From the Institute of Biochemistry, University of
Fribourg, CH-1700 Fribourg, Switzerland, ¶ Ludwig Institute for
Cancer Research, London W1W 7BS, United Kingdom,
INSERM U-145,
IFR 50, Faculté de Medecine, 06107 Nice Cedex 2, France, and
** Department of Biochemistry and Molecular Biology, University College
London, London WCE 6BT, United Kingdom
Received for publication, December 15, 2000, and in revised form, February 9, 2001
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ABSTRACT |
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Phosphoinositide 3-kinases (PI3Ks) are dual
specificity lipid and protein kinases. While the
lipid-dependent PI3K downstream signaling is well
characterized, little is known about PI3K protein kinase signaling and
structural determinants of lipid substrate specificity across the
various PI3K classes. Here we show that sequences C-terminal to
the PI3K ATP-binding site determine the lipid substrate specificity of
the class IA PI3K Phosphoinositide 3-kinase lipid products play a central role in
the regulation of a number of cellular processes; the
monophosphorylated PtdIns
3-P1 is a key lipid regulator
of vesicle trafficking in all eukaryotic organisms, including yeast
(1). The polyphosphoinositides PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 are transiently produced in multicellular organisms in response to receptor activation by extracellular stimuli
and, by acting as second messengers, orchestrate a large array of
mitogenic, metabolic, and antiapoptotic responses (reviewed in Refs.
2-4).
Production of 3-phosphorylated phosphoinositides is dependent on the
activity of phosphoinositide 3-kinases (PI3Ks) (5). According to their
substrate specificity and mode of activation, PI3Ks are divided into
three classes (6). Class I PI3Ks phosphorylate, in vitro,
PtdIns, PtdIns 4-P, and PtdIns(4,5)P2 and are placed into
two subclasses depending on their coupling to cell surface receptors.
Class IA heterodimeric PI3Ks, to which the prototypic PI3K In addition to their function as lipid kinases, PI3Ks have an intrinsic
protein kinase activity, of which the physiological significance is
less well characterized. Initial studies showed that p110 The lipid substrate specificity of PI3Ks, as well as of other members
of the phosphoinositide kinases superfamily, appears to be dependent on
activation loop sequences as determined by the following approaches:
(a) sequence swapping on PI3K In this study, we show that swapping of foreign sequences into the
activation loop of p110 Expression Plasmids and Site-directed Mutagenesis--
To
generate hybrid PI3Ks, silent PflMI and KpnI
restriction sites were introduced by the unique site elimination method
(21) into wild type p110 Protein Expression and Transient Transfections--
Human
embryonic kidney (HEK) 293 and COS-7 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
(v/v) fetal calf serum (FCS; Life Technologies, Inc.). HEK 293 cells
were seeded (2 × 106/10-cm dish) 24 h prior to
transfection by the calcium phosphate method (typically with 1 µg of
p85
Heterodimers of p85 Antibodies and Immunoblotting--
Rabbit polyclonal antibodies
directed against wortmannin, p110 Lipid and Protein Kinase Assays--
Lipid kinase assays were
basically carried out as previously described (31). Immobilized
proteins were suspended in a presonicated mixture of 10 µg of
phosphatidylserine and 10 µg of PtdIns, PtdIns 4-P, or
PtdIns(4,5)P2 in 40 µl of reaction buffer (20 mM HEPES, 5 mM MgCl2, pH 7.4).
Sodium cholate (2.3%, w/v) was included in mixtures containing
PtdIns(4,5)P2. The reaction was initiated by the addition
of 10 µl of 50 µM ATP supplemented with 10 µCi of
[
Protein serine kinase activity of PI3K was assayed as in Ref. 14 with
modifications given in Ref. 23. The activities of Myc-PKB and
Myc-p70S6k were determined by using 30 µM
Crosstide peptide as substrate as described (33).
Cellular PI3K Activity--
Transiently transfected COS-7 were
starved overnight in DMEM without FCS and then rinsed with
phosphate-free DMEM (Life Technologies, Inc). Subsequently, cells were
incubated with 250 µCi of [32P]orthophosphate (Hartmann
Analytic) in 1.8 ml of phosphate-free DMEM per 6-cm dish for 3 h
at 37 °C, 5% CO2. Total lipids were extracted according
to Ref. 34 and deacylated for HPLC analysis (32).
Wortmannin Labeling of PI3K and Substrate
Competition--
Glutathione-Sepharose with immobilized
GST-p110 Two-dimensional Phosphopeptide Mapping and Phosphoamino Acid
Analysis--
PI3K
For phosphoaminoacid analysis, the radioactively labeled IRS-1 was
separated as above; HCl hydrolysis and phosphoaminoacid separation were
performed as described (36).
PI3K Structure Modeling--
The model of p110
Differences in accessibility were calculated using the
program NACCESS (76) and where calculated for all residues within 15 Å from the PtdIns(4,5)P2 ligand accessed with a 1.4-Å probe.
Construction and Expression of p110
To establish whether the DFG C-terminal region plays a role in
directing the substrate specificity of the various PI3K classes, we
produced p110 Lipid Kinase Activity of the p110
To assess whether the insertion of sequences from different PI3K
classes affects the substrate specificity of the p110
To investigate their in vivo behavior, p110 Modification of Lipid Kinase Activity Does Not Affect Other
Properties of the p85/p110
GST-p110 Site-directed Mutagenesis Defines Key Residues of the p110
Catalytic Subunit as Determinants of Lipid Substrate
Specificity--
Once we defined the DFG C-terminal region as
the determinant for lipid substrate specificity in p110
The importance of the positive charges provided by Lys973
and Lys980 (Lys942 and Arg949 in
p110 p110
To determine whether p110
Our experiments in 293 cells lead us to conclude that p110 While the catalytic region of PI3Ks is located in the conserved
kinase domain, members of different PI3K classes display a typical
substrate specificity. Indeed, in vitro, class IA
p85/p110 We previously demonstrated that wortmannin inhibits class I PI3Ks
p85/p110 The primary sequence of p110 The above finding and the fact that manipulations of the
activation loop switch p110 Swapping of foreign sequences into p110 The fact that the cII hybrid, opposed to its in vitro
behavior, was unable to produce PtdIns(3,4)P2 in
vivo could not permit us to investigate the different roles played
in signaling cascades by PtdIns(3,4)P2 and
PtdIns(3,4,5)P3, yet the cII and cIII hybrids were
exploited as tools to investigate the contribution that PI3K plays in
signaling either as a lipid kinase or as a protein kinase.
The requirement for PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 is definitely established for the
regulation of the PI3K-PDK-PKB/Akt-p70S6k mitogenic
signaling pathway (66, 67); PtdIns(3,4,5)P3 binds to
and activates phosphoinositidedependent kinases, which in turn phosphorylate and activate PKB/Akt (68-70) and p70S6k
(28). In addition, PKB itself, upon binding to
PtdIns(3,4)P2 via its PH domain (27, 70, 71) provides
further activation signals to p70S6k (72). We studied here
the effect of constitutively activated wild-type p110 The requirement for 3'-phosphoinositides in some PI3K-mediated
signaling pathways does not rule out an involvement of this kinase as a
protein kinase in others. In fact, PI3K The data described here clearly demonstrate that the two activities of
PI3Ks (i.e. the lipid- and protein-phosphorylating activities) are generated by different structural kinase motifs. Moreover, the p110 (p85/p110
). Transfer of such activation loop
sequences from class II PI3Ks, class III PI3Ks, and a related
mammalian target of rapamycin (FRAP) into p110
turns the lipid
substrate specificity of the resulting hybrid protein into that of the
donor protein, while leaving the protein kinase activity unaffected.
