Download The p101 subunit of PI3Kγ restores activation by Gβ mutants

Biochem. J. (2012) 441, 851–858 (Printed in Great Britain)
851
doi:10.1042/BJ20111664
The p101 subunit of PI3Kγ restores activation by Gβ mutants deficient in
stimulating p110γ
Aliaksei SHYMANETS*†, Mohammad R. AHMADIAN†, Katja T. KÖSSMEIER†, Reinhard WETZKER‡, Christian HARTENECK*
and Bernd NÜRNBERG*†1
*Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and
Interfaculty Centre of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany, †Institute of Biochemistry and Molecular Biology II,
Medical Faculty, Heinrich Heine University, 40225 Düsseldorf, Germany, and ‡Department of Molecular Cell Biology, Centre for Molecular Biomedicine, Jena University Hospital, 07745
Jena, Germany
G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ ) plays
a crucial role in inflammatory and allergic processes. PI3Kγ ,
a dimeric protein formed by the non-catalytic p101 and
catalytic p110γ subunits, is stimulated by receptor-released Gβγ
complexes. We have demonstrated previously that Gβγ stimulates
both monomeric p110γ and dimeric p110γ /p101 lipid kinase
activity in vitro. In order to identify the Gβ residues responsible
for the Gβγ –PI3Kγ interaction, we examined Gβ 1 mutants for
their ability to stimulate lipid and protein kinase activities and to
recruit PI3Kγ to lipid vesicles. Our findings revealed different
interaction profiles of Gβ residues interacting with p110γ or
p110γ /p101. Moreover, p101 was able to rescue the stimulatory
activity of Gβ 1 mutants incapable of modulating monomeric
p110γ . In addition to the known adaptor function of p101, in
the present paper we show a novel regulatory role of p101 in the
activation of PI3Kγ .
INTRODUCTION
dimer, resulting in abrogation of p85-mediated inhibition of the
catalytic p110 subunits [28,29,31–34]. Molecular biological and
crystal structure studies argue that the mode and strength of
the interaction between the p85 adaptor and catalytic subunits
provides the basis for the autoinhibitory function of the p85
subunit, which is responsible for the differences in activity and
regulation within class IA PI3K enzymes [7,13,35–37].
In contrast with the class IA PI3Ks, the mechanism of PI3Kγ
regulation is poorly understood. Initially, PI3Kγ was discovered
as a Gβγ -sensitive monomer [15,20], but shortly afterwards p101
was described as an indispensable complex partner of p110γ
responsible for sensitizing PI3Kγ to Gβγ [14]. In fact, the
data suggest that Gβγ may stimulate PI3Kγ solely through
interaction with p101, which somewhat resembles the scenario
known for the activation of class IA PI3Ks. To elucidate how p101
sensitizes Gβγ for PI3Kγ , we and others have studied PI3Kγ
in its monomeric or heterodimeric form in vitro and in vivo [16–
18,22,24,38,39].
We have found evidence that Gβγ interacts with both PI3Kγ
subunits in a selective manner in order to stimulate PtdIns(3,4,5)P3
formation. This prompted us to propose a model of Gβγ -induced
activation of PI3Kγ , in which Gβγ has to bind to the non-catalytic
p101 subunit for translocation of the enzyme to the membrane,
enabling PtdIns(3,4,5)P3 formation by direct interaction of Gβγ
with p110γ [17]. The underlying data suggest that the latter
step is independent of p101; however, we could not exclude the
possibility that p101 may be involved in Gβγ -induced stimulation
of membrane-attached p110γ . Therefore Gβγ may interact with
p101 and p110γ through individual or common binding sites.
In order to validate our hypothesis and to identify the structural
determinants of Gβγ involved in the membrane recruitment
and regulation of PI3Kγ enzymatic activity, we addressed this
Class I PI3Ks (phosphoinositide 3-kinases) are secondmessenger-generating enzymes, which transform extracellular
signals into the principle product PtdIns(3,4,5)P3 in order to
control a plethora of fundamental cellular responses, including
proliferation, differentiation, growth and chemotaxis [1–8]. On
the basis of their structural features and modes of regulation,
class I PI3Ks have been grouped into the class IA and class
IB subfamilies. Class IA PI3Ks are heterodimeric lipid kinases
composed of one out of five non-catalytic p85-type adaptor
subunits and a catalytic subunit classified as p110α, p110β or
p110δ [2,7,9–12]. Members of the class IA PI3K subfamily are
recognized by the nature of their catalytic subunit, which currently
appears to be more important for assigning signalling specificity
than the adapter subunit [7,11,13]. All class IA enzymes are
tightly and directly regulated by RTKs (receptor tyrosine kinases),
other tyrosine kinases and Ras GTPases, whereas class IB PI3Ks
are under the control of GPCRs (G-protein-coupled receptors)
via direct interaction with Gβγ [14–19]. Only one class IB
catalytic subunit, p110γ , is known [20]. It forms dimers with
one of two non-catalytic subunits, p101 or p87 (also known as
p84) [14,18,21,22]. Although the p110γ subunit defines PI3Kγ
as a GPCR-controlled Gβγ -dependent effector, recent evidence
suggests that Ras proteins, together with the non-catalytic subunits
p101 and p87, also contribute to signalling specificity [23–25].
