(Received for publication, August 29, 1994; and in revised form, November 11, 1994)
From the
In neurons cAMP-dependent protein kinase II (PKAII
) is
sequestered in the dendritic cytoskeleton because the regulatory
subunit (RII
) of the enzyme is tightly bound by A Kinase Anchor Proteins (AKAPs). The prototypic neuronal
anchor protein AKAP75 has a COOH-terminal 22-residue RII
binding
(tethering) site. A key feature of the tethering site is that several
amino acids with large aliphatic side chains mediate the high-affinity
binding of RII
.
Mutagenesis, recombinant protein expression,
and physicochemical characterization were used to investigate the
structural basis for the homodimerization and AKAP75 binding activities
of RII. Several crucial residues are located in an
NH
-terminal region that encompasses amino acids
13-36. Substitution of Ala for Leu
or Phe
generates monomeric RII
subunits that cannot bind AKAP75.
The results are not due to general misfolding since mutant RII
monomers bind cAMP and inhibit the catalytic subunit of PKAII
with
the same affinity and efficacy as wild-type RII
dimers. Moreover,
substitution of Ala for Leu
, Val
,
Leu
, Phe
, Leu
, or Leu
and replacement of Leu
with Ile or Val did not
impair the dimerization reaction. Evidently, large hydrophobic side
chains of Leu
and Phe
play pivotal roles in
stabilizing RII
-RII
interactions. A secondary consequence of
destabilizing RII
dimers is the loss of intracellular
targeting/anchoring capacity because monomers fail to bind AKAP75.
Other NH
-terminal residues directly modulate the affinity
of RII
dimers for the AKAP75 tethering site. Replacement of
Val
-Leu
with Ala-Ala produced a dimeric
RII
protein that binds AKAP75
4% as avidly as wild-type
RII
. It is possible that the aliphatic side chains of Val
and Leu
interact with the essential Leu and Ile
residues in the AKAP75 tethering region.
Cyclic AMP-dependent protein kinase II (PKAII
) (
)is the predominant PKA isoform and principal mediator of
cAMP action in the central nervous system (reviewed in (1, 2, 3) ). More than 70% of PKAII
is
tethered to specific sites in the cytoskeleton or organelles of neurons
via the high-affinity binding of RII
by A Kinase Anchor Proteins
(AKAPs)(1, 4, 5, 6) . Bovine AKAP75,
human AKAP79, and rat AKAP150 are novel, homologous proteins that
contain a conserved 22-residue domain near the carboxyl terminus that
mediates the binding of RII
(7, 8, 9) .
RII
is also bound at this site, whereas RI
and RI
are
not ligands. Several amino acid residues with long aliphatic side
chains (Leu
, Leu
, Ile
,
Leu
, see (6) for sequence) contribute to a
hydrophobic environment that stabilizes AKAP75
RII
complexes(9) . Contributions of hydrophobic residues to the
RII
binding activity are dependent on both the size of the side
chains and their position in the primary sequence(9) .
Recent studies indicate that AKAPs direct signals carried by cAMP to
specific effector sites. In rat forebrain neurons AKAP150 and RII
(PKAII
) are enriched and co-localized along microtubules in the
cytoskeleton of dendrites(5) . In cultured rat hippocampal
neurons an AKAP
RII
(PKAII
) complex is necessary for the
maintenance of kainate-stimulated ion currents through a
glutamate-gated channel (10, 11, 12) . In
addition, overexpression of AKAP75 in a model cell system depleted
RII
, RII
, and catalytic subunits of PKA from the cytoplasm
and sequestered PKAII isoforms in the cytoskeleton(13) .
The
cited studies support a modification of a model originally proposed in
Glantz et al.(5) and Hirsch et
al.(6) : AKAPs place PKAII in proximity with (a) neurotransmitter-activated adenylate cyclase in the plasma
membrane, (b) plasma membrane substrates such as
neurotransmitter receptors (for desensitization) and ion channels, and (c) substrates in the cytoskeleton such as
microtubule-associated proteins. This arrangement generates target
sites for the reception and propagation of signals carried by cAMP.
Moreover, the scheme permits the modulation of neuronal function over
longer distances via alterations in the cytoskeleton and the electrical
properties of synapses.
Residues 1-50 in RII and RII
subunits are crucial for binding with AKAP75 and microtubule-associated
protein 2, an RII
selective binding
protein(14, 15, 16) . This
NH
-terminal region of RII subunits also mediates
homodimerization(14, 17) . At present, little is known
about individual amino acid residues at the NH
terminus of
RII isoforms that promote dimerization and/or the tethering of RII
subunits with AKAPs. Central questions are: which amino acids are
essential for dimerization of RII
or RII
? Which residues are
crucial for high-affinity binding with AKAPs? Are the AKAP binding and
dimerization regions identical, or are they overlapping domains that
may be partially resolved?
These questions were investigated by
pursuing the observation that hydrophobic interactions are important
factors in establishing stable AKAP75RII
complexes(9) . We now report that certain amino acids with
large aliphatic side chains and an aromatic residue (Phe
)
play prominent roles in linking PKAII
to AKAPs. Several of these
residues are also involved in RII
dimerization. However, some
mutated RII
dimers bind AKAPs poorly, whereas one monomeric mutant
RII
is an effective competitor for the binding site on AKAP75.