All resulting hybrids lacked the ability to produce
phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino
acid substitutions and structure modeling showed that two conserved
positively charged (Lys and Arg) residues in the activation loop are
crucial for the functionality of class I PI3Ks as phosphatidylinositol
4,5-bisphosphate kinases. By transient transfecion of 293 cells,
we show that p110
hybrids, although unable to support
lipid-dependent PI3K signaling, such as activation of
protein kinase B/Akt and p70S6k, retain the
capability to associate with and phosphorylate insulin receptor
substrate-1, with the same specificity and higher efficacy than wild
type PI3K
. Our data lay the basis for the understanding of the class
I PI3K substrate selectivity and for the use of PI3K
hybrids to
dissect PI3K
function as lipid and protein kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(p85
/p110
) belongs, are composed of an adaptor subunit of 50-85
kDa containing two SH2 domains and a tightly bound 110-kDa catalytic
subunit of either the p110
, p110
, or p110
Type. Upon receptor tyrosine kinase activation, the SH2 domains of the adaptor bring the heterodimer to the cell surface, thereby allowing the p110
catalytic subunit to phosphorylate its lipid substrate(s). The class IB
PI3K
is associated to a p101 adapter, which was proposed to mediate
activation via G
subunits of trimeric G proteins (7). The
most recently described class II PI3Ks are monomeric enzymes (~170
kDa in size) containing a C-terminal C2 domain. To date, three
mammalian isoforms have been identified: the ubiquitously expressed
PI3K-C2
(also referred as m-Cpk, p170, or Dm_68D, (8,
9)), PI3K-C2
(10), and the liver-specific isoform PI3K-C2
(11).
Class II PI3Ks preferentially phosphorylate PtdIns and PtdIns 4-P,
while class III PI3K only produces PtdIns 3-P, which is central to
vesicle trafficking (12). Besides, several PI3K-related enzymes exist
that do not have demonstrated lipid kinase activity but are functional
as protein kinases. These include enzymes involved in DNA repair such
as the target of rapamycin (mTOR/FRAP), the catalytic subunit of the
DNA-dependent protein kinase (DNAPK), and
others (for a review, see Ref. 13).
-mediated
phosphorylation on the p85
adapter reduces the lipid kinase activity
of the heterodimer (14). Likewise, a shut-off of activity has been
observed in p110
upon phosphorylation on serine 1039 (15). Recently,
it has also been shown that the protein kinase activity of the class IB
PI3K
is sufficient to activate the mitogen-activated protein kinase
Erk-2, thus overcoming the requirement for 3'-phosphorylated lipids in
this pathway (16). An involvement for protein autophosphorylation in
the activation of the class III PI3K has also been described, via an
intrinsic serine/threonine kinase activity of Vps34 (17). The function of the different PI3Ks is thus determined by their class-specific lipid
substrate specificity and protein kinase activities.
(16), (b)
activation loop swapping between type 1 and type 2 phosphatidylinositol phosphate kinases (18), and (c) molecular modeling studies
on the x-ray-solved structure of PI3K
(19). The activation loop of
phosphoinositide kinases is located C-terminally to the conserved DFG
motif of the catalytic domain and can be viewed as the topological counterpart of the activation loop of protein kinases (20).
permits us to switch its lipid substrate
specificity into that of class II or class III PI3Ks or even to fully
abolish its lipid kinase activity, while leaving the protein kinase
functionality unaltered. We then use the manipulated p110
to dissect
the functionalities of the PI3K
heterodimer as a lipid kinase and as
a protein kinase. Further, we identify by site-directed mutagenesis key
residues of class I PI3K required for phosphorylation of PtdIns(4,
5)P2. Our biochemical data provide a better understanding
of the PI3K substrate specificity, and the chimeric construct described
here should provide a powerful tool to distinguish lipid kinase
versus protein kinase functions of PI3K in signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(22) within codons for GHFLD and RVP,
respectively (see Fig. 1A). Oligonucleotide cassettes coding
for selected amino acid sequences of class I, II, and III PI3Ks and
FRAP as depicted in Fig. 1 were then ligated into the
PflMI/KpnI-digested plasmids. All insertions were
confirmed by DNA sequencing (Microsynth, Balgach, Switzerland) and
transferred as PstI-HindIII fragments to
cytomegalovirus promoter-driven expression vectors pSCT1 (23) and
pSTC-tkGST (24) carrying the p110
gene. Mutated regions were equally
inserted into a p110
with an N-terminal Myc tag and an
extension for a C-terminal isoprenylation sequence from Ha-Ras
(Myc-p110
-CAAX in pSG5; see Ref. 25). The pMT2-based expression
vector for the p85
regulatory subunit was described before (23, 26). Point mutations in the previously described GST-PI3K
wild type and
cII hybrid (16, 24) were obtained by inserting appropriate nucleotide
cassettes into the AvrII and KpnI silent sites
and confirmed by sequencing. The expression plasmids
Myc-p70S6k in pRK5 and Myc-iSH2 in pCMV6 were previously
described (27, 28). Mouse insulin receptor substrate-1 (IRS-1) (29) was
subcloned into pcDNA3 as a HindIII-HindIII fragment.
and 10 µg of p110
expression vectors or 10 µg of
GST-PI3K
expression vector). A medium change 12 h later was
followed by cell lysis at 48 h (23). COS-7 cells (3 × 105/6-cm dish) were seeded 24 h before transfection by
the DEAE-dextran method. One microgram of expression vector was added
to 10 µl of 20 mg/ml DEAE-dextran (Amersham Pharmacia Biotech), and
the volume was brought to 600 µl with PBS. Cells were incubated with the resulting mixture for 30 min, and then 3 ml of DMEM supplemented with 171 µM chloroquine was added. After 3 h, the
medium was exchanged with DMEM supplemented with 10% FCS (v/v). Cells
were lysed or metabolically labeled 48-60 h later.
/p110
were immunoprecipitated from cell
lysates with anti-p85
antibodies and immobilized on protein A-Sepharose (CL-4B; Amersham Pharmacia Biotech). Alternatively, glutathione-Sepharose (4B; Amersham Pharmacia Biotech) was used to
isolate GST-p110
·p85
complexes and GST-PI3K
s.
Sepharose beads were washed twice with lysis buffer (20 mM
Tris·HCl, 138 mM NaCl, 2.7 mM KCl, pH 8.0, supplemented with 5% glycerol, 1 mM MgCl2, 1 mM CaCl2, 1 mM
sodium-o-vanadate, 20 µM leupeptin, 18 µM pepstatin, 1% Nonidet P-40, 5 mM EDTA,
and 20 mM NaF), twice with 0.1 M Tris-HCl plus
0.5 M LiCl (pH 7.4), and twice with the respective reaction
buffers (see below).
, and p85
were used as
previously described (23). Monoclonal anti-Myc antibodies (9E10) were
from Babco, rabbit anti-IRS-1 antibodies were from Upstate
Biotechnology, Inc., and rabbit anti-phospho-PKB Ser473
antibodies were from New England Biolabs. Monoclonal
anti-phosphotyrosine antibodies were raised from hybridoma cells (clone
PY72). Proteins were separated on standard SDS-PAGE and transferred to
polyvinylidene difluoride (Millipore Corp.) according to Ref. 30.
Primary antibodies were detected with species-specific goat anti-rabbit
or anti-mouse peroxidase-conjugated secondary antibodies (Sigma) and
enhanced chemiluminescence (ECL; Pierce).
-32P]ATP (3000 Ci/mmol; Hartmann Analytic) and kept
at 30 °C for 10 min. Reactions were stopped by adding 50 µl of 1 M HCl and 200 µl of chloroform/methanol (1:1, v/v).