Class IA PI3K regulation depends on tyrosine phosphorylation
of activated RTK recognised by the SH2 (Src homology
2) domains of p85 subunits of the PI3K dimers which
mediate translocation of the enzyme from the cytosol to the
plasma membrane [10,11,19,26–30]. The recognition process
is associated with conformational changes within the PI3K
Key words: Gβγ , G-protein, p101, phosphoinositide 3-kinase γ
(PI3Kγ ), signal transduction.
Abbreviations used: ACII, adenylyl cyclase II; C12 E10 , polyoxyethylene 10-lauryl ether; GIRK, G-protein-activated inward rectifier potassium channel;
GPCR, G-protein-coupled receptor; NTA, nitrilotriacetic acid; PI3K, phosphoinositide 3-kinase; PLCβ, phospholipase Cβ; RTK, receptor tyrosine kinase;
Tos-Phe-CH2 Cl, tosylphenylalanylchloromethane.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
852
A. Shymanets and others
question by using Gβ 1 mutants where the amino acids involved in
interactions with GDP-bound Gα or downstream effectors were
substituted by alanine [40–43].
EXPERIMENTAL
Expression and purification of recombinant proteins
Sf9 (Fall Armyworm Ovary; Gibco) cells were cultured in
suspension with TNM-FH medium (Sigma) supplemented with
10 % (v/v) FBS (fetal bovine serum; Gibco), lipid medium
supplement (1:100 dilution; Sigma), penicillin (100 units/ml)
and streptomycin (0.1 mg/ml). For protein expression, Sf9 cells
(1.5×106 cells/ml) were infected with viruses encoding the
subunits of PI3Kγ and/or wild-type or mutant Gβ together
with Gγ [42,44]. After 48 (PI3Kγ ) or 60 (Gβγ variants) h of
infection, the cells were collected by centrifugation at 1000 g for
5 min and washed twice with PBS. Subsequent purification of
lipidated recombinant Gβ 1 γ 2 variants was performed as detailed
previously [44]. Expression and purification of recombinant His6 tagged PI3Kγ were carried out according to protocols published
previously [16,45] with some modifications. After elution from
a Resource 15Q 5/5 column, fractions containing PI3Kγ were
pooled, concentrated and loaded on to a gel filtration Superdex
HR 10/30 column. Proteins were eluted using a buffer containing
20 mM Tris/HCl, pH 8, 150 mM NaCl, 2 mM dithiothreitol
and 0.033 % C12 E10 (polyoxyethelene-10-lauryl ether). Purified
proteins were quantified by Coomassie Brilliant Blue staining
following SDS/PAGE (10 % or 15 % gel) with BSA as the
standard. The proteins were stored at − 80 ◦ C.
Copurification of Gβ 1 γ 2 with PI3Kγ subunits
For the copurification experiments [24], viruses encoding His6 fused subunits of PI3Kγ , p101 or p110γ were co-infected with
viruses encoding Gβ 1 γ 2 . After 55 h, the cells were harvested
and lysed by forcing the Sf9 cell suspension through a 22gauge needle five times and subsequently through a 26-gauge
needle 10 times. The suspension was incubated for 30 min with
a buffer containing 20 mM Hepes/NaOH, pH 7.5, 150 mM NaCl,
10 mM 2-mercaptoethanol and 0.5 % C12 E10 , and incubated with
Ni2 + -NTA (nitrilotriacetic acid) Superflow for 2 h. After several
washing steps, the proteins of interest were eluted using a buffer
containing 20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 10 mM 2mercaptoethanol, 0.1 % C12 E10 and 200 mM imidazole.
Gel electrophoresis, immunoblotting and antibodies
Generation and characterization of the antiserum against Gβ 1
subunit (AS 398) are detailed elsewhere [46]. Monoclonal
anti-PI3Kγ antibodies directed against intact p110γ were
described previously [15]. Preparations containing Gβ 1 and
p110γ proteins were fractionated by SDS/PAGE (10 % or 15 %
gel) and transferred on to nitrocellulose membranes (HybondTM -C
Extra, Amersham Biosciences). Visualization of antibodies was
performed using an ECL (enhanced chemiluminescence) system
(Amersham Biosciences) or the SuperSignal® West Pico Chemiluminescent Substrate (Pierce) according to the manufacturers’
instructions.
Proteolysis of Gβ 1 γ 2 variants with trypsin
The digestion assay, with some modifications, was performed as
detailed previously [47]. Proteins were cleaved with Tos-Phe
c The Authors Journal compilation c 2012 Biochemical Society
CH2 Cl (tosylphenylalanylchloromethane, also known as TPCK)treated trypsin. The assays were conducted in a final reaction
volume of 30 μl containing 20 mM Tris/HCl, pH 8.0, 150 mM
NaCl, 2 mM dithiothreitol and 0.033 % C12 E10 .
The G-protein concentration in the reaction mixture was
167 μg/ml. Tos-Phe-CH2 Cl-treated trypsin was diluted in the
same buffer and added to the sample at a 1:25 trypsin/substrate
ratio. The samples were incubated for 40 min at 30 ◦ C. Proteolysis
was terminated by the addition of 4× Laemmli sample buffer and
the samples were boiled for 1 min. The reactions were analysed
by SDS/PAGE (15 % gel).