AKAP binding and RII
dimerization domains may be partially
overlapping structures composed of both shared and distinct elements.
Wild-type and mutant cDNAs (474
base pairs) encoding residues 1-158 of RII (designated
RII
-N158) were excised from pGRII
by digestion with NdeI and BamHI and cloned into the expression plasmid
pET-14b (Novagen) which was digested with the same restriction enzymes.
The partial RII
cDNA is preceded by plasmid DNA that encodes an
initiator Met and 19 additional amino acids (MGSSHHHHHHSSGLVPRGSH).
Transcription of the chimeric gene is governed by a promoter sequence
for bacteriophage T7 RNA polymerase.
Recombinant pET-14b plasmids
were introduced into Escherichia coli BL21 (DE3)(2) .
The host bacterium contains a chromosomal copy of the phage T7 RNA
polymerase gene under the control of the lac promoter(21) . Cultures (200 ml) of transformed E.
coli BL21 were grown to A = 0.8.
Subsequently, isopropyl-1-thio-
-D-galactopyranoside was
added to a final concentration of 1 mM and the incubation was
continued for 2 h at 37 °C. Next, bacteria were harvested by
centrifugation at 8,000
g for 10 min at 4 °C. All
further operations were performed at 0-4 °C. Pelleted cells
were washed once with 20 ml of 20 mM potassium phosphate
buffer, pH 7.0. The bacteria were then resuspended in 3 ml of 20 mM potassium phosphate, pH 7.0, containing lysozyme (0.1 mg/ml),
aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin (1
µg/ml), and benzamidine (10 mM). After a 30-min incubation
at 0 °C bacteria were disrupted by a combination of freezing in
liquid N
and thawing at 4 °C (two cycles) and
subsequent sonication for 60 s at half-maximal power in a Heat Systems
ultrasonic disintegrator. The bacterial lysate was centrifuged at
40,000
g for 30 min and the resulting supernatant
solution was applied to a column (1 ml) of iminodiacetic acid Sepharose
6B (Pharmacia Biotech Inc.) that was charged with Ni(II) by
pre-equilibration with 50 mM NiSO
. The column was
washed sequentially with 20 ml of 20 mM Tris-HCl, pH 7.9,
containing 0.5 M NaCl and 5 mM imidazole, and then 30
ml of Tris-HCl, pH 7.9, containing 0.5 M NaCl and 60 mM imidazole. Fusion proteins were eluted with 8 ml of Tris-HCl, pH
7.9, 0.5 M NaCl, 1 M imidazole. Purified proteins
were dialyzed against 3 changes (one liter each) of 10 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl. After a
final dialysis against phosphate-buffered saline containing 50% (v/v)
glycerol the samples were stored at -20 °C. AKAP75 binding
activity and/or physicochemical properties of the expressed proteins
were maintained for more than 1 year under these conditions.
We
described the cloning of the intron-less gene for AKAP75 into a
mammalian expression vector (pAKAP75) in a previous
publication(6) . The AKAP75 coding region was released from
pAKAP75 by digestion with NotI and ApaI and subcloned
into pGEM5Z that was cleaved with the same restriction enzymes. An NdeI site that includes the initiator ATG was created by
site-directed mutagenesis as described above. DNA encoding full-length
AKAP75 was prepared by cutting with XbaI (a unique XbaI site in the 3`-untranslated sequence precedes the ApaI site used for cloning into pGEM5Z, see (6) ),
filling in the recessed terminus with Klenow DNA polymerase I, and then
cleaving with NdeI. The resulting DNA fragment was cloned into
pET-14b which was digested with BamHI, made blunt with Klenow
DNA polymerase I and then cleaved with NdeI. Induction and
purification of (His)-AKAP75 were carried out as described
above.
Intact, unmodified RII and several RII
mutants (see
``Results'') were expressed in the baculovirus/Sf9 cell
system. The baculovirus transfer vector pVL1392 and baculovirus DNA
containing a lethal deletion were obtained from PharMingen (San Diego,
CA). A cDNA insert that encodes the entire RII
polypeptide was
obtained from the plasmid pGRII
(see above) by digestion with NdeI. After incubation with Klenow DNA polymerase I and dNTPs,
the blunt-ended insert was cloned to the SmaI site of the
transfer plasmid pVL1392. Recombinant baculovirus was produced and used
to infect Sf9 cells according to the manufacturer's (PharMingen)
protocols. Three days after infection, Sf9 cells from three T75 flasks
were harvested by centrifugation at 1000
g. After
washing twice with phosphate-buffered saline at 4 °C, pelleted Sf9
cells were disrupted as described previously(22) . The cell
lysis buffer (22) was modified by (a) substituting 20
mM sodium phosphate, pH 7.4, for 20 mM Tris-HCl, pH
7.5, and (b) omitting non-ionic detergent. Cell homogenates
were centrifuged at 40,000
g for 15 min at 4 °C
and the supernatant solution (cytosol) was collected. The cytosol was
supplemented with 0.15 M NaCl and 5% (v/v) glycerol prior to
gel filtration analysis (see below).