Extracted lipids were separated on oxalate-treated silica 60 TLC plates
(Merck) with chloroform/acetone/methanol/acetic acid/water
(80:30:26:24:14, v/v/v/v/v). Phosphorylated lipids were detected by
autoradiography on Kodak X-Omat films or phosphorimaging (GS-525 from
Bio-Rad). The identity of lipid spots was verified by deacylation and
HPLC analysis on a Partisphere SAX column as described by Arcaro and Wymann (32).
·p85 complexes was washed twice with PBS supplemented with
0.5% Triton X-100 and suspended in PBS plus 0.1% Triton X-100.
Wortmannin (1000× stock in Me2SO) was added to a
final concentration of 200 nM and incubated for 15 min on
ice. For substrate competition experiments, immobilized PI3Ks were
incubated with 1 mM ATP or 0.1 mg/ml presonicated PtdIns(4,5)P2 for 15 min at 37 °C before the sample was
put on ice and exposed to wortmannin. Excess inhibitor was removed by two PBS plus 0.5% Triton X-100 washes. SDS-PAGE and immunodetection of
covalently bound wortmannin was carried out as in Ref. 23. Wortmannin
sensitivity of PI3K hybrids was assayed by measuring lipid or protein
serine kinase activity of glutathione Sepharose-bound GST-p110
·p85
after a 15-min incubation with the indicated concentrations of
wortmannin at 37 °C.
·IRS-1 immune complexes subjected to a PI3K
protein kinase assay were separated on a 7.5% SDS-PAGE. After
separation, the gel was thoroughly rinsed with water and exposed
overnight to a Kodak X-Omat film. The portion of the gel containing the radioactive IRS-1 band was excised and digested for 36 h with trypsin as described (35). Tryptic digests (>3000 cpm) were spotted on
a cellulose plate and the two-dimensional separation of the
phosphopeptides consisted of a high voltage electrophoresis (900 V,
2.5 h in 30% formic acid) followed by a TLC separation with
water/2-butanol/acetic acid/pyridine (48:60:12:40, v/v/v/v).
was generated
using QUANTATM with the x-ray structure of PI3K
(19) as the
template. The location and orientation of ATP was directly transferred
from the PI3K
structure to the model of p110
. The activation loop
(not resolved in the PI3K
structure) was modeled by searching
through a proprietary high resolution data base of fragments. The loop
was then energetically minimized locally to alleviate steric
hindrances. This was followed by Steepest Descent global minimization
until the energy leveled out. PtdIns(4,5)P2 was modeled and
docked manually using the SURFACE and GAPS programs of
Laskowski (75). Each orientation was examined subsequently for
putative hydrogen bonds, salt bridges, and bad contacts. Once a
satisfactory initial orientation was obtained, the orientation of the
activation loop was modeled to allow for contacts to be formed with the
PtdIns(4,5)P2. This required only minor changes in
and
angles and an adjustment of the Arg949 side chain. The
new model was then subjected to a steepest descent minimization.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Hybrids--
While the
ATP-binding site of PI3K
was biochemically localized by the
identification of Lys802 as the residue taking part in the
phosphate transfer reaction (23) and most recently confirmed by
crystallographic studies (19), little is known about the productive
interaction of PI3Ks with phosphoinositides. We have shown previously
that wortmannin covalently binds to Lys802 in PI3K
and
that this binding can be prevented by preincubation of PI3Ks with
PtdIns(4,5)P2. This suggests a spatial overlap of the
wortmannin and the PtdIns(4,5)P2-binding site. Putative
PI-interacting residues were found C-terminal to the highly conserved
DFG motif within homology region 1. In p110
, this region contains a
polybasic stretch, 941KKKKFGYKRER951,
characterized by two basic boxes, 941KKKK and
947KRER; class II PI3Ks retain a KRDR box, similar
to the 947KRER of p110
, but do not display homology to
the 941KKKK box; class III members and PI3K-related
proteins (FRAP/mTOR, DNAPK, ATM, and RAD 3 (13)) possess,
in the same region, sequences unrelated to the sequences observed in
the PI3K classes I and II; and, inside the PI3K-related protein group,
these sequences are also unrelated to each other.
hybrids in which the p110
amino acid sequence comprised between 933DFGHF and the conserved
Leu956 was exchanged with corresponding sequences from
class II p170/Cpk (8, 9), class III human Vps34 (37), and the
PI3K-related protein kinase FRAP (38). We hereby refer to these p110
hybrids as cII, cIII, and cIV upon insertion of p170/Cpk, Vps34, and
FRAP sequences, respectively (Fig. 1).
Since the alignment between p110
and Vps34 presents a 5-amino acid
gap and the Vps34 region is rich in prolines, which may have disrupting
effects on the secondary structure of the protein, several cIII hybrids
were constructed; the entire region between 933DFGHFL and
Leu956 was replaced by the sequence from Vps34 (cIII.2) or
shorter pieces (cIII.1 and cIII.3), and arbitrary amino acids (AVAAV or
AV; cIII.4 and cIII.5, respectively) were added to fill the 5-amino
acid gap of the sequence alignment. Due to the absence of significant gaps among p110
, p170/Cpk, and FRAP and from the information gained
in preliminary experiments with the cIII hybrids, a single cII and cIV
hybrid was constructed (Fig. 1B). The p110
hybrids were
transiently expressed in 293 cells as PI3K
heterodimers; p110
was
either expressed untagged or as a GST fusion. The integrity of the
heterodimer was not affected by the insertion of sequences from other
PI3K classes, and the intact heterodimer was recovered either by
immunoprecipitation with anti-p85
antibodies (see Fig. 5A) or glutathione beads fishing of GST-p110
(data not
shown).
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Fig. 1.
PI3K classes and p110
hybrid constructs. A, schematic alignments of
PI3Ks and related enzymes. The homology regions (HR1-HR4)
are shown in gray, and the binding sites for Ras (on class I
PI3K only), wortmannin (Wm), and ATP are indicated by
triangles. B, amino acid sequences of members of
classes I-III and related members (IV, e.g. FRAP) are
aligned with hybrid constructs obtained by manipulation of the
nonconserved region following the DFG motif of the ATP-binding site of
p110
. Class IV included e.g. mTOR and other
PI3K-related protein kinases. The triangle shows the
conserved Leu956.
Hybrids--
We first used
PtdIns as a substrate to evaluate the lipid kinase activity of the
p110
hybrids, since it is an in vitro substrate common to
the three PI3K classes (5, 39). The lipid kinase activity of cIII.1,
cIII.4, and cIII.5 p110
hybrids was 40% with respect to the control
p110
WT, suggesting a minor affinity for the substrate of the
Vps34 sequences inserted. Insertion of the whole Vps34 region comprised
in the 933DFGHFL/Leu956 stretch, although
corresponding to the best matched alignment, gave rise to a protein
almost unable to support PtdIns phosphorylation, equal to for
the hybrid carrying a 2-amino acid deletion (cIII.2 and cIII.3,
respectively). The higher activities were thus observed in the hybrids
maintaining the p110
VPFV sequence N-terminal to Leu956,
as in the cIII.1 hybrid, regardless of further insertion of the AVAAV
or AV sequences (cIII.4 and cIII.5 hybrids). Based on these results and
on the lost specificity toward PtdIns 4-P and PtdIns(4,5)P2
phosphorylation (see below), it appears that the minimal Vps34 amino
acid sequence required to transform p110
into a class III-like lipid
kinase is the 8-amino acid stretch GRDPKPLP (Fig. 1B). The
cII hybrid was more active toward PtdIns phosphorylation (143 ± 38% of the PtdIns (Fig. 2A),
and the cIV hybrid was lipid kinase-inactive. Insertion of the short
region from FRAP (a PI3K-related protein with no lipid kinase activity assigned thus far), rendering p110
unable to behave as a lipid kinase, confirms the idea that the swapped region is involved in lipid
substrate recognition and phosphorylation (Fig. 2).