In vitro assay for lipid kinase activity
The assays were conducted in a final volume of 50 μl
containing 40 mM Hepes/NaOH, pH 7.4, 0.1 % BSA, 1 mM
EGTA, 7 mM MgCl2 , 120 mM NaCl, 1 mM dithiothreitol
and 1 mM β-glycerophosphate (vesicle buffer) as described
previously [24,45] with some modifications. A 30 μl lipid vesicle
mixture, containing 320 μM phosphatidylethanolamine, 300 μM
phosphatidylserine, 140 μM phosphatidylcholine and 30 μM
sphingomyelin supplemented with 40 μM PtdIns(4,5)P2 was
dried using N2 gas and sonicated in vesicle buffer. Subsequently,
the phospholipid vesicles were mixed with Gβ 1 γ 2 and incubated
on ice for 10 min. The samples containing different amounts of
Gβ 1 γ 2 were adjusted to identical detergent concentrations, such
as 0.002 % of C12 E10 . Thereafter 10 ng of PI3Kγ was added,
and the mixture was incubated for another 10 min at 4 ◦ C in a
final volume of 40 μl. Then, the assay was started by adding
40 μM ATP (1 μCi of [γ -32 P]ATP, Hartmann Analytic) in 10 μl
of the above-mentioned assay buffer at 30 ◦ C. After 15 min,
the reaction was stopped with 150 μl of ice-cold 1 N HCl and the
tubes were placed on ice. The lipids were extracted by vortexing
the samples with 500 μl of a 1:1 chloroform/methanol solution.
After centrifugation (4000 g for 1 min and 4 ◦ C), the organic phase
was washed with 200 μl of 1 N HCl. Subsequently, 25–70 μl of
the organic phase was resolved on a potassium oxalate-pretreated
TLC plate (Whatman) with 35 ml of 2 N acetic acid and 65 ml of
n-propyl alcohol as the mobile phase. Dried TLC plates were
exposed to Fuji imaging plates, and autoradiographic signals
were quantified with a Fujifilm FLA-5000 imaging system
(Raytest).
Differences in the lots of the phospholipids used and variability
in various experimental parameters made it difficult to assure the
precision of reproducibility necessary for analysing the results of
the different Gβ 1 γ 2 variants. For that reason, the ability of Gβ 1 γ 2
variants to simulate PI3Kγ in all experiments was studied in sideby-side experiments using wild-type Gβ 1 γ 2 . The simultaneous
determination of PI3Kγ activity induced by wild-type Gβ 1 γ 2
enabled the calculation of the correlation coefficient of PI3Kγ
stimulation between each tested concentration of the Gβ 1 γ 2
variants and wild-type Gβ 1 γ 2 . On the other hand, the different
data sets of PI3Kγ activity induced by wild-type Gβ 1 γ 2 allowed
us to determine the maximal stimulation (V max ) and EC50 values
of our experimental set-up. The results are means +
− S.D. from
at least three independent experiments. The maximal stimulation
(V max ) of p110γ and p110γ /p101 in the presence of wild-type
Gβ 1 γ 2 was 3.2 +
− 1.3 and 28.1 +
− 7.6 nmol PtdIns(3,4,5)P3 per mg
of protein/min respectively. The EC50 values were 202.6 +
− 28.3
and 8.7 +
2.8
nM
for
p110γ
and
p110γ
/p101
respectively.
On the
−
basis of the mean values, the data for the Gβ 1 γ 2 variants were
normalized by replotting the data corresponding to the template
curve using the mean of the correlation coefficients estimated in
individual experiments.
Differential modulation of PI3Kγ activities
853
In vitro assay for protein kinase activity
The protein kinase activity of PI3Kγ was measured as described
previously for the lipid kinase activity with some modifications
[45]. The assay volume was 25 μl (2 μCi of [γ -32 P]ATP per
tube). The phospholipid vesicles were prepared without
PtdIns(4,5)P2 . The reaction was stopped after an incubation period
of 30 min at 30 ◦ C by adding 10 μl of 4× Laemmli sample buffer.
Following separation by SDS/PAGE (10 % gel), the proteins were
transferred on to nitrocellulose membranes. Dried membranes
were exposed to Fuji imaging plates, and autoradiographic
signals were measured using a FLA-5000 Fuji-Imager (Raytest).
Generation and presentation of the dose-response curves was done
as described for the lipid kinase activity.
Lipid vesicle pull-down assay
The experimental conditions for the determination of Gβ 1 γ 2
and PI3Kγ association in phospholipid vesicles were similar
to the measurements of the enzymatic activity of PI3Kγ [24].
The assay did not contain radioactively labelled ATP and had
a higher amount of PI3Kγ (200 – 400 ng). After an incubation
period of 15 min at 30 ◦ C, the mixture was put on ice and centrifuged at 12 000 g for 2 min at 4 ◦ C. The supernatant and pellet
were separated. The supernatant was supplemented with 4×
Laemmli sample buffer. The pellet was resuspended and washed
twice with vesicle buffer. Subsequently the pellet was resolved
in 1× Laemmli sample buffer. The samples were subjected
to SDS/PAGE (10 % gel) and transferred on to nitrocellulose
membranes. Semiquantitative analysis of immunoblots was
performed using specific antisera against p110γ and Gβ 1 subunits.