AKAP75 was
phosphorylated by employing a similar procedure. Three modifications
were made in the reaction mixture(1) : phosphate buffer was
replaced with 25 mM Tris-HCl, pH 8.0; the catalytic subunit of
PKA was replaced with homogeneous subunit of C. elegans casein kinase II(23) ; and NaCl was added to a final
concentration of 0.1 M.
In this paper we
demonstrate that the binding assay can be executed successfully when
the immobilized and soluble ligands are interchanged (see
``Results''). Western blots containing wild-type and mutant
RII-N158 proteins were screened with 0.3 nM
P-labeled AKAP75 (70,000 cpm/ml) in the overlay
buffer. Conditions of the assay and procedures for the visualization
and quantification of bound ligand were identical with those used for
P-labeled
RII
(1, 2, 9, 25) .
Stokes radii for
full-length, wild-type, and mutant RII subunits were estimated by
gel filtration on a column (1.5
80 cm) of Sephacryl S-300
(Pharmacia) equilibrated with the column buffer described above. The
flow rate was 11 ml/h and 110 fractions (1.25 ml) were collected. The
elution patterns of RII
subunits were determined by performing
[
H]cAMP binding assays as described
previously(27) . Approximately 80-85% of the applied
binding activity was recovered in each case. The column was calibrated
with apoferritin (a = 6.10 nm), aldolase (a = 4.81 nm), albumin (a = 3.55 nm), and
ovalbumin (a = 3.05 nm). Standards were obtained from
Sigma.
Figure 9:
Cyclic AMP
and catalytic subunit binding activities of wild-type RII and
monomeric RII
mutants. A, the concentration dependence of
[
H]cAMP binding activity was determined for
RII
(
), Ala
-Ala
RII
(
), and Phe
-RII
(
) as described under
``Experimental Procedures.'' Assays were performed with 40
nM wild-type RII
; the concentration of mutant RII
subunits was 35 nM. B, the ability of wild-type and
mutant monomeric RII
subunits to inhibit the phosphotransferase
activity of the catalytic subunit (8 nM) of PKAII
was
assayed as described under ``Experimental Procedures.''
Percent maximum enzymic activity is reported as: (
P
radioactivity incorporated into the synthetic peptide substrate
Kemptide in the presence of the indicated concentrations of RII
or
RII
mutant)
(
P radioactivity incorporated
into Kemptide in the absence RII subunits)
100. Since the
wild-type and mutant RII
subunits produced very similar plots only
a single line is drawn through the data points. The symbols used are the same as those in A. Experiments for A and B were replicated three times. Typical results are
shown.
Candidate hydrophobic residues in RII were substituted with Ala
or other amino acids by site-directed mutagenesis, as described under
``Experimental Procedures.'' A list of RII
mutants that
were produced and studied is provided in Table 1. Following
mutagenesis, cDNAs encoding partial (NH
-terminal) and
full-length RII
polypeptides were subcloned into the E. coli expression plasmid pET-14b. These constructs direct the synthesis
of fusion proteins in which the RII
sequences are preceded by 20
amino acid residues encoded by DNA in the plasmid (see
``Experimental Procedures''). When transformed E. coli were incubated with isopropyl-1-thio-
-galactopyranoside the
highest levels of fully soluble, recombinant proteins were observed for
chimeric polypeptides that contained residues 1-158 of RII
(Fig. 1A). Moreover, the presence of the oligopeptide
sequence (His)
, which corresponds to residues 5-10 in
the fusion protein, enabled the single-step purification of recombinant
RII
(hereafter designated RII
-N158) to near-homogeneity by
chromatography on Ni
-saturated iminodiacetic acid
Sepharose 6B (Fig. 1B). In contrast, full-length
RII
fusion protein (438 amino acid residues) was expressed at a
lower level and was partially (
50%) insoluble. The soluble portion
of full-length fusion protein bound the metal chelate resin with very
low efficiency, thereby precluding facile purification of the various
mutant proteins. Presumably, this is due to interactions between the
NH
-terminal (His)
region and a sequence in the
central or COOH-terminal region of RII
.
Figure 1:
Expression and purification of
NH-terminal segments of RII
. Wild-type and mutant
polypeptides that contain 158 residues of the NH
-terminal
sequence of RII
were expressed in E. coli BL21 as
described under ``Experimental Procedures.'' A,
samples of total soluble proteins derived from 0.1 ml of an E. coli culture were fractionated in a 0.1% SDS-10% polyacrylamide gel.
The transformed bacteria were incubated for 2 h in the absence (odd-numbered lanes) or presence of
isopropyl-
-D-thiogalactopyranoside (even numbered
lanes). The recombinant RII
-N158 proteins contained either
all wild-type residues (lanes 1 and 2) or Ala
substitutions for the following pairs of residues:
Leu
-Leu
(lanes 3 and 4),
Val
-Leu
(lanes 5 and 6),
Leu
-Leu
(lanes 7 and 8),
Phe
-Leu
(lanes 9 and 10),
and His
-Phe
(lanes 11 and 12). The gel was stained with Coomassie Blue. Induced,
recombinant polypeptides exhibit an apparent M
of
25,000 (calculated M
= 19,600). This
parallels the overestimation of the M
value for
full-length RII
under the same
conditions(32, 33) . B, RII
-N158 fusion
proteins were purified as described under ``Experimental
Procedures.'' Purified proteins were analyzed by electrophoresis
in a 0.1% SDS-15% polyacrylamide gel and stained with Coomassie Blue. Lane W received the NH
-terminal segment of
wild-type RII
; lanes 2-8 and 10-15 contained the RII
-N158 mutants listed in Table 1. The
number of the lanes corresponds to the numbering of the mutants in Table 1. The protein content of the lanes varied over the range
0.4-1.4 µg.