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Fig. 2.
PI3K activities of
p110 hybrid constructs. The GST or GST
fusion proteins produced from constructs shown in Fig. 1 by
transfection of 293 cells are indicated at the bottom. The
numbers correspond to various constructs of cIII hybrids.
A, GST-p110
·p85 complexes were immobilized on
glutathione beads and subjected to a PI3K assay using PtdIns as a
substrate (see "Experimental Procedures"; mean ± S.E.,
n = 3). B, a typical experiment of PtdIns
3-P formation by the hybrid constructs. C, expression
pattern of GST-p110
hybrids probed with anti-p110
antiserum.
hybrids, in vitro lipid kinase assays were performed using the three
substrates accepted by class I PI3K
. As previously shown (22),
PI3K
WT phosphorylated PtdIns, PtdIns 4-P, and
PtdIns(4,5)P2. The cII hybrid phosphorylated PtdIns and
PtdIns 4-P but not PtdIns(4,5)P2, as described for p170/Cpk
and other class II PI3Ks (8, 9, 40). The lipid kinase-active cIII
hybrids (cIII.1 (Fig. 3) and cIII.4 and
cIII.5 (data not shown)) acquired the substrate specificity of class
III Vps34 (17, 37), only phosphorylating PtdIns. The cIV hybrid and the
cIII.2,3 hybrids, which do not phosphorylate PtdIns (Fig.
2A), were also unable to support PtdIns 4-P and
PtdIns(4,5)P2 phosphorylation (Fig. 3 and data
not shown). The identity of 32P-labeled 3'-phosphorylated
products of Fig. 3 was confirmed by HPLC analysis of the deacylated
radioactive spots. Notably, the two slowly migrating radioactive
products obtained when using PtdIns as substrate both represent a
3'-monophosphorylated phosphoinositide (lyso-PtdIns 3-P and not
PtdIns(3,4)P2 or PtdIns(3,4,5)P3), since deacylation and HPLC separation of each radioactive spot give a single
peak eluting at the same retention time of the upper PtdIns 3-P.
Likewise, the higher Rf signal produced by the cII hybrid upon
presentation of PtdIns 4-P as substrate is PtdIns 3-P. This probably
arises from a PtdIns contamination in the PtdIns 4-P substrate, and it
is produced only by the cII hybrid and only marginally by p110
WT
notwithstanding the higher activity of the former. The nature of the
radioactive spots thus analyzed has been established by comparison with
standards produced by phosphorylation of the lipid substrates with
baculovirus-produced PI3K
WT (data not shown).
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Fig. 3.
Top, altered PI substrate
specificity of p110 hybrid constructs. GST fusion proteins of the
type indicated at the bottom were subjected to PI3K assays
using PtdIns, PtdIns 4-P, or PtdIns(4,5)P2 as substrates.
The arrows on the right indicate the migration
distance of PtdIns 3-P, PtdIns(3,4)P2, or
PtdIns(3,4,5)P3 standards. The origin is indicated. The
asterisk represents an ATP degradation product separating on
an oxalate-treated TLC plate. Bottom, quantitation of the
relative activity of the hybrids compared with the WT for the three
different substrates. (Data for PtdIns as substrate are as in Fig. 2;
data for PtdIns 4-P and PtdIns(4,5)P2 as substrates are
from the shown TLC and typical of at least three independent
experiments).
hybrids were
constitutively activated by recloning into a Myc-tagged p110
bearing a carboxyl-terminal farnesylation signal from Ha-Ras that localizes the
protein to the plasma membrane (Myc-p110
-CAAX (25, 41, 42)). COS-7
cells were transiently transfected with CAAX-boxed p110
hybrids
(Fig. 4B) and serum-starved
overnight to suppress the activity of endogenous PI3Ks. Following
[32P]orthophosphate metabolic labeling, TLC analysis of
extracted lipids from cells transfected with wild type p110
-CAAX
showed production of PtdIns(3,4,5)P3, while, consistent
with the in vitro data, any transfected p110
-CAAX hybrid
failed to produce it (Fig. 4A); subsequent HPLC analysis of
deacylated lipids also demonstrated accumulation of
PtdIns(3,4)P2 in WT p110
-CAAX-transfected cells but not
in cells transfected with any of the hybrids (Fig. 4C). By
HPLC analysis, we never detected any increase in PtdIns 3-P levels
(data not shown), and this is in agreement with the accepted idea that
PtdIns is not an in vivo substrate for class I PI3Ks (see
"Discussion").
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Fig. 4.
PI production in COS-7 cells. COS-7
cells were transfected with the indicated Myc-tagged p110 constructs
(M, mock transfection; KR, Lys802 to
Arg; for chimera, see Fig. 1B) with C-terminal
isoprenylation sequences (CAAX box). A, cells were
metabolically labeled with 32Pi, and lipids
were extracted and analyzed by TLC. The arrows indicate the
location of standards. PI, phosphatidylinositol; PIP,
phosphatidylinositol phosphate; PIP2, phosphatidylinositol
4,5-bisphosphate; PIP3 phosphatidylinositol
3,4,5-trisphosphate. B, in parallel, cells were lysed and
probed with anti-Myc antibodies to evaluate Myc-p110
-CAAX
expression. C, cells treated as for A were
extracted, and lipids were deacylated and subjected to HPLC separation.
Traces from cells transfected with wild type p110
-CAAX are displayed
as a solid line, and traces from cells
transfected with a cII-CAAX construct are shown as a broken
line.
Hybrid Heterodimers--
To prove that
the alteration in lipid substrate specificity of the p110
hybrids is
due to a specific effect of the exchanged activation loops and not to
major structural alterations of the polypeptide caused by the
manipulations performed, we analyzed the autokinase activity of the
hybrids (14, 43) and interaction with the PI3K-specific inhibitor
wortmannin (32). The cII and cIII hybrids efficiently supported p85
serine 608 phosphorylation; a lower, but still significant as compared
with the kinase-dead K802R mutant (23), p85 phosphorylation was
mediated by the cIV hybrid (Fig. 5).
Serine phosphorylation on p85 leads to a feedback negative control on
the lipid kinase activity of the heterodimer (14, 43). Here we observed
the same extent of feedback inhibition on the wild type heterodimer and
p85-p110
heterodimers bearing cII or cIII.1 hybrid catalytic
subunits (Fig. 6).
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Fig. 5.
Protein kinase activity of
p110 hybrids. PI3K
was
immunoprecipitated with anti-p85 antibodies from mock-transfected cells
(M) or cells transfected with p85
and kinase-dead
(KR, Lys802 to Arg mutation), WT, or hybrid
p110
(as indicated at the bottom). A,
expression of the p85
/p110
complex was detected by Coomassie Blue
staining. B, immobilized PI3K
was subjected to a protein
kinase assay (see "Experimental Procedures"), and incorporated
32P was analyzed after reducing SDS-PAGE by
autoradiography.
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Fig. 6.