Figure 1 Characterization of purified recombinant Gβ 1 γ 2 variants by partial
trypsin digestion
The integrity of purified recombinant Gβ 1 γ 2 variants harbouring alanine mutations within the
Gα binding cluster of the Gβ 1 subunit was validated by analysing the sensitivity of the protein
to trypsin digestion. Each protein (5 μg) was incubated with 0.2 μg of Tos-Phe-CH2 Cl-treated
trypsin for 40 min at 30 ◦ C in a total volume of 30 μl. Proteolysis was terminated by adding
10 μl of 4× Laemmli sample buffer. Proteins were subjected to SDS/PAGE and analysed by
Coomassie Brilliant Blue staining. The occurrence of 26 kDa and 14 kDa bands is indicative of
properly folded Gβ 1 γ 2 proteins. Heat denaturation (1 h at 95 ◦ C) of the Gβ 1WT γ 2 protein prior
to tryptic digestion resulted in the appearance of additional bands and served as a negative
control. Molecular masses and the positions of Gβ 1 and His-Gγ 2 are indicated. Molecular
mass is given in kDa on the right-hand side. WT, wild-type.
RESULTS AND DISCUSSION
Sensitivity of Gβ 1 γ 2 variants to trypsin digestion
Gβ 1 mutants co-expressed with the His6 -tagged and isoprenylated
Gγ 2 subunit formed heterodimers and were purified from Sf9
cells as described previously [44]. Proper protein folding of the
purified mutants was confirmed by a partial trypsin digestion
assay. This approach has been described previously as a useful tool
to examine protein integrity as a surrogate for correct folding of
mutant proteins [42,47]. The tryptic digestion of the intact Gβ 1 γ 2
dimer yielded only two proteolytic Gβ 1 fragments of 26 kDa and
14 kDa, despite the presence of 32 potential tryptic sites in the
primary sequence (Figure 1). In contrast, thermal denaturation of
the protein prior to tryptic digestion resulted in a protein smear
with a multiplicity of protein fragments indicative of proteolysis
of the unfolded Gβ 1 protein (Figure 1, WT panel, middle lane).
Following this approach, we checked all purified isoprenylated
Gβ 1 γ 2 variants for correct folding and dimerization with Gγ
prior to further functional analysis (Figure 1). Only the Gβ 1K78A γ 2
mutant showed more than two proteolytic fragments (Figure 1,
K78A panel). Nevertheless, purified Gβ 1K78A γ 2 was able to fully
stimulate PI3Kγ activities (see below), including protein kinase
activity. This may indicate that the K78A mutation is not directly
involved in PI3Kγ interactions, regardless of whether or not the
K78A mutation destabilizes the overall Gβ 1 structure. Despite its
enhanced sensitivity towards trypsin treatment, the conformation
of Gβ 1K78A γ 2 may be stabilized upon interaction with PI3Kγ .
The purified Gβ 1 γ 2 variants underwent a first round of
evaluation to determine PI3Kγ lipid kinase activity under
maximal stimulatory conditions. The extent of stimulation of
dimeric p110γ /p101 activity by the different variants was
plotted against the Gβγ -stimulated activity of monomeric p110γ
Figure 2 Ability of Gβ 1 γ 2 variants to stimulate p110γ /p101 or p110γ lipid
kinase activity
The efficiency (V max ) of Gβ 1 γ 2 variants to stimulate lipid kinase activity of p110γ /p101
compared with the efficiency to stimulate p110γ is shown. Maximal stimulation of the enzyme
was tested in the presence of 400 nM Gβ 1 γ 2 for p110γ /p101 or 1000 nM Gβ 1 γ 2 for p110γ .
Assays were performed as described in the Experimental section. The distribution of the Gβ 1 γ 2
variants in the graph allowed description of at least four functionally defined groups.
Gβ 1 γ 2 variants indicated by grey symbols (L55A, K57A, K78A, I80A, K89A, S98A, N119A,
T143A and D186A) stimulate PI3Kγ more or less the same as wild-type (WT) Gβ 1 γ 2 . The
efficiency of W99A, M101A, Y59A, D228A and W332A variants to stimulate p110γ was
significantly reduced (black circle and square symbols), whereas the efficiency to stimulate
p110γ /p101 was unaltered. The L117A and Y145A variants (black triangle symbols) exhibited
reduced efficiency to stimulate both p110γ and p110γ /p101.
(Figure 2). Each symbol reflects an individual Gβ 1 mutant. Their
distribution allowed the assignment of the Gβ 1 γ 2 variants to
different groups, highlighted by grey or black colour coding. The
grey symbols represent the group of Gβ 1 γ 2 variants showing more
or less wild-type features, which can be clearly distinguished from
the mutants represented by black symbols.
c The Authors Journal compilation c 2012 Biochemical Society
854
Figure 3
A. Shymanets and others
Gβ 1 γ 2 variants leave PI3Kγ enzymatically unaltered
The stimulation of lipid kinase activity of p110γ (A) and p110γ /p101 (B) in response to increasing concentrations of Gβ 1 γ 2 variants. The assays were performed as described in the Experimental
section. Stimulation of PI3Kγ enzymatic activities by wild-type (WT) Gβ 1 γ 2 is indicated by continuous line. Gβγ -induced activation of PI3Kγ lipid kinase activity was normalized, as described
in the Experimental section, and illustrated as the percentage of the maximal stimulation by wild-type Gβ 1 γ 2 . (C) Association of PI3Kγ with phospholipid vesicles was increased by Gβ 1 γ 2
variants. Purified recombinant Gβ 1L55A γ 2 and Gβ 1K57A γ 2 variants were tested for their ability to recruit p110γ /p101 to phospholipid vesicles as described in the Experimental section. Assays were
performed in the presence of 400 ng of PI3Kγ . Aliquots of sedimented phospholipid vesicles were subjected to SDS/PAGE followed by immunoblotting using specific antisera. (D) Wild-type Gβ 1 γ 2
significantly enhanced the association of p110γ /p101, but not p110γ , with phospholipid vesicles. The assays were performed in the presence of 400 ng of PI3Kγ . Aliquots of supernatants and
sedimented phospholipid vesicles were subjected to SDS/PAGE followed by immunoblotting using specific antisera as indicated. Note that for a better presentation of the relative protein distribution
within the phospholipid vesicles and aqueous phase, only one third of the supernatant was subjected to SDS/PAGE. Nevertheless, the blots demonstrate a decrease in the signal in the supernatant,
whereas immunoreactivity increased in the pellet with increasing Gβ 1 γ 2 concentrations (p110γ /p101, right-hand panel).