The availability of
purified wild-type and mutant RII-N158 polypeptides allowed direct
characterization of their AKAP binding activities and physical
properties in the absence of interfering proteins. To verify the
validity of this approach, wild-type RII
-N158, which contains the
PKAII
``autophosphorylation'' site (Ser
),
was labeled by incubation with [
-
P]ATP and
the PKA catalytic subunit and then incubated with a Western blot
containing AKAP75. Immobilized anchor protein bound
P-RII
-N158 and intact
P-RII
(prepared as described in (24) ) with the same affinity.
Typical results are shown in Fig. 2A. PhosphorImager
analysis revealed that the molar ratio of bound RII
-N158:bound
RII
remained at
1 as the concentration of the two proteins
was varied over the range of 0.1-10 nM (data not shown).
Binding of full-length and partial RII
polypeptides was blocked by
excess nonradioactive RII
-N158 or RII
(Fig. 2A). In competition binding experiments, both
RII
-N158 and RII
had IC
values of
2
nM. Dimerization of RII
-N158 was demonstrated by gel
filtration chromatography, sedimentation analysis, and nondenaturing
electrophoresis (see below).
Figure 2:
Characterization of recombinant AKAP75 and
the AKAP75 binding activity of RII-N158. A, Western blot
that contained 20 ng of recombinant AKAP75 in each of 6 lanes was
prepared. Overlay binding assays were performed (see
``Experimental Procedures'') using 0.2 nM
P-labeled RII
-N158 (lanes 1, 3, and 5) or 0.2 nM
P-labeled intact RII
(lanes 2, 4, and 6). Each lane from the filter was
incubated with 3
10
cpm of probe in 3 ml; probes
were labeled to the same specific activity. Lanes 3 and 4 were probed in the presence of 100 nM nonradioactive
RII
-N158; lanes 5 and 6 were probed in the
presence of 100 nM nonradioactive RII
. An autoradiogram
is shown. B, full-length AKAP75 was expressed as a (His)
fusion protein and was purified by Ni
chelate
chromatography. Samples from 3 different preparations (lanes
2-4) were analyzed by electrophoresis in a 0.1% SDS-8.5%
polyacrylamide gel. Lanes 2-4 received 0.4, 1, and 2
µg of recombinant protein. Lane 1 contained the marker
protein transferrin (M
= 78,000). A
Coomassie Blue-stained gel is presented. Minor amounts of proteolytic
fragments were occasionally observed.
Mutations in RII that disrupt AKAP
binding activity can be detected rapidly and efficiently by reversing
the standard RII
binding ``overlay'' procedure (see
``Experimental Procedures''). To perform this screening assay
DNA encoding full-length AKAP75 (6) was subcloned into pET-14b
and the resulting AKAP75 fusion protein was purified to homogeneity by
Ni
-chelate chromatography (Fig. 2B).
Purified AKAP75 was labeled with
P by incubation with
casein kinase II and [
-
P]ATP.
Figure 3:
Screening mutant RII-N158 proteins
for AKAP75 binding activity. Samples (25 ng) of wild-type and variant
RII
-N158 proteins were electrophoresed in a 0.1% SDS-10%
polyacrylamide gel and then transferred to a polyvinylidene difluoride
(Immobilon-P) membrane. The membrane was probed with
P-labeled AKAP75 as described under ``Experimental
Procedures'' and exposed to x-ray film at -70 °C. The
resulting autoradiogram is shown. Lane 1 received wild-type
RII
-N158. Other proteins assayed were RII
-N158 mutants with
Ala substituted for: Leu
-Leu
(lane
2), Val
-Leu
(lane 3),
Leu
-Leu
(lane 4),
Phe
-Leu
(lane 5),
His
-Phe
(lane 6),
Leu
-Gln
(lane 7), and
Gly
-Thr
(lane
8).
Properties of the
mutated NH termini were characterized further in
competition binding assays (Fig. 4). Both intact RII
(not
shown) and wild-type RII
-N158 potently inhibited the binding of
P-RII
to AKAP75 and yielded IC
values of
2 nM under standard assay conditions. Replacement of
Leu
-Leu
and Val
-Leu
with Ala-Ala diminished the avidity of the NH
terminus of RII
for AKAP75 substantially, yielding IC
values that increased
20-25-fold. Variants of the
RII
NH
terminus that contained Ala at positions 12 and
13 or 35 and 36 were unable to compete for the high-affinity binding
site on AKAP75. Mutant polypeptides containing Ala-Ala substitutions
for Gly
-Thr
(Fig. 4),
Leu
-Gln
(not shown), and
Glu
-Ile
(not shown) exhibited IC
values that were only 1.5-2.5-fold higher than that
exhibited by the wild-type protein.
Figure 4:
Competitive inhibition of the binding of
RII to AKAP75 by wild-type and mutant RII
-N158 proteins.