Feedback control of PI3K
lipid kinase activity by p85 phosphorylation. A,
PI3K
heterodimers were immunoprecipitated from transfected 293 cells
with anti-p85 antiserum and incubated in protein kinase buffer in the
presence (+) or absence (
) of 1 mM ATP. Samples were
subsequently split for a kinase assay in the presence of
[
-32P]ATP (B) or a lipid kinase assay
(mean ± S.E., n = 2) (C). The type of
p110
cotransfected with p85 is indicated at the bottom
(cIII represents cIII.1).
hybrids were subsequently analyzed for their interaction
with wortmannin (Fig. 7A). The
cII and cIII hybrids were covalently modified by wortmannin.
Unexpectedly, the cIV hybrid was not covalently attacked by wortmannin,
both under standard assay conditions (200 nM wortmannin in
PBS plus 0.1% Triton X-100; Fig. 7A) and even in the
absence of detergent in the incubation mixture (data not shown). Once
we demonstrated that wortmannin binding still takes place in the cII
and cIII hybrids, we further investigated the inhibitor-protein
interaction by performing inhibitor-substrate competition experiments.
Preincubation of p85·GST-p110
WT and the cII and cIII.1
hybrids with either ATP or PtdIns(4,5)P2 prior to
wortmannin treatment totally prevented the covalent binding of
wortmannin to the catalytic subunit (Fig. 7C). Effective ATP competition for wortmannin binding, associated with the retained catalytic activity of the heterodimer, both as a lipid and protein kinase, demonstrates that the cII and cIII hybrids were not altered in
the ATP binding site. The observation that PtdIns(4,5)P2
inhibits covalent wortmannin binding to the cII and cIII hybrids was
less expected, these hybrids being unable to phosphorylate this
substrate, and demonstrates that PtdIns(4,5)P2 binding to
the cII and cIII hybrids still takes place on other regions of p110
also required for wortmannin binding. This is in keeping with
structural data obtained on the PI3K
isoform in which the
lipid-binding surface comprises, in addition to the activation loop,
the crevice between the C- and N-terminal lobes of the catalytic domain
(19). Thus, PtdIns(4,5)P2 efficiently competes for
wortmannin binding in the cII and cIII hybrids despite not being
phosphorylated to PtdIns(3,4,5)P3 due to the exchanges made
in the activation loop. Wortmannin binding experiments, although
informative on the specificity of the protein-inhibitor interaction, do
not allow us to test the inhibitory effect of wortmannin on the
enzymatic activity of the protein. To address this point, the lipid
kinase activity of cII and cIII hybrids and autophosphorylation of the
cIV hybrid were measured after preincubation with
increasing wortmannin concentrations; the inhibition curves for cII and
cIII.1 lipid kinase activities and cIV autokinase activity were
identical to the inhibition curve obtained for the WT protein with an
IC50 in the low nanomolar range (Fig. 7D).
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Fig. 7.
Interaction of hybrid PI3Ks with inhibitor
and substrate. PI3K and mutants were isolated as
p85 ·GST-p110
complexes and immobilized on
glutathione-Sepharose. A, immobilized PI3K
preparations
(type indicated below panel B) were
incubated with 200 nM wortmannin (+ Wm) or
Me2SO (
) and analyzed after SDS-PAGE with anti-wortmannin
antiserum on immunoblots (
Wm). B, expression
of p110
was verified by reprobing the membranes with anti-p110
antisera (
p110). C, competition of PI3K
substrates with wortmannin. ATP (1 mM; left) or
presonicated PtdIns(4,5)P2 (0.1 mg/ml; right)
was added to p85
·GST-p110
before the complexes were incubated
with 200 nM wortmannin. Detection of covalently bound
wortmannin was carried out as in A. D, PtdIns 3-P
formation in the presence of the given wortmannin concentrations was
tested for wild type (closed circles), cII
(closed squares), and cIII.1 (closed
triangles) PI3Ks (left panel).
Phosphorylation of immunoprecipitated p85 by coexpressed wild type or
cIV PI3K is shown to the right.
, we
sought to determine which are the basic residues necessary for
discriminating the lipid substrate by inserting single/double amino
acid substitutions. To this end, we employed PI3K
instead of p110
because it has a less positively charged 972YKSF box, which
aligns with the 941KKKK box of p110
(Figs. 1A
and 8A). The conservation of
the second lysine throughout the class I PI3Ks suggests that this is
the key positive residue in this box. Lys973 of PI3K
was
therefore replaced by an aspartic acid, and Lys980 (located
in the 979NKER sequence, which aligns to the
948KRER of p110
) was also replaced by an aspartic acid.
Finally, a lysine-serine motif was reinserted in the PI3K
-cII hybrid
(see Fig. 8A and Ref. 16), thus restoring the positively
charged character of the 972YKSF box in a cII background.
The three mutants (PI3K
K973Q (equivalent to p110
K942),
PI3K
K980Q (equivalent to p110
R949), and cIIQM/KS; Fig.
8A) were transiently expressed as GST fusions in 293 cells. The expression level of the mutants was comparable with that of wild-type GST-PI3K
, and the mutants could undergo
autophosphorylation (Fig. 8B). When tested for lipid kinase
activity, the three mutants were effective in phosphorylating PtdIns
and (albeit to a lesser extent) PtdIns 4-P, as expected for wild-type
p110 and the cII hybrid in both
and
isoforms (Fig. 3).
Modifications in the lipid kinase behavior were instead observed when
PtdIns(4,5)P2 was presented as substrate; both the K973Q
and K980Q mutations yielded a protein unable to produce
PtdIns(3,4,5)P3, while restoration of the positively
charged character of the first basic box lost in the cII background
(cIIQM/KS) restored the ability of the protein to phosphorylate
PtdIns(4,5)P2 (Fig. 9). Such
discrimination of the mutants toward PtdIns(4,
5)P2 phosphorylation was also observed in
reactions were a mixture of PI lipids was used as substrate (data not
shown).
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Fig. 8.
Construction, expression, and protein kinase
activity of GST-PI3K point mutants.
A, sequence alignment highlighting 1) the basic residues in
the activation loop conserved among class I PI3Ks (gray
shading) and 2) the single (or double) amino acid mutation
produced (boldface letters). The GST-PI3K
cII
hybrid is from Ref. 16. B, GST-PI3K
variants were
immobilized on glutathione-Sepharose and subjected to an
autophosphorylation assay. The autophosphorylation pattern of the
mutants is shown at the top (32P), and
the Coomassie stained protein expression pattern is shown at the
bottom (St.).
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Fig. 9.
Altered PI substrate specificity of
GST-PI3K point mutants. The mutants of
the type indicated at the top were subjected to PI3K assays
using PtdIns, PtdIns 4-P, or PtdIns(4,5)P2 as substrates as
indicated. The arrows on the left indicate the
migration distance of PtdIns 3-P, PtdIns(3,4)P2, or
PtdIns(3,4,5)P3 standards. A, quantification of
lipid kinase activity (mean ± S.E., n = 3).
B, typical TLC showing the production of 3-phosphorylated
phosphoinositides.
, respectively) for PtdIns(4,5)P2 turnover was
further supported by model calculations on a structure of p110
(see
"Experimental Procedures") derived from the recently published
crystal structure of the PI3K
catalytic subunit (19).
PtdIns(4,5)P2 could be easily docked into the catalytic
groove of p110
to mediate a close contact of the 3-OH group of the
inositol head group and the
-phosphate group of ATP. The unresolved
activation loop (19) could be rearranged to make contact with the PI
head group; K942p110
ended up interacting with the 5-phosphate
group, and R949p110
interacted with the 4-phosphate group. Energy
minimization did not disrupt this arrangement. The assembled
ATP·PtdIns(4,5)P2·p110
complex tightly incorporated
the two substrates (Fig. 10). This structural complex was then used to estimate the solvent accessibility of various residues in the presence and absence of ATP and
PtdIns(4,5)P2. Calculations were carried out using a 1.4-Å
probe (see "Experimental Procedures"). As positively charged,
PtdIns(4,5)P2-interacting residues Lys776
p110
(19.9% contribution), Lys941 (10.4%),
Lys942 (30.5%), and Arg949 (13.4) were
identified (Table I). Lys776
is conserved in PI3K
but substituted in p110
and p110
by a Met. This Met is, however, embedded between two Lys residues, so that
these might compensate for the missing positive charge. A positively
charged patch at this position is not present in class II and III
PI3Ks. Within the activation loop, only the positive charges
Lys942 and Arg949 (Lys973 and
Lys980 in PI3K
) are present throughout the class I
PI3Ks. Together with the mutational analysis, this clearly illustrates
that these residues provide an important charge compensation upon
PtdIns(4,5)P2 binding.
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Fig. 10.
Model of the p110
substrate complex. A, space-filling model of
PtdIns(4,5)P2 (PIP2) and ATP docked into the
catalytic pocket of p110
, which is shown as a sliced surface plot
(red). Lys942 (K942) and
Arg949 (R949) are shown as space-filling
residues and interact with the 5- and 4-phosphate groups
(numbered) of PtdIns(4,5)P2, respectively.
B, PtdIns(4,5)P2, ATP, Lys942, and
Arg949 are represented as sticks embedded in a
ribbon diagram of p110
(green). The
activation loop is highlighted in red, and the sequence of
the activation loop is given in the same color
(C). Green, carbon; blue, nitrogen;
orange, phosphate; red, oxygen; pink,
hydrogen.
Percentage accessibility of the catalytic pocket of p110
that become accessible to
a 1.4-Å probe when either PtdIns(4,5)P2 (PIP2) or
PIP2 and ATP are removed from a p110
substrate complex are
shown. The columns give the percentage accessibility for the indicated
side chains. The PIP2/ATP column gives the accessibility for
the pocket when both PIP2 and ATP are present. The ATP column
shows the accessibility when PIP2 has been removed, while "No
ligand" represents values in the absence of both ATP and
PIP2. The columns marked
PIP2 and
ATP indicate
inferred changes by removal of the respective ligand (
PIP2 = ATP
PIP2/ATP;
ATP = No ligand
ATP).
Increasing values in
PIP2 thus characterize
PtdIns(4,5)P2-interacting residues.
Hybrids Lose Lipid Kinase but Retain Protein Kinase
Signaling Capabilities--
To study the effect of p110
hybrids on
downstream signaling, we co-transfected COS-7 cells with a Myc-tagged
PKB and p110
-CAAX wild type/hybrids, and PKB activities were assayed
in anti-Myc immunoprecipitates 36-60 h post-transfection. We observed
that Myc-PKB kinase activity was elevated only in cells co-transfected with p110
-CAAX WT and not by the cII and cIII hybrids. Accordingly, Western blot with anti-active PKB Ser473 antibodies showed
a strong activation of Myc-PKB as well as of the endogenous PKB (Fig.
11A). Likewise, we
co-transfected 293 cells with Myc-p70S6k, p110
wild
type/hybrids, and a Myc-tagged inter-SH2 (iSH2) domain of p85, which
constitutively activates the p110
catalytic subunit (44).
Immunoprecipitated Myc-p70S6k from starved cells was
assayed for kinase activity using crosstide peptide as substrate (33).
In this experiment, iSH2-p110
wild type induced a 6-fold activation
of p70S6k, while any other transfected form of PI3K was
unable to activate p70S6k (Fig. 11B). These
data, associated with the previously described in vivo lipid
measurements (Fig. 4), confirm that production of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 is an
absolute requirement for PI3K-mediated activation of PKB/Akt and the
downstream p70S6k, and loss of lipid kinase activity by the
p110
hybrids renders them unable to support PKB/Akt and
p70S6k activation.
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Fig. 11.
Signaling properties of PI3K
hybrids. A, COS-7 cells were co-transfected with the
indicated Myc-tagged p110 CAAX constructs and Myc-PKB. PKB activity
was assayed on anti-Myc immunoprecipitates from overnight starved cells
and is expressed as -fold increase relative to cells transfected with
kinase-dead p110
-CAAX-KR (mean ± S.E., n = 3).
In parallel, PKB activation was monitored by anti-active PKB
Ser473 Western blot on cell lysates. B,
catalytic p110
WT, kinase-dead p110
, and chimeras (as
indicated) were co-transfected with Myc-p70S6k and Myc-iSH2
in 293 cells. Protein kinase activity of Myc-p70S6k was
assayed in anti-Myc immunoprecipitates of starving cells 24-36 h
post-transfection and is displayed relative to p70S6k
activity in cells not transfected with p110
·iSH2 complex
(mean ± S.E., n = 3, top). Expression
levels of Myc-p70S6k and p110
were evaluated by Western
blotting on cell lysates and anti-Myc-iSH2 immunoprecipitates by
anti-Myc and anti-p110
antibody, respectively (bottom).
C, 293 cells were cotransfected with p85
, p110
s, and
IR in an IRS-1 background as indicated, and lysates were
immunoprecipitated with
-p85 antiserum. p85 immunoprecipitates were
subjected to an in vitro protein kinase assay. IRS-1
phosphorylation was quantified after SDS-PAGE separation
(32P, middle) by phosphor imaging
(mean ± S.E., n = 3, bottom).
Expression levels of immunoprecipitated p85 (Coomassie stain,
St.) and associated IRS-1 (
-IRS-1) and
tyrosine phosphorylation of IRS-1 (
-pY) are shown at the
top.
hybrids retain protein kinase-associated
signaling capabilities, we co-transfected 293 cells with the insulin
receptor, IRS-1, and various PI3K
s as described in the legend
to Fig. 11C. In cells transfected with the insulin receptor, p85 immunoprecipitates contained associated IRS-1 in a
tyrosine-phosphorylated form, while no IRS-1 association to p85 (nor
IRS-1 tyrosine phosphorylation) was detected in cells not transfected
with the insulin receptor (Fig. 11C) or transfected with a
catalytically inactive form of the receptor (data not shown). In
vitro PI3K protein kinase assay on p85 immunoprecipitates showed
that the hybrids cII and cIII.1 can phosphorylate IRS-1, as it was
previously known for the PI3K
(45). The tested hybrids were even
more effective than the wild type in phosphorylating IRS-1. We exclude
an occurrence of IRS-1 phosphorylation by other protein kinases such as
mitogen-activated protein kinase (46), GSK-3 (47), or PKB (48) for two
reasons: first, immunoprecipitates from cells transfected with the
catalytically inactive p110
K802R yield a lower IRS-1
phosphorylation; second, IRS-1 phosphorylation was inhibited by 100 nM wortmannin preincubation (data not shown). To evaluate
whether the increase in IRS-1 phosphorylation observed in cells
transfected with the cII and cIII.1 hybrid reflects a higher
phosphorylating activity of the hybrid PI3K toward IRS-1 or the
utilization of random phosphorylation sites, we performed phosphoamino
acid analysis and two-dimensional tryptic phosphopeptide analysis on
phosphorylated IRS-1. Phosphoamino acid analysis indicated that p110
wild type as well as cII (Fig.
12B) or cIII.1 phosphorylate IRS-1 exclusively on serine and threonine residues. The phosphopeptide pattern observed for IRS-1 phosphorylated by PI3K
wild type, cII
(Fig. 12A) and cIII.1 (not shown), is superimposable,
indicating that the more extensive phosphorylation reflects a higher
activity of the cII and cIII.1 hybrids. A difference in the intensity
of the signal is, in fact, observed, thus indicating that certain serine/threonine residues on IRS-1 are more effectively phosphorylated by the cII and cIII.1 hybrids.