Gβ 1 γ 2 variants with wild-type phenotype
Gβ 1 γ 2 variants with an alanine residue within the Gα-binding
region of Gβ 1 at the positions Leu55 , Lys57 , Lys78 , Ile80 , Lys89 ,
Ser98 , Asn119 or Thr143 stimulated the lipid kinase activity of both
p110γ and p110γ /p101 in a similar manner to the experiments
performed in parallel using wild-type Gβ 1 γ 2 (Figures 3A and
3B). Alanine mutations of these amino acids did not affect the
Gβ 1 γ 2 -dependent recruitment of p110γ /p101 to phospholipid
vesicles (Figure 3C and results not shown). Since the Gβ 1 γ 2 dependent translocation of monomeric p110γ to phospholipid
vesicles (in the pellet) was weak (Figure 3D), we excluded this
aspect from the present study. It is remarkable that this group
of Gβ 1 residues are apparently not essential for the interaction
with PI3Kγ , as they are integral elements for binding to both
the N-terminal (Lys57 , Ser98 , Asn119 and Thr143 ) and switch II
interfaces (Leu55 , Lys78 , Ile80 and Lys89 ) of the GDP-bound Gα
subunit [40,41]. Alanine mutations of these residues have been
shown to inhibit the formation of the heterotrimeric complex with
the Gα subunit and/or modulate the activity of effector proteins,
including ACII (adenylyl cyclase II), PLCβ 2 (phospholipase Cβ 2 ),
GIRK (G-protein-activated inward rectifier potassium channel)
and the Ca2 + channel Cavα1B [42,48,49]. Taken together, these
residues cluster into an interacting surface on Gβ 1 that is involved
in the modulation of effectors other than PI3Kγ .
c The Authors Journal compilation c 2012 Biochemical Society
In addition to the Gβ 1 γ 2 variants showing more or less
indistinguishable effects from wild-type Gβ 1 γ 2 , we identified
variants which differed in their capacity to stimulate PI3Kγ
activity, suggesting that these Gβ 1 residues are important for the
Gβ 1 γ 2 -dependent regulation of PI3Kγ (Figure 2). Within this
group of regulatory relevant mutants, two mutants represented
in Figure 2 by black triangles (L117A and Y145A) can be
distinguished from the mutant groups represented in Figure 2
by black circles (W99A and M101A) and black squares (Y59A,
D228A and W332A) based on their mode of PI3Kγ activation.
Key residues of Gβ 1 necessary for PI3Kγ regulation
Within the panel of mutants tested, two variants attracted the
most attention (Figures 4A and 4B, black triangle symbols).
These mutants, Gβ 1L117A γ 2 and Gβ 1Y145A γ 2 , failed to activate
p110γ at any of concentrations tested (Figure 4A). This finding
points to a direct interacting role of residues Leu117 and Tyr145 in
the activation process of the catalytic p110γ subunit of PI3Kγ .
Similar observations for Gβ 1L117A γ 2 were reported previously for
the regulation of ACII, PLCβ 2 and PLCβ 3 [42,48]. Additionally,
Gβ 1 residues Leu117 and Tyr145 are involved in interactions with
the GRK2 PH (GPCR kinase 2 pleckstrin homology) domain
[50]. Surprisingly Gβ 1Y145A γ 2 regained its ability to stimulate
p110γ when complexed to p101, albeit with significantly lower
Differential modulation of PI3Kγ activities
Figure 4
855
Gβ 1 γ 2 variants with altered characteristics of PI3Kγ lipid kinase activation
Stimulation of lipid kinase activity of p110γ (A) or p110γ /p101 (B) in response to increasing concentrations of Gβ 1 γ 2 variants. The assays were performed as described in the Experimental
section. Intermediate stimulations of PI3Kγ enzymatic activities by Gβ 1Y59A γ 2 , Gβ 1D228A γ 2 and Gβ 1W332A γ 2 variants are indicated by square symbols and grey broken lines. Stimulation of
PI3Kγ enzymatic activities by wild-type (WT) Gβ 1 γ 2 is indicated by a star symbol. Gβγ -induced activation of PI3Kγ lipid kinase activity was normalized, as described in the Experimental
section, and illustrated as the percentage of maximal stimulation by wild-type Gβ 1 γ 2 . (B, inset) Stimulation of lipid kinase activity of p110γ /p87 in response to increasing concentrations
of Gβ 1 γ 2 and Gβ 1W99A γ 2 was determined. (C) Association of PI3Kγ with phospholipid vesicles induced by Gβ 1 γ 2 variants. Shown is the recruitment of p110γ /p101 to phospholipid
vesicles induced by recombinant Gβ 1W99A γ 2 , Gβ 1M101A γ 2 , Gβ 1L117A γ 2 and Gβ 1Y145A γ 2 variants. (D) Binding of Gβ 1WT γ 2 , Gβ 1W99A γ 2 and Gβ 1L117A γ 2 to PI3Kγ subunits. Non-tagged versions
of recombinant Gβ 1 γ 2 variants were co-expressed with His–p110γ or His–p101 in Sf9 cells and purified as described in the Experimental section. Following purification on Ni2 +-NTA Superflow
resin, bound proteins were separated by SDS/PAGE and analysed by immunoblotting using anti-His6 and anti-Gβ 1–4 antisera. Whereas the W99A variant resulted in reduced interaction with the
non-catalytic p101 subunit, substitution of Leu117 for an alanine residue impaired the interaction of Gβ 1 γ 2 dimers with both p110γ and p101 subunits of PI3Kγ .