Multiple Western blots, which contained 30 ng of AKAP75 in each lane,
were prepared on Immobilon-P membranes. Individual excised lanes were
incubated with 0.3 nM
P-labeled RII
(2
10
cpm in 2.5 ml) in the presence or absence of the
indicated concentrations of nonradioactive competing polypeptides. The
samples were processed according to the standard overlay binding
procedure as indicated under ``Experimental Procedures.'' The
amounts of
P radioactivity bound to AKAP75 were quantified
in a PhosphorImager (Molecular Dynamics) as described
previously(13) . The data are presented as % maximal
P binding activity. This represents: (amount of
P bound in the presence of competitor protein
the
amount
P bound in the absence of the competitor)
100. These studies were replicated three times and yielded very similar
results in each case. A typical set of results is shown. In addition,
the same results were also obtained when nondenatured AKAP75 was
directly applied to Immobilon-P and processed as described above. The
competing proteins were: wild-type RII
-N158 (
) and
RII
-N158 mutants in which Ala replaced Leu
and
Leu
(
), Val
and Leu
(
), Leu
and Leu
(
),
Gly
and Thr
(
), or His
and Phe
(
).
Figure 5:
AKAP75 binding activity of RII-N158
proteins that contain mutations at residues 12, 13, and 36. Samples (25
ng) of mutant RII
-N158 proteins were applied to a polyvinylidene
difluoride membrane and probed with radiolabeled AKAP75 as described in
the legend for Fig. 3. The resulting autoradiogram is presented.
The mutated NH
-terminal segments of RII
contained the
following amino acid substitutions: Glu for Leu
(lane
1), Ala for Leu
(lane 2), Ala for Leu
(lane 3), Ile-Ile for Leu
-Leu
(lane 4), Val-Val for Leu
-Leu
(lane 5), and Ala for Phe
(lane
6).
Substitution of
Phe with Ala (mutant 15, Table 1) in RII
yielded the most striking result. AKAP75 binding activity was totally
abrogated (Fig. 5, lane 6). Thus, the bulky aromatic
side chain subserves an essential and pivotal role in linking RII
to AKAP75 and hence, the cytoskeleton.
Figure 6:
Determination of the Stokes radius for
wild-type and mutant RII-N-158 proteins. Samples of wild-type and
variant proteins were labeled with
P and characterized by
gel filtration on a column of Sephacryl S-100 as described under
``Experimental Procedures''. A, superimposed
representative elution patterns for wild-type RII
-N158 (
)
and mutants having Ala substituted for either Leu
and
Leu
(
) or Val
-Leu
(
). Only the relevant fractions are shown. B, the
column was calibrated with standards (open circles) as
described under ``Experimental Procedures.'' The data were
plotted according to Siegel and Monty(26) . K
= (elution volume of protein peak - void volume)
(total column volume - void volume). Stokes radii
measured for Group I and Group II RII
-N158 proteins (see text) are
indicated with solid circles. C, a correlation between
oligomerization state and mobility in a nondenaturing polyacrylamide
gel was established for RII
-N158 proteins. Samples (1.5 µg of
protein) of wild-type (lane 1) and mutant RII
-N158
proteins were electrophoresed in a 7.5% polyacrylamide gel as described
under ``Experimental Procedures.'' The variant polypeptides
had Ala substituted for Leu
and Leu
(lane
2), Leu
and Leu
(lane 3),
Val
and Leu
(lane 4), Phe
and Leu
(lane 5), Gly
and
Thr
(lane 6), and Phe
(lane
8). The mutant RII
-N158 protein in lane 7 contained
Ile in place of Leu
and Leu
. A gel that was
stained with Coomassie Blue is shown. Proteins applied to lanes 1 and 4-7 were determined to be dimers (D)
on the basis of their hydrodynamic properties; polypeptides in lanes 2, 3, and 8 are monomers (M).
Figure 7:
Determination of sedimentation
coefficients for wild-type and mutant RII-N158 proteins. Samples
of wild-type and variant proteins were labeled with
P,
mixed with internal standards, centrifuged in a 5-20% (w/v)
sucrose gradient, and assayed as described under ``Experimental
Procedures.'' A, distribution of
Ala
-Ala
RII
-N158 after sucrose density
gradient centrifugation; B, distribution of
Ala
-Ala
RII
-N158 after centrifugation.
Only the relevant fractions are shown for A and B.
Fraction 1 is at the top of the gradient. C, determination of s
, values for Group I and
Group II proteins from the calibration
curve.
Molecular weights (M) were then calculated (26) from the
equation M
=
[6
N/(1-
)]
[a
s], where a =
Stokes radius; s = sedimentation coefficient; n = Avogadro's number;
= viscosity of
water;
= density of water;
= 0.731, the
partial specific volume of wild-type (32) and mutant
RII
-N158. (
)The experimental results for Stokes radius
and s
yielded molecular weights of
36,000 for Group I proteins and
19,000 for Group II proteins.
These values are within 10% of molecular weights established for
RII
-N158 dimer (39,200) and monomer (19,600) on the basis of
derived amino acid sequence data(18) . Thus, most RII
-N158
polypeptides that retain partial or maximal AKAP75 binding activity
undergo dimerization. Conversely, mutant RII
-N158 proteins that
are either incapable of binding AKAP75 (Leu
-Leu
to Ala
-Ala
, Phe
to
Ala
mutants) or exhibit an extremely low affinity for the
anchor protein (Leu
to Ala
mutant) are
monomers. The variant monomeric protein in which both Leu
and Leu
are replaced with Ala is an exception to
these generalizations.
An independent analysis of the dimerization
state of the variants was performed by nondenaturing electrophoresis.
In this system proteins are resolved on the basis of their overall size
and their charge to mass ratio(34) . In RII variants that
contain Ala for Leu or Phe substitutions the charge to mass ratio is
expected to be virtually constant. Thus, monomers should be readily
separated from dimers by virtue of their higher mobilities.
Electrophoretic analysis on a 7.5% acrylamide gel resolved the
RII
-N158 mutant proteins into two classes (Fig. 6C). The rapidly migrating group included the
polypeptides that behaved as monomers in hydrodynamic experiments; the
slowly migrating group corresponded to the homodimers.
In order to address these points cDNAs encoding
selected full-length mutant and wild-type RII subunits were
inserted into baculovirus (see ``Experimental Procedures'').
RII
and RII
variants were then expressed as native
(non-fusion) proteins in infected Sf9 cells. When Sf9 cytosol
containing wild-type human RII
was analyzed by gel filtration on a
calibrated column of Sephacryl S-300, a large peak of
[
H]cAMP binding activity emerged at fraction 73 (Fig. 8A). The Stokes radius for the cAMP-binding
protein was 4.9 nm, a value that is in excellent agreement with that
determined for RII
from bovine brain(35) . No cAMP binding
activity was observed in cytosol from Sf9 cells infected with
baculovirus lacking the cDNA insert. In contrast, RII
containing
Ala
-Ala
(Fig. 8B), Ala
alone (not shown), or Ala
(not shown) eluted at
fractions 78 or 79 and had Stokes radius of
3.9 nm. Western
immunoblot analysis disclosed that intact RII
proteins
(approximate M
54,000) were produced in each
instance, thereby indicating that the results were not attributable to
proteolysis (Fig. 8C). After determining sedimentation
coefficients as described above (data not shown), molecular weights of
51,000 to 53,000 were calculated for the RII
variants. The M
of wild-type RII
dimer is 97,000. Thus, as
expected from the characterization of the NH
-terminal
partial peptides, the mutant proteins were monomeric.
Figure 8:
Determination of the Stokes radius for
wild-type RII and an RII
variant. RII
and RII
containing Ala-Ala in place of Leu
-Leu
were
expressed in the baculovirus/Sf9 cell system (see ``Experimental
Procedures'') and characterized by gel filtration on a column of
Sephacryl S-300 as described under ``Experimental
Procedures.'' Samples containing 1 mg of total cytosol protein
(including
15 µg of RII
or mutant RII
) were applied
to the column. Aliquots (50 µl) of the fractions were assayed for
[
H]cAMP binding activity. A, elution
pattern for wild-type RII
. Only the relevant fractions are shown. B, elution pattern for Ala
-Ala
RII
. C, samples (15 µl) of the peak fractions
were characterized by electrophoresis in a 0.1% SDS-10% polyacrylamide
gel and subsequent Western immunoblot analysis with anti-RII
IgGs(5, 13) . Lanes 1 and 2 contained wild-type RII
; lanes 3 and 4 received Ala
-Ala
RII
; lanes 5 and 6 received a sample of the Ala
RII
peak that was isolated in separate gel filtration experiment. The even-numbered lanes were probed with anti-RII
IgGs in the
presence of excess (300 ng) RII
. The apparent M
of RII
is 54,000. The Western blot was developed by an
enhanced chemiluminescence procedure (ECL, Amersham) as described
previously(9, 13) . Chemiluminescence signals that
were recorded on x-ray film are presented.
Despite the
difference in oligomerization state the mutant and wild-type RII
polypeptides were equipotent in binding cAMP (Fig. 9A)
and inhibiting the activity of the catalytic subunit of PKAII
(Fig. 9B). Hence, domains proximal and distal to the
NH
terminus of RII
are correctly folded and function
normally.
A combination of mutagenesis, recombinant protein expression,
binding analysis, and hydrodynamic characterization revealed a group of
amino acids in RII that mediate subunit dimerization and promote
the binding of RII
with AKAP75. The crucial residues are located
in a dimerization/AKAP binding region that includes amino acids
13-36 at the NH
terminus of RII
. Mutations
introduced immediately upstream and downstream from the core region had
little effect on the properties of partial polypeptides.
Leu, Phe
, and the amino acid pairs
Val
-Leu
and Leu
-Leu
play central roles in establishing a high-affinity binding site
in RII
that couples with AKAP75. Each of these amino acids has a
bulky side chain that can participate in structure-stabilizing
hydrophobic interactions(36, 37) . However, amino
acids in this group exert their actions by two different mechanisms.