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Fig. 12.
A, tryptic phosphopeptide mapping of
IRS-1 in vitro phosphorylated by p110 WT (top)
and cII hybrid (bottom). Electrophoretic separation is on
the horizontal axis, and TLC is shown on the
vertical axis. The origin is marked by a
white spot. B, phospho-amino
acid analysis of phosphorylated IRS-1 showing the presence of
both Ser(P) and Thr(P) residues.
hybrids,
although unable to signal via 3'-phosphorylated lipid products, may
retain signaling capabilities as protein kinases with respect to IRS-1
serine/threonine phosphorylation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -
, and -
and class IB p101·PI3K
heterodimers
phosphorylate PtdIns, PtdIns 4-P, and PtdIns(4,5)P2 (22,
49-51); C2 domain-containing class II PI3Ks are commonly considered to
phosphorylate PtdIns and PtdIns 4-P but not PtdIns(4,5)P2
(8, 9, 11, 40, 52), although a report exists indicating that
PtdIns(4,5)P2 phosphorylation occurs if the substrate is
presented in PtdSer-containing micelles (53), and class III PI3Ks of
the Vps34 type only phosphorylate PtdIns (17, 37). Moreover, protein
kinases belonging to the PI3K superfamily (yeast and mammalian
TOR/FRAP, the ATM gene product, and DNA-dependent protein
kinase among them (see Ref. 54 for a review) have not been shown to
possess any lipid kinase activity (13). The aim of our investigation
was to understand the basis for such different substrate specificity
among the members of the PI3K superfamily and to determine whether
signaling capabilities can be retained if the PI3K p110
catalytic
subunit is manipulated in such a way that its substrate specificity is altered.
and p101·PI3K
at low nanomolar concentration by
binding covalently to a lysine residue required for the phosphate transfer reaction to the lipid substrate (Lys802 in p110
and Lys833 in PI3K
; (23, 24)). Although this lysine is
conserved among PI3Ks, some PI3Ks are less sensitive to wortmannin
inhibition (e.g. IC50 for yeast Vps34 is 3 µM), indicating that noncovalent wortmannin-protein
interactions directing the inhibitor to the conserved lysine residue
occur and determine the effectiveness of nucleophilic attack on the
lysine
-amino group. Moreover, wortmannin binding on p110
and
PI3K
is effectively competed for by preincubation of the enzyme with
its substrates PtdIns(4,5)P2 and ATP, indicating that
wortmannin and the substrates interact within the same region of the
PI3K catalytic subunit, namely the kinase domain. We therefore
hypothesized that the affinity for a specific subset of
phosphoinositides as substrates observed in the various PI3K classes is
driven by specific substrate-recognizing motifs in the kinase domain.
displays, C-terminally to the conserved
933DFG motif, a 941KKKKFGYKRER951 positively
charged stretch resembling a KXnKXKK
(n = 3-7) motif that was proposed to be the
PtdIns(4,5)P2 binding site of gelsolin and
phosphoinositide-specific phospholipases C (55, 56). This region may
therefore be involved in recognizing the polar head group of the
phosphoinositide. Sequence alignment of the C-terminal region to the
DFG motif of PI3Ks shows a basic profile for the class I members,
represented in p110
by the sequences 941KKKK and
948KRER and in PI3K
by the sequences 972YKSF
and 979NKER (Figs. 1B and 8A). Class
II members retain the homology with the KRER sequence but no
relationship to the KKKK sequence, and class III members do not display
any similarity in this region and do not have basic amino acid
stretches (Fig. 1B). Insertion of DFG C-terminal sequences
from class II and III PI3Ks into p110
conferred to p110
the
substrate selectivity of the cII and cIII hybrids (see "Results"
and Fig. 3). Phosphorylation of PtdIns and PtdIns 4-P by the cII hybrid
was increased, and phosphorylation of PtdIns by the cIII.1, cIII.4, and
cIII.5 hybrids lowered, as compared with p110
WT. We speculate that
the swapped activation loops can be responsible for the variation in
the lipid kinase activity of the hybrids. Consistently, the reported
specific activity of the mammalian class II PI3K p170 toward PtdIns is
3 nmol min
1 mg
1 (9)
as compared with 2 nmol min
1
mg
1 for p110
(40). Also, in a direct
comparative study, the PtdIns phosphorylating activity of the human
class III PI3K Vps34 has been shown to be lower than that of PI3K
(37). The complete loss of lipid kinase activity of the cIV hybrid
further demonstrated that the region investigated is specifically
required for recognition and phosphorylation of the lipid substrate
(Fig. 2). Based on the results obtained from the in vitro
lipid kinase assays, we speculate that the 941KKKK and
948KRER boxes are both required for the productive binding
of the 5- and 4-phosphate groups of PtdIns(4,5)P2.
Therefore, elimination of the 941KKKK p110
positively
charged box in the cII hybrid accounts for its inability to
phosphorylate PtdIns(4,5)P2 and is in agreement with
results obtained in a similar mutant of PI3K
(16). The subsequent
production of the two point mutants, PI3K
K973Q and PI3K
K980Q,
confirmed the need for two positively charged amino acids in specific
positions within these boxes in order to achieve PtdIns(4,5)P2 turnover. In keeping with this, the restoring
of a positive charge in the cII background (cIIQM/KS mutant) yielded a
cII variant with reacquired functionality toward
PtdIns(4,5)P2 phosphorylation. Our biochemical data are in
agreement with structure modeling performed on the basis of a p110
substrate complex generated with the recently reported x-ray structure
of a class IB PI3K
(19) as a template. In the PI3K
structure, the
activation loop sequence is not ordered. Together with the mutational
analysis, our modeling data suggest that K942p110
interacts with the
5-phosphate and R949p110
with the 4-phosphate group of
PtdIns(4,5)P2. A simulation of solvent accessibility
further supports the importance of K942p110
and R949p110
. Within
the activation loop, these two positively charged residues seem to be
crucial and sufficient to mediate PtdIns(3,4,5)P3
production. It can therefore be speculated that the activation loop
becomes ordered upon PtdIns(4,5)P2 binding and that solvent
molecules are removed from the 5- and 4-phosphate groups when these
interact with K942p110
and R949p110
. By this process, the
activation loop seals the catalytic pocket off from solvent access,
which is a prerequisite for a successful transfer of the
-phosphate
group of ATP to the 3-OH group of PtdIns(4,5)P2.
substrate specificity indicate that PI
kinase activation loops function in general as dynamic structures to
limit access to the correct substrate among the PIs present in the
cell's membranes. Accordingly, Kunz et al. (18), by
reciprocally exchanging the activation loops of type 1 and type 2 phosphatidylinositol phosphate kinases, where able to invert their
lipid substrate specificities. In a different manner, selective
polyphosphoinositide head group discrimination is observed for the PH
domains of Grp1, Btk, and PDK-1, which exclusively bind
PtdIns(3,4,5)P3, versus the PH domains of DAPP1
and PKB, which bind to both PtdIns(3,4,5)P3 and
PtdIns(3,4)P2 (70), and in the FYVE domains of yeast Vps27p and human early endosome autoantigen 1, which exclusively recognize PtdIns 3-P and not PtdIns 5-P or PtdIns (71, 72).
could, in principle, alter
the structure of the hybrid protein, and the observed variations in
substrate specificity could therefore be due to nonspecific structural
changes. To exclude this possibility, we sought to establish whether
other properties of the enzyme, namely its intrinsic protein kinase
activity (14, 43) and interaction with the inhibitor wortmannin (23),
are unaffected in the p110
hybrids. In vitro
p110
-mediated p85
serine 608 phosphorylation was unaffected in
the cII and cIII hybrids; cIV showed a reduced protein kinase activity
but still significantly higher than the negative control provided by
the p110
K802R kinase-dead catalytic subunit (23). Serine
phosphorylation on the p85
subunit is functionally relevant, since,
by decreasing the lipid kinase activity of the heterodimer, it provides
a feedback control mechanism on the activity of the protein (14, 43).
