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Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021 [M-M. G., K. H. K., H. H.]; Department of Medicine [P. K.] and Cellular Biochemistry and Biophysics Program [H. Y., N. P. P.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021; and Osaka Bioscience Institute, Osaka 565-0874, Japan [H. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The PTEN gene encodes a 403-amino acid phosphatase that acts
on both polypeptide and
PIP33
substrates (6
, 7)
. The generation of
PIP3 by the PI-3 kinase is the key event
in activating multiple downstream pathways (reviewed in Ref.
8
). These include the activation of PKB/Akt
promoting survival signals, of p70S6-kinase involved in
G1 cell cycle transition, of Tec-family
nonreceptor tyrosine kinases inducing the activation of PLC-
and calcium release from intracellular stores, and of the small G
protein Rac mediating cytoskeletal rearrangements. The
inactivation of both PTEN alleles in glioblastoma causes
constitutive activation of the PKB/Akt downstream effector of the PI-3
kinase (9)
. The degree of PKB/Akt activation correlates to
the transformed phenotype in glioblastoma cells (10)
and
use of a constitutively active PKB/Akt form in PTEN-suppressed breast
cancer cells rescues these cells from apoptosis (11)
,
suggesting that PTEN exerts its tumor-suppressor function by negatively
regulating the PI-3 kinase-PKB signaling pathway.
The biological effects of PTEN on tumor cells are different depending on the cell type. In glioblastoma cells, PTEN induces cell-cycle arrest in G1 (12 , 13) , whereas in LNCaP prostate cancer cells, it induces apoptosis (14) ; both of these effects depend on the phosphatase activity of the protein.
In addition to the amino-terminal phosphatase domain, the PTEN crystal
structure revealed the presence of a C2 COOH-terminal domain
(15)
. C2 domains bind phospholipid membranes
(16)
, and we have shown that the PTEN C2 domain that is
formed by two anti-parallel ß-sheets associates with lipids by a
Ca2+-independent novel mechanism involving
stretches of basic residues situated in two regions (CBR3 and
C
2) connecting the ß-strands (15)
. The mutagenesis of
clusters of these residues impaired the tumor-suppressor function of
PTEN supporting the lipid-binding role of the C2 domain
(15)
. Because the disruption of the lipid-binding function
would require multiple hits, there are no reported combined mutations
of these residues in tumors. However, a subset of the tumor-derived
mutations are situated in the loops interconnecting the ß strands of
the C2 domain, and in this study we present a detailed analysis of the
functional and biological effects of these mutations. We also propose a
role for the PTEN C2 domain in the productive positioning of the
phosphatase active site with respect to the phosphoinositide
substrate.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PTEN-L345Q, PTEN-351,
PTEN-H93A, M-C2, and M-CBR3 in the pCX retroviral vector were
described previously (10
, 15)
. In M-C2, the amino acid
sequence 327-KANKDKANR-335 is replaced with 327-AAGADAANA-335 and in
M-CBR3 the sequence 263-KMLKKDK-269 is changed to 263-AAGAADA-269. The
myristoylation signal of v-Src, MGSSKSKPKDPSQR
(17)
, was added by PCR at the NH2
terminus of wild-type PTEN and of M-CBR3, resulting in the Myr-PTEN and
Myr-CBR3 constructs, respectively. The mutants PTEN-Y68H, PTEN-L186V,
PTEN-P204S, PTEN-S227F, PTEN-G251C, PTEN-K289E, and PTEN-D331G were
obtained by PCR with mutated primers and cloned with an
N-terminal Myc tag in the pCX vector. The L345Q
mutation was introduced by PCR in the mutant M-CBR3 cDNA, resulting in
the double mutant CBR3-L345Q. In the mutants PTEN-T319,
PTEN-G251C, and PTEN-D331G, the COOH-terminal PEST
sequences were deleted by inserting a stop codon after the amino acid
351. This truncation resulted in the deletion constructs T319-351,
G251C-351, and D331G-351. For transient transfections, the point
mutants PTEN-P204S and PTEN-G251C were expressed in pFLAG-CMV-2 vector
(Kodak).
Cell Growth Assays.
U-87 MG glioblastoma cell line (American Type Culture Collection),
COS-7, and Bosc23 cells were grown in DMEM growth medium with
10% FCS. The LNCaP prostate cancer cell line (gift of Makoto Sumitomo)
was maintained in RPMI 1640 supplemented with 10% FCS.
The protocols for transfection, retroviral infection with amphotropic defective retroviruses, stable expression of PTEN proteins, cell proliferation, and soft agar colony assays were described in detail elsewhere (10) .
Protein Analysis.
Cell lysis, immunoprecipitation, immunoblotting, and pulse-chase assays
were performed as described previously (10
, 18)
.
Antibodies were obtained as follows: anti-PTEN A2B1 monoclonal antibody
from Chemicon; M2 anti-FLAG from Kodak; anti-Myc from
Invitrogen; anti-Myc 9E10 from Calbiochem; and
anti-phosphoS473 Akt/PKB and anti-Akt/PKB from New England Biolabs.
Phosphatase Assay.
The phosphatase reactions with water-soluble
diC8-PIP3 (Echelon) were
performed with immunoprecipitated proteins from cells as described
(10)
.
Immunofluorescence.
U87-MG cells (2 x 104) were
plated on poly-D-lysine-coated glass coverslips (Becton
Dickinson) in growth medium and left to adhere overnight. The cells
were fixed in 3% formaldehyde for 30 min, permeabilized with 0.2%
Triton X-100 in PBS for 5 min, blocked with 50 mM glycine
in PBS for 10 min, incubated with the anti-PTEN antