ice-cold ether, and centrifuged to isolate the precipitate. The
precipitates were air-dried, dissolved in DMSO, and purified using
HPLC (3% to 40% acetonitrile gradient against water with 0.1%
TFA over 40 min). The peak corresponding to the peptide was
collected, freeze-dried, and characterized by electrospray ionization
mass spectrometry: 1 Cou-Aoc-GRTGRRFSYP-amide [Exact Mass
calculated: 1579.84, found: 1580.84 (M + H)+], 2 Atto425-Aoc-
GRTGRRFSYP-amide [Exact Mass calculated: 1719.98, found:
1720.97 (M + H)+], 3 Cou343-Aoc-GRTGRRFSYP-amide [Exact
Mass calculated: 1603.82, found: 1604.85 (M + H)+], 5 Cou-Aoc-
GRTGRRFAYP-amide [Exact Mass calculated: 1562.8, found:
782.6 (M + 2H)2+, 522.2 (M + 3H)3+], 6 Cou-Aoc-GATGRRF-
SYP-amide [Exact Mass calculated: 1494.69, found: 1495.80 (M
+ H)+], 7 Cou-Aoc-GRTGARFSYP-amide [Exact Mass calculated:
1494.69, found: 1495.77 (M + H)+], 8 Cou-Aoc-GRTGRAFSYP-
amide [Exact Mass calculated: 1495.69, found: 1495.78 (M + H)+],
9 Cou-Aoc-GATGRAFSYP-amide [Exact Mass calculated: 1409.58,
found: 1409.72 (M + H)+], 10 Cou-Aoc-GATGARFSYP-amide
[Exact Mass calculated: 1409.58, found: 1409.73 (M + H)+], 11
Cou-Aoc-GRTGAAFSYP-amide [Exact Mass calculated: 1409.58,
found: 1409.72 (M + H)+], and 12 Cou-Aoc-GATGAAFSYP-
amide [Exact Mass calculated: 1323.6, found: 1324.5 (M + H)+].
Identification of Lead Quencher Dye 4. The concentration of
peptide 1 was adjusted using a molar excitation coefficient of 60,000
M-1 cm-1 at 430 nm. GST-tagged 14-3-3τ (purified to a single
band at 56 KDa on 12.5% SDS PAGE) was dialyzed four times in
50 mM Tris pH 7.5, prior to use, and its concentration was
determined using the Bradford assay. Concentrations of each of
the 47 assembled dyes (See Table S1, Supporting Information) were
adjusted on the bais of weight. PKA enzyme (2.08 mg/mL) was
purchased from Promega. Peptide 1 (1 µM) was incubated with 10
µM GST-tagged 14-3-3τ, 1 mM ATP, 2 mM DTT, 5 mM MgCl2,
and 50 mM Tris-HCl at pH 7.5, in a quartz 96-well plate (Hellma).
Each dye was added to a separate well at 5 µM. PKA (10 nM) was
In summary, we have developed a protein kinase sensing
system with a robust dynamic range and used it to characterize
the compartmentalized location of PKA activity in mitochondria.
Given the important role of PKA in mediating the dynamics of
mitochondrial biochemistry, the ability to monitor protein kinase
activity should prove useful in assessing the mitochondrial
response from both healthy individuals and from patients with
mitochondrial-based disorders.35
Materials and Methods
Materials. General reagents and solvents were purchased from
Fisher or Sigma-Aldrich. Novasyn TGR Resin and all natural Fmoc-
protected amino acids were purchased from EMD Biosciences Inc.
HCTU [1H-benzotriazolium-1-[bis(dimethylamino)methylene]-5-
chloro-hexafluorophosphate (1),3-oxide] was purchased from Pep-
tides International (Louisville, KY, U.S.A.). Fluorescent dyes (7-
(diethylamino)coumarin-3-carboxylic acid, Coumarin 343 [11-oxo-
2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-
(35) Carlucci, A.; Lignitto, L.; Feliciello, A. Trends Cell. Biol. 2008, 18,
604–613.
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A R T I C L E S
Agnes et al.
added to each well, and the enzyme-dependent increase in
fluorescence (λex 420 nm, λem 475 nm) was determined with a plate
reader (Molecular Devices Spectra Max Gemini EM). Dyes D1,
D6, D18, D33, and D39 showed >2-fold enhancements in
fluorescence.
Phosphorylation of Sensor 1 and Dephosphorylation of
Phospho Sensor Peptide. PKA-catalyzed phosphorylation of sensor
1 was performed using the conditions in “Optimization of Enzyme-
Dependent Fold-Change” described below. The mass of the
resulting phosphorylated peptide was found to be 830.6 (M +
2H)2+, 554.1 (M + 3H)3+ (Exact Mass calculated 1658.8). The
phosphorylated lead peptide was dephosphorylated using Protein
Phosphatase 1 (NEB) in the presence of 50 mM HEPES pH 7.5,
100 mM NaCl, 2 mM DTT, 0.01% Brij 35, and 1 mM MnCl2. The
mass of the resulting dephosphorylated peptide was found to be
789.5 (M + 2H)2+ (Exact Mass calculated 1577.8).
Acquisition of Apparent Km and Vmax Values. Phosphorylation-
dependent increase in coumarin fluorescence intensity of peptides
1-3 was monitored on a Photon Technology QM-1 spectrofluo-
rimeter at 30 °C. Standard curves were used to correlate fluores-
cence intensity with concentration of product formed. For generating
the standard curve, various substrate concentrations were incubated,
in duplicate, with 5 nM PKA at 25 °C over 30 h (in the presence
of 320 µM dye, 20 µM 14-3-3τ, 1 mM ATP, 5 mM MgCl2, 2 mM
DTT, and 50 mM Tris HCl pH 7.5). The fluorescence intensity
after complete phosphorylation was plotted against concentration,
and parameters obtained from a linear regression of the data were
used to convert the fluorescence intensity to product concentration.