Here we show that both the hybrids cII and cIII.1 did not display any
variation in this kind of feedback control with respect to the wild
type heterodimer. Wortmannin binding was also unaffected in the cII and
cIII hybrids. Our biochemical observations relative to wortmannin
binding can be rationalized with respect to the reported structure of
PI3K
bound to wortmannin (60). In this recent study, it is shown that wortmannin binds in the ATP binding site of the enzyme with one
face directed toward the N-terminal lobe of the ATP binding site and
the other face packed against the C-terminal lobe, where an interaction
with residues 950, 953, 961, 963, and 964 is occurring. Interestingly,
these residues are placed immediately before the DFG motif, and it is
shown in the same study that mutagenesis of residues N-terminal to the
DFG motif modulates the sensitivity of the enzyme to wortmannin. On the
contrary, our manipulations on the C-terminal side of the DFG motif did
not affect wortmannin binding to the hybrids cII and cIII. Although the
cII and cIII hybrids retained wortmannin binding, the cIV hybrid was
unable to covalently bind wortmannin, even in the absence of detergent in the incubation mixture, which provides a less stringent assay condition. This may be due to the lower wortmannin sensitivity of FRAP
(61). In addition, wortmannin-binding competition experiments showed
that 1) the ATP binding site remains intact after construction of the
hybrid proteins, since ATP effectively competes for wortmannin binding,
and 2) the exchanged regions, although responsible for the differential
phosphorylation of the phosphoinositide polar head group, do not
preclude the binding of phosphoinositides to the surface of the enzyme,
as demonstrated by the competition of PtdIns(4,5)P2 toward
wortmannin binding in the hybrids cII and cIII.1. This is in accord
with structural data on PI3K
in which the C- and N-terminal lobes of
the catalytic domain are described as taking part in the binding of
both lipid substrate and wortmannin (19, 60). To complement the
in vitro experiments, we next explored the in
vivo behavior of the p110
hybrids. To eliminate interference
from endogenous PI3Ks, we took advantage of an agonist-independent
constitutively active form of p110
(Myc-p110
-CAAX; Ref. 25); WT
and K802R kinase-dead as well as cII, cIII.1, cIII.2, and cIV
Myc-p110
-CAAX were tested for the in vivo production of
3'-phosphorylated phosphoinositides in serum-starved COS-7 cells. Cells
transfected with WT Myc-p110
-CAAX accumulated
PtdIns(3,4)P2 and PtdIns(3,4,5)P3, while these
lipids were not produced in cells transfected with any of the p110
hybrids. In our 32P labeling experiments, we never observed
changes in PtdIns 3-P levels, irrespective of the transfected
p110
-CAAX. Thus, class I PI3Ks do not behave, in vivo, as
PtdIns kinases, although PI3K activities are often measured in
vitro by presenting PtdIns as substrate. The fact that the cII
hybrid was unable to produce PtdIns(3,4)P2 implies that
also PtdIns 4-P may be not an in vivo substrate for class I
PI3Ks, and the appearance of PtdIns(3,4)P2 upon
p110
-CAAX WT overexpression may be due to SHIP-dependent 5' dephosphorylation of PtdIns(3,4,5)P3 (73) rather than
phosphorylation of PtdIns 4-P. Accordingly, several lines of evidence
place PtdIns(4,5)P2 as the unique in vivo
substrate for class I PI3Ks (7, 27, 63-65), and our data from
metabolic labeling experiments are consistent with this idea, since
none of the hybrids could produce PtdIns(3,4,5)P3 or the
less phosphorylated PtdIns(3,4)P2. Nevertheless, the reason why class I PI3Ks can phosphorylate PtdIns, PtdIns 4-P, and
PtdIns(4,5)P2 in vitro but exclusively
PtdIns(4,5)P2 in vivo is not yet understood. A
report indicates that this may be due to the fact that in
vivo substrate presentation to PI3Ks is carried out by a
phosphatidylinositol transfer protein, which may thus regulate the
in vivo preferential utilization of
PtdIns(4,5)P2 (74).
and p110
hybrids on PKB and p70S6k activation, in transiently
transfected COS-7 and 293 cells, respectively, and found that none of
the hybrids tested could activate p70S6k, in keeping with
the above.
protein kinase activity has
been implicated in IRS-1 serine phosphorylation in insulin-treated
adipocytes (45) and in STAT3 and IRS-1 phosphorylation upon activation
of the type 1 IFN receptor by IFN-
(73, 74). In addition, previous
work from our laboratory demonstrated that PI3K
-mediated activation
of the mitogen-activated protein kinase Erk-2 relies solely on PI3K
protein kinase action (16). Now, we show here that p110
hybrids,
although unable to activate PKB and p70S6k, still retain
the capability to associate and in vitro phosphorylate the
insulin receptor substrate IRS-1 to an even bigger extent than the
p85/p110
wild type. By two-dimensional phosphopeptide mapping, we
also show that the increased IRS-1 phosphorylation supported by the cII
and cIII.1 hybrid as compared with p110
wild type reflects a higher
activity of the hybrids rather than a utilization of nonspecific
phosphorylation sites. Finally, by phosphoamino acid analysis, we show
that phosphorylation takes place on serine and threonine residues (Fig.
12B).
hybrids that we have designed will allow further investigation of whether PI3K
protein kinase activity is per se sufficient to sustain known PI3K signaling pathways and
open the possibly of uncovering new PI3K-dependent
signaling events not requiring production of 3'-phosphorylated phosphoinositides.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Downward for the Myc-p110-CAAX
construct; T. Franke, B. Hemmings, and G. Thomas for PKB/Akt and
p70S6k expression plasmids; S. Keller for the IRS-1
cDNA; and B. Hemmings for crosstide peptide and help with the
PKB/Akt assay. We thank S. Bonnafous and C. Filloux for help with
two-dimensional map experiments and Piers Gaffney and Roger Williams
for advice during the modeling of the
PtdIns(4,5)P2·PI3K
complex.
![]() |
FOOTNOTES |
---|
* This work was supported by Swiss Cancer League Grant SKL 780-2-1999 and a Human Frontier Science Program grant (to M. P. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of "Swiss National Science Foundation" predoctoral fellowship (3100-50506.97; to M. P. W.) and postdoctoral fellowship. Present address: INSERM U-145, Faculté de Médecine, Av. de Valmbrose, 06107 Nice Cedex 2, France.
To whom correspondence should be addressed: Inst. of
Biochemistry, Rue du Musée 5, CH-1700 Fribourg, Switzerland.
Tel.: 41 26 300 86 55; Fax: 41 26 300 9735; E-mail:
MatthiasPaul.Wymann@UniFR.ch.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011330200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PtdIns 3-P, phosphatidylinositol 3-phosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns 4-P, phosphatidylinositol 4-phosphate; PtdIns(4, 5)P2 or PIP2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphoinositide 3-kinase; SH2, Src homology 2; iSH2, inter-SH2; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; IRS, insulin receptor substrate; PKB, protein kinase B; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PI, phosphatidylinositol; HPLC, high pressure liquid chromatography; S6k, S6 kinase; cI to cIV, class I to IV, respectively.
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