potency and efficiency (Figure 4B). Correspondingly Gβ 1Y145A γ 2
recruited p110γ /p101 to phospholipid vesicles, although with
less efficiency than wild-type Gβγ (Figure 4C). In contrast, the
Gβ 1L117A γ 2 mutant, which also failed to stimulate p110γ , only
revealed a very weak ability to stimulate it (up to ∼20 %) in
the presence of p101 (Figure 4B). The strong reduction in the
PI3Kγ -stimulatory activity of Gβ 1L117A γ 2 correlated with an
impaired ability to recruit p110γ /p101 to phospholipid vesicles
(Figure 4C). Furthermore in the copurification experiment
(Figure 4D), Gβ 1L117A γ 2 showed remarkably decreased interactions with p101 as compared with wild-type Gβ 1 γ 2 , and did not
apparently copurify with the p110γ subunit of PI3Kγ .
These results readily suggest that the severe reduction in Gβγ
mutant stimulatory capacity was caused by an impairment in the
interaction with PI3Kγ subunits rather than specific interference
in the activation process. This conclusion may be premature
since the significance of both the recruitment and copurification
analysis is limited, since these methods detect static rather than
dynamic protein–protein interactions and their semi-quantitative
character. Therefore we decided to check the interaction of Gβγ
mutants with PI3Kγ by testing the ability of Gβγ to stimulate
the protein kinase activity of PI3Kγ . Although the physiological
role of PI3Kγ autophosphorylation is still unclear, this feature
is attractive to exploit as a read-out because the complexity
of the assay is dramatically reduced due to the identity of the
enzyme and substrate. Previously, we found that Gβγ enhances
the autophosphorylation of Ser1101 of p110γ in a dose-dependent
manner [45,51]. Although basal autophosphorylation is visible in
the absence of lipid vesicles, Gβγ -dependent stimulation requires
their presence [45]. Using this approach, we tested the monomeric
p110γ protein first (Figure 5A). Interestingly, it exhibited a clearly
visible basal phosphate incorporation, which was in the same
stoichiometric range as reported previously [16]. Gβγ stimulated
autophosphorylation of the monomer less than 2-fold (Figure 5A).
In contrast, p101 suppressed the basal autophosphorylation of
PI3Kγ , but also enabled wild-type Gβγ to stimulate protein
kinase activity in a concentration-dependent manner by more
than 12-fold (Figures 5A and 5B). All mutants stimulated the
autophosphorylation of heterodimeric PI3Kγ (Figure 5B and
results not shown). In particular, the Gβ 1L117A γ 2 variant increased
the protein kinase activity of PI3Kγ . This clearly demonstrates
that Gβ 1L117A γ 2 , similar to all other Gβγ mutants, still physically
interacts with PI3Kγ .
Gβ 1 γ 2 variants depend on p101 to stimulate PI3Kγ activity
The mutants Y59A, W332A, D228A, M101A and W99A formed
a set of Gβ 1 γ 2 variants exhibiting decreasing efficiency to
c The Authors Journal compilation c 2012 Biochemical Society
856
Figure 5
A. Shymanets and others
Gβ 1 γ 2 variants stimulating PI3Kγ protein kinase activity
(A) Stimulation of the protein kinase activity of p110γ or p110γ /p101 in response to increasing
concentrations of wild-type Gβ 1 γ 2 was tested. The assays were performed as described in the
Experimental section. Shown are representative autoradiographs from at least three independent
experiments. In contrast with dimeric PI3Kγ , wild-type Gβ 1 γ 2 did not significantly increased
autophosphorylation of p110γ in the absence of p101. Wild-type Gβ 1 γ 2 led to only a 1.6-fold
stimulation above the basal level p110γ protein kinase activity, whereas a 12-fold stimulation
of p110γ /p101 protein kinase activity was found in the presence of Gβ 1 γ 2 . (B) Protein kinase
activity of p110γ /p101 in response to increasing concentrations of Gβ 1WT γ 2 , Gβ 1W99A γ 2 ,
Gβ 1M101A γ 2 , Gβ 1L117A γ 2 and Gβ 1Y145A γ 2 variants. The assays were done in the presence of
40 ng of p110γ /p101, as detailed in the Experimental section, and illustrated as the percentage
of maximal stimulation by wild-type (WT) Gβ 1 γ 2 . Although the ability of the Gβ 1L117A γ 2 variant
to stimulate lipid kinase activity of PI3Kγ is drastically reduced, the efficiency of protein kinase
activity in the presence of the mutant reaches a similar extent as compared with wild-type
Gβ 1 γ 2 .
stimulate the catalytic p110γ subunit in its monomeric form
(Figure 4A, square and circle symbols). Compared with wild-type
Gβ 1 γ 2 , the maximum effect ranged between 48 % (Y59A) and
3 % (W99A). The mutants Y59A, W332A and D228A showed
intermediate efficacy, whereas the potency of stimulation (EC50 )
was similar to wild-type Gβ 1 γ 2 (Figure 4A). The results with the
Gβ 1M101A γ 2 and Gβ 1W99A γ 2 mutants were most striking. In fact,
Gβ 1W99A γ 2 failed to significantly stimulate lipid kinase activity
at all, although it still bound to p110γ (Figures 4A and 4D).
However, we noted that binding of the mutant to p101 was
blunted (Figure 4D). In order to examine whether this finding
has an impact on the membrane recruitment of dimeric PI3Kγ ,
we assessed the recruitment of p110γ /p101 to phospholipid
vesicles by Gβ 1 γ 2 variants (Figure 4C). Gβ 1W99A γ 2 showed the
most diminished recruitment capability, whereas all other Gβ 1 γ 2
mutants of this group (Y59A, M101A, D228A and W332A) were
more efficient (Figure 4C and results not shown).
Much to our surprise, the Gβ 1W99A γ 2 mutant was still able to
efficiently stimulate PI3Kγ in the presence of p101 (Figure 4B).
Actually, under the experimental conditions employed, the noncatalytic p101 subunit fully rescued maximal enzymatic activity
in the presence of Gβ 1W99A γ 2 as well as all other Gβ 1 γ 2 variants of
this set. However, the potencies of the mutants to stimulate PI3Kγ
activity was reduced (from 22.3 nM for D228A to 98.6 nM for
W99A). In order to strengthen the true rescue effect of the p101
subunit, rather than an indirect effect due to the presence of an
extra protein in the reaction mixture, we analysed the stimulation
of lipid kinase activity of p110γ /p87 by the Gβ 1W99A γ 2 mutant.
It is known that the p87 subunit binds the catalytic p110γ
subunit, but is not directly involved in its activation by the Gβγ
dimer [21,22,24]. In contrast with p110γ /p101 (Figure 4B), the
Gβ 1W99A γ 2 mutant almost completely lost its ability to stimulate
p110γ /p87 (Figure 4B, inset).
These results not only validate our current understanding of
the Gβγ -dependent activation of PI3Kγ , but also highlight novel
unexpected aspects. The largely hydrophobic interaction of Gβ 1
with Gα is provided by Trp99 of Gβ 1 [40,41,52]. The fact that Gα
c The Authors Journal compilation c 2012 Biochemical Society
Figure 6
Gβ 1 fingerprint in the regulation of PI3Kγ enzymatic activities
The crystal structure of the Gβγ dimer shows that Gβ exhibits a seven-blade
propeller configuration harbouring seven WD-40 repeats [40,41]. Amino acids within the
Gα–GDP-binding region of Gβ 1 are differently involved in regulation of PI3Kγ and are clustered
in three functionally defined sectors. Sector 1 (Tyr59 , Asp228 and Trp332 , shown in orange) has an
auxiliary role in the p101-dependent stimulation of PI3Kγ . Sector 2 (Trp99 and Met101 , shown
in red) has an essential role in the p101-dependent stimulation of PI3Kγ . Sector 3 (Leu117 and
Tyr145 , shown in blue) is involved in the p101-independent regulation of PI3Kγ . Amino acids
with activity indistinguishable from wild-type are shown in white.
and effectors share a common binding surface on Gβ suggests that
mutation of Trp99 will also disturb the interaction between Gβγ
and its effectors. Indeed, it has been shown that the mutation
of Trp99 to an alanine residue affects the regulation of PLCβ 2 ,
ACII and GIRK1/GIRK4 [42]. Consistently, the catalytic p110γ
subunit of PI3Kγ represents a prototypical Gβγ effector, which is
insensitive to stimulation by the Gβ 1W99A γ 2 mutant. Remarkably
p101 resolved the incapability of the disabled Gβ 1 γ 2 variant to
restore maximal stimulatory PI3Kγ activity. This phenomenon
may be explained by a co-regulatory function of the p101 subunit.
Moreover, the assumed regulatory function of p101 appears to
depend on Gβ 1 γ 2 , even if the G-protein is unable to directly
stimulate the catalytic subunit. These conclusions refine our
current understanding of how Gβ 1 γ 2 regulates PI3Kγ activity
via p101, i.e. in addition to its adapter function which enables the
membrane-bound Gβ 1 γ 2 to recruit cytosolic PI3Kγ , p101 should
also be considered as a Gβ 1 γ 2 -sensitive regulatory subunit.
Specific PI3Kγ fingerprint on the surface of Gβ 1 γ 2
All of the tested Gβ 1 γ 2 variants harbouring mutations within the
Gα–GDP binding region could be divided into two main groups
(Figure 6): (i) variants with the phenotype of wild-type Gβ 1 γ 2 ,
which are not involved in the regulation of PI3Kγ (white coloured
residues, L55A, K57A, K78A, I80A, K89A, S98A, N119A,
T143A and D186A); and (ii) variants with an impact on PI3Kγ
regulation (Y59A, W99A, M101A, L117A, Y145A, D228A and
W332A). Amino acid residues phenotypically resembling wildtype PI3Kγ stimulation (Figure 6, amino acids shown in white)
are integral elements of both the N-terminal (Leu55 , Lys78 , Ile80 and
Lys89 ) and switch interfaces (Lys57 , Ser98 , Asn119 and Thr143 ) on
the surface of Gβ 1 interacting with the GDP-bound Gα subunit
[40,41,53]. Despite the impact of these Gβ 1 γ 2 variants in Gprotein-dependent signalling [42,48,49], they were not critical in
the modulation of PI3Kγ .