For determining the initial velocities, different concentrations of
the peptide substrate was equilibrated with 20 µM 14-3-3τ, 1 mM
ATP, 5 mM MgCl2, 2 mM DTT, in 50 mM Tris buffer pH 7.5, for
10 min. PKA (5 nM) was added, and the reaction progress curves
were obtained. Reaction rates were determined from the slope under
conditions where 5-8% substrate had been converted to product
in duplicate (typically from 200 to 300 s). The resulting slopes
(initial velocity, Vo) for each of the progress curves were plotted
versus the concentration of substrate. A nonlinear regression analysis
(SigmaPlot version 8.02 software) was used to fit the data to the
rectangular hyperbola model.
mM Tris HCl pH 7.5 buffer. Fluorescence data (λex 420 nm, λem
475 nm) was acquired using a Photon Technology QM-4 spectrof-
luorimeter at 30 °C. Correction for the inner filter effect was made
as previously reported.36 Molar absorbtivities (λex 420 nm, λem 475
nm) were calculated from single absorbance spectra at [4] ) 16
µM. After correcting for the inner filter effect, the percentage of
quench was plotted against the concentration of the dye. Concentra-
tion of the dye-peptide complex was determined on the basis of
the change in fluorescence emission intensity. The data were fit
using the nonlinear regression mode of SigmaPlot ver.8.02. The
calculated KD values are reported in Table 2.
Job Plot for Determination of Stoichiometry. Fluorescence
emission (λex 420 nm, λem 475 nm) of varying concentrations of
peptide 1 (0.2, 0.6, 1.0, 1.4, 1.8 µM) and dye 4 (65% purity) (1.8,
1.4, 1, 0.6, 0.2 µM) at a fixed total concentration of 2 µM was
acquired in duplicate, on a Photon Technology QM-4 spectrofluo-
rimeter at 30 °C (Figure 3).
Mitochondria-Based Experiments. A standard curve for PKA
concentration versus activity was generated using 1 µM peptide 1,
32 µM dye 4, 10 µM 14-3-3τ, 1 mM ATP, 5 mM MgCl2, 2 mM
DTT, 1 mM cAMP, in 50 mM Tris HCl at pH 7.5. Assays were
conducted in duplicate at two concentrations of total mitochondria
protein (14 µg and 55 µg) using intact mitochondria (MitoSciences),
trypsin-treated mitochondria (mitochondria samples incubated with
1:50 trypsin/total mitochondrial protein for 1 h at 37 °C, followed
by treatment with 20-fold excess of soybean trypsin inhibitor) and
sonicated mitochondria, [mitochondria sonicated (Ultrasonic Pro-
cessor, Tekmar, Cincinnati, OH, U.S.A.) for 30 s on ice]. Initial
rates were converted into pg of active PKA per µg of mitochondria
using the standard curve. Rates in the presence of Ser/Thr
phosphatase inhibitor cocktail (P2850 Sigma) were acquired at the
recommended 1:100 dilution.
Trypsin Treatment of Mitochondria. Mitochondria were
incubated with trypsin (1:50 trypsin/total mitochondrial protein) for
1 h at 37 °C followed by treatment with a 20-fold excess of soybean
trypsin inhibitor. The outcome of the trypsin digestion was validated
by Western blot using an outer membrane marker, Tom 20, and a
matrix marker, Hsp60.
Western Blot Analyses. Twenty micrograms of total protein was
loaded onto 4-12% bis-tris-polyacrylamide gels, separated by
electrophoresis, and electroblotted onto PVDF membranes. The
membranes were then blocked in 0.5% nonfat dry milk followed
by incubation with the appropriate primary antibody (C subunit of
PKA 1:1000, Tom 20 1:1000, and Hsp60 1:5000) for 10 min. The
membranes were then washed three times with 0.1% Tween-20 in
PBS followed by incubation with a goat antimouse secondary
antibody conjugated to horseradish peroxidase (1:2000) for 10 min.
The membranes were washed with 0.1% Tween-20 in PBS (3×),
PBS (3×), and 0.5% NaCl (3×) and detection of horseradish
peroxidase performed using the ECL Plus reagent (GE).
Optimization of Enzyme-Dependent Fold-Change. Peptide 1
(1 µM) was incubated with 1 mM ATP, 2 mM DTT, 5 mM MgCl2,
50 mM Tris-HCl at pH 7.5, in different wells of a quartz 96-well
plate. To each well, were added systematically varied concentrations
of dye 4 (0-600 µM) and GST-tagged 14-3-3τ (0-40 µM)
followed by 10 nM PKA. Enzyme-dependent enhancement in
fluorescence (λex 420 nm, λem 475 nm) was determined with a plate
reader (Molecular Devices Spectra Max Gemini EM). The maxi-
mum fluorescence increase was observed with 320 µM dye 4 and
20 µM GST-tagged 14-3-3τ. These optimized conditions were used
to assay peptides 1-3 using a Photon Technology QM-4 spectrof-
luorimeter. The enzyme-dependent fold changes obtained under
these conditions are reported in Table 1 and are shown in Figure
2.
Acknowledgment. We thank the NIH (CA79954 and NS048406)
for financial support.
Acquisition of Apparent KD Values for 4 with Peptides 1
and 6-12. Varying concentrations of 4 ranging from 0.02 to 200
µM, were added to 1 µM of the coumarin-labeled peptides in 100
Supporting Information Available: This material is available
free of charge via the Internet at http://pubs.acs.org.
(36) Levine, R. L. Clin. Chem. 1977, 23, 2292–2301.
JA909652Q
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