Replacement of residues at the NH and COOH termini of
the core region (Leu
, Phe
) with Ala markedly
diminished or extinguished AKAP75 binding activity (Fig. 3Fig. 4Fig. 5). The loss of binding activity
was associated with a change in oligomerization potential. The
Ala
RII
-N158 and Ala
RII
-N158
mutants are monomeric proteins ( Fig. 6and Fig. 7). When
the same mutations were introduced into full-length RII
, monomeric
proteins were again generated. However, the abilities of mutant
RII
monomers and wild-type RII
dimer to bind cAMP and inhibit
the catalytic subunit were indistinguishable (Fig. 9). Thus, the
reduction in the size of the hydrophobic side chain at either residue
13 or 36 selectively disrupts dimerization, but has no effect on the
folding of either proximal or distal segments of the RII
polypeptide.
The deletion of NH-terminal segments
(45-90 residues) of R subunits produces monomeric proteins that
retain two cAMP binding sites and the capacity to inhibit the catalytic
subunit of PKA(14, 15, 17) . These
observations on truncated R subunits and the present results on intact
RII
that contains a contiguous, but dimerization-defective
NH
-terminal sequence, preclude subunit oligomerization as a
key (positive or negative) factor in the generation of functional
domains that regulate kinase activity.
Substitution of Leu with Ala is a conservative mutation. The introduction of
Ala
should neither alter the net charge of the protein nor
affect substantially the propensity of the NH
-terminal
sequence to fold into an
-helical or
-strand
conformation(38) . The hydrophobic character of the side chain
is also preserved. Nevertheless, the functional outcome of this single
mutation is both striking and illuminating: RII
is unable to
dimerize. An apparent consequence of the monomerization of RII
is
that the high-affinity binding site for AKAP75 is lost. The importance
of the size of the aliphatic side chain at residue 13 was analyzed
further by constructing additional RII
-N158 variants. Mutant
proteins that contained Ile
-Ile
,
Val
-Val
(Fig. 5), or Ile
alone
were dimers and readily formed complexes with
AKAP75, although their affinities for the anchor protein were
2-3-fold lower than that exhibited by the wild-type protein.
Thus, a bulky aliphatic side chain at residue 13 is sufficient to
ensure the stabilization of dimeric RII
and support binding with
the anchor protein. Both conservative (Ala) and non-conservative (Glu)
substitutions for the contiguous Leu
residue failed to
abolish either the dimerization or AKAP binding functions of the
NH
terminus. Evidently, the position of the long-chain
aliphatic amino acid in the primary sequence and presumably, its
precise orientation in the folded, higher order structure of the
NH
-terminal domain, play pivotal roles in conferring the
properties of RII
oligomerization and indirectly, the
intracellular targeting of PKAII
to the cytoskeleton. It seems
likely that similar considerations apply to the role Phe
.
Further mutational analysis is underway to test this proposition.
In
an earlier study Scott et al.(14) employed truncation
analysis to investigate the interaction of murine RII with
microtubule-associated protein 2, an RII
-selective anchor protein (16, 25) . Deletions of 35, 30, or 14 residues from
the NH
terminus of RII
disrupted dimerization and
abolished microtubule-associated protein 2 binding activity. The
properties of the truncated RII
subunits and the characterization
of loss-of-function mutations in RII
(Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8)
suggest that subunit dimerization may be a pre-requisite for subsequent
interactions with PKAII anchoring proteins. Key findings relevant to
the model (14) are that ``native'' RII
monomer
(produced in eukaryotic cells) binds cAMP and inhibits PKA catalytic
activity, yet it cannot be sequestered by AKAP75. Furthermore, the
discovery of central roles for Phe
and Leu
in
promoting RII
-RII
interactions provides an initial insight
into the structural basis for dimerization.
While this paper was in
review Hausken et al.(39) reported that deletion of
10 residues at the NH terminus of RII
generates a
monomeric protein which is unable to bind an AKAP. Deletion of residues
1-5 did not compromise dimerization or AKAP binding activity.
These data suggest that the sequence PPGLT contains a residues(s) that
contributes significantly to the stabilization of RII
(and
perhaps, RII
) dimers. Alternatively, the elimination of 10
NH
-terminal residues may alter the orientation of the side
chain of Leu
(which becomes residue 3), thereby
diminishing its ability to promote RII
-RII
interaction. These
possibilities can be evaluated by site-directed mutagenesis.
Hausken
and co-workers (39) also showed that a small fusion protein
containing residues 1-30 of RII dimerized although it lacks
Phe
. The reason for this difference between RII
and
RII
is not known. The limited segment of RII
sequence may not
be an exact analog of a large RII
subunit containing a single
amino acid substitution. Alternatively, since the
NH
-terminal segments (residues 1-30) of RII
and
RII
are only
67% identical, it is possible that non-conserved
residues in RII
facilitate dimerization in an isoform-specific
manner.
Certain hydrophobic amino acid residues that are positioned
between Leu and Phe
subserve an essential,
but distinctly different role in the tethering of RII
by AKAP75.
Characterization of a prototypic RII
-N158 mutant, in which
Val
and Leu
were replaced with Ala, provided
evidence for this conclusion. Ala
-Ala
RII
-N158 exhibits the hydrodynamic properties of a dimeric
protein. However, AKAP75 binds wild-type RII
25-fold more avidly
than the Ala
-Ala
mutant protein (Fig. 4). Studies on Ala
-Ala
RII
-N158 yielded similar results. Evidently, the large
aliphatic side chains of residues 20, 21, 31, or 33 facilitate the
high-affinity tethering of RII
to AKAP75. In contrast,
reduction in the size of the hydrophobic groups at these positions has
little or no impact on the homodimerization of RII
. Likewise,
substitution of Ala for Ile
and Ile
in RII
results in a dimeric protein with reduced affinity for
AKAPs(39) . Thus, the dimerization of RII
, although
necessary, is not sufficient to generate an optimal binding site for
AKAP75. Aliphatic hydrophobic residues within the binding region may
provide a specific hydrophobic surface that interacts with a
complementary surface generated by Leu and Ile residues previously
implicated in the tethering site on AKAP75(9) .
The
properties of Ala-Ala
RII
-N158 are
anomalous. The expressed protein is monomeric, suggesting that
Leu
and/or Leu
participate in the
stabilization of the dimeric configuration. On the other hand, the
protein binds AKAP75, albeit with modest affinity ( Fig. 3and Fig. 4). A possible explanation is that contact with the AKAP75
tethering region induces the dimerization of the mutant polypeptide.
Alternatively, substitution of Ala at residues 28 and 29 may elicit
local changes in structure that partially mimic the arrangement of
Val
-Leu
, Phe
, and Leu
in the RII
dimer, thereby generating a modest binding
capacity. Further mutational analysis and physicochemical studies are
needed to address this issue.
The hydrophobic residues that drive
the dimerization of RII and its coupling with AKAP75 are conserved
in the NH
terminus of human and bovine RII
, with the
exception of a Val for Leu replacement at residue
29(40, 41) . This conservation is consistent with the
dimeric nature of RII
and RII
subunits and the ability of the
two RII isoforms to share binding sites on a variety of AKAPs (14, 15) . Divergent sequences elsewhere in the
NH
termini of the two proteins must account for the
exclusive homodimerization of RII isoforms and their differential
binding affinity for certain AKAPs (29, 42) .
Further structural divergence is evident in the NH terminus of mouse RII
(43) , which contains Val-Gly
instead of Val
-Leu
. This raises the
possibility that the conserved Val
residue plays a central
role in mediating the avid binding of RII
with AKAP75 while
Leu
makes a lesser contribution. Such a situation would
parallel the discovery that the branched aliphatic side chain of
Leu
is crucial for RII
dimerization, whereas,
substitution of adjacent Leu
with Ala or Glu has little
effect on oligomerization and AKAP75 binding activity ( Fig. 5and text of ``Results''). An alternative
possibility is that mouse RII
may bind AKAP75 with lower affinity
than RII
isoforms from other species. Further mutational analysis
and AKAP binding studies are needed to address this issue.
Roles for bulky hydrophobic side chains in the stabilization of protein structures and protein-protein interactions are well-established(36, 37) . The fundamentally important functions subserved by ``leucine zippers'' in the oligomerization of transcription factors illustrate the latter point(44, 45) . Our results can be rationalized in the context of the elegant studies of Matthew's laboratory (31) on the functions of Leu and Phe residues in bacteriophage T4 lysozyme. Biochemical, biophysical, and x-ray crystallographic measurements document that the replacement of selected Leu and Phe residues with Ala can destabilize protein structure as a function of two parameters. One component is the difference in energy required to desolvate Ala relative to the larger hydrophobic moieties. Second, the decrease in hydrophobic surface creates solvent-free cavities of varying size in the folded structure that cannot be fully compensated by the local rearrangement of hydrophobic side chains. The generation of relatively large cavities minimizes favorable van der Waals contacts and substantially weakens the classical hydrophobic effect(31) .
Finally, the binding of partially unfolded proteins by the molecular chaperone BiP, provides a relevant paradigm for protein-protein association driven by long aliphatic side chains(46) . BiP sequesters proteins by coupling with a linear stretch of 7 amino acid residues. Leu, Ile, Met, and Val are highly preferred at several internal positions in such sequences. Moreover, thermodynamic analysis indicates that the binding of proteins to BiP is mediated by same types of interactions that stabilize hydrophobic domains of T4 lysozyme (31) and other proteins.
Overall, our
data suggest the speculation that the principal roles of the side
chains of Leu and Phe
are to interact with
and bury otherwise exposed hydrophobic moieties, thereby providing a
favorable free energy increment for dimerization. The dimerization of
RII
would, in turn, alter the disposition of hydrophobic side
chains within the NH
-terminal region (e.g. Val
-Leu
) to create a characteristic
exposed binding surface that is sequestered by the concerted actions of
Leu
, Leu
, Ile
, and
Leu
in AKAP75(9) . Ultimately determination of
the three-dimensional structure of the relevant domains of RII
,
AKAP75, and RII
AKAP75 complexes will be required to
thoroughly evaluate this scheme and provide detailed molecular
mechanisms for dimerization and AKAP binding. However, the
structure-function studies described herein elucidate the identities
and functions of amino acid residues that mediate the dimerization and
cytoskeletal targeting of RII
. Certain crucial amino acids
(Val
-Leu
, Phe
-Leu
)
that are essential for optimal binding of RII
with AKAP75 play no
major role in the dimerization reaction. In contrast, the dominant
effects of Phe
and Leu
seem to be due to
their indispensable contributions to dimerization. Thus, higher-order
structural elements that govern RII
-RII
interactions and
AKAP75
RII
complex formation may be partially
shared and partially distinct.