Amino acids (Figure 6, shown in colour: Tyr59 , Trp99 , Met101 ,
Leu117 , Tyr145 , Asp228 and Trp332 ) belonging to the Gα/Gβγ switch
interface of Gβ 1 clustered at the Gβ 1 γ 2 –PI3Kγ interaction site.
On the basis of the functional results shown above, the interaction
site is composed of at least three sectors constituted by adjacent
amino acids, which represent functionally defined rather than
structurally defined groups.
Differential modulation of PI3Kγ activities
Sectors 1 and 2 (Figure 6)
59
857
ACKNOWLEDGEMENTS
99
101
228
332
Exchange of Tyr , Trp , Met , Asp or Trp for an alanine
residue results in stimulation of p110γ /p101 with less potency
(Figure 4B), whereas the efficiency of stimulating the p110γ
monomer is reduced (Figures 2 and 4A). The amino acids of this
group can be divided into two functionally defined clusters. Sector
1 clusters amino acids (Tyr59 , Asp228 and Trp332 ) with auxiliary
roles determined by their intermediate effects in the stimulation
of monomeric p110γ (Figure 4A, square symbols and Figure 6,
amino acids shown in orange). In contrast, Sector 2 residues
exchanged for an alanine (Trp99 and Met101 ) lose their ability to
stimulate p110γ (Figure 4A, circle symbols and Figure 6, amino
acids shown in red). All of the residues share an interesting feature:
although alanine mutations partially or completely abrogate their
capability to stimulate p110γ lipid kinase activity, p101 rescued
their stimulating activity on heterodimeric PI3Kγ , arguing for a
coregulatory function of p101 (Figure 4B).
Sector 3 (Figure 6)
The exchange of Leu117 or Tyr145 for an alanine residue allows
for clear phenotypic discrimination from the effects mediated by
the other amino acids (Figures 4A and 4B, triangle symbols, and
Figure 6, amino acids shown in blue). Leu117 or Tyr145 mediate
the stimulation of PI3Kγ , largely independently of p101, thus
underlining the crucial role of these amino acids in the activation
process of p110γ .
Conclusion
We studied the molecular mechanism of Gβ 1 γ 2 -mediated stimulation of PI3Kγ . In our approach, several parameters important
for Gβγ -induced stimulation of PI3Kγ were analysed, including
stimulation of monomeric p110γ , dimeric p110γ /p101 and
phospholipid vesicle recruitment of dimeric p110γ /p101. Using
mutation of amino acids localized in the Gα/Gβγ switch interface,
we determined the fingerprint of those amino acids relevant to
Gβ 1 –PI3Kγ interaction. Accordingly, three amino acid sectors
were defined with distinct impacts on PI3Kγ regulation. The
amino acids of Sectors 1 and 2 all exert p101-dependent PI3Kγ
stimulation. Exchange of these amino acids with alanine resulted
in reduced (Sector 1) or complete loss (Sector 2) of efficacy to
stimulate monomeric p110γ , whereas the efficacy of stimulating
dimeric p110γ /p101 was maintained and only the potency was
reduced, which argues for a Gβγ -dependent regulatory function
of p101. Leu117 and Tyr145 in Sector 3 are involved in the
stimulation of p110γ , which when mutated to an alanine residue
was insufficiently rescued by p101. The results of the present
study validate and extend our earlier observations demonstrating
a direct interaction of Gβ with p110γ and rebut the view of a linear
activation chain, in which p101 solely mediates signals from Gβγ
to p110γ . More interestingly, we have provided the first evidence
that within this dual interaction of Gβγ with PI3Kγ subunits,
p101 functions not only as a sole Gβγ adapter for membrane
anchoring of PI3Kγ , but also exhibits regulatory functions by
participating in the Gβγ -induced activation process of p110γ .
AUTHOR CONTRIBUTION
Aliaksei Shymanets and Bernd Nürnberg designed the research. Aliaksei Shymanets
performed the research. Aliaksei Shymanets, Mohammad R. Ahmadian, Katja T. Kössmeier
and Reinhard Wetzker contributed new reagents/analytical tools. Aliaksei Shymanets,
Mohammad R. Ahmadian, Katja T. Kössmeier, Christian Harteneck and Bernd Nürnberg
analysed the data. Aliaksei Shymanets, Mohammad R. Ahmadian, Christian Harteneck,
Reinhard Wetzker and Bernd Nürnberg wrote the paper.
We thank Professor Heidi Hamm (Vanderbilt University, Nashville, U.S.A.) for providing
the baculoviruses encoding the Gβ 1 mutants. The expert technical assistance of Sonja
Weinmann and Renate Riehle is greatly appreciated. We also thank Dr Roger Williams,
Dr Len Stephens and Dr Phill Hawkins (Babraham Institute, University of Cambridge,
Cambridge, U.K.) for fruitful discussions. All members of the Nürnberg laboratory
previously located in Düsseldorf and in Tübingen are thanked for discussion and support.
FUNDING
The study was financed in part by the Deutsche Forschungsgemeinschaft (DFG).
Mohammad R. Ahmadian and Katja T. Kössmeier were supported by the NGFNplus
program of the German Ministry of Science and Education (BMBF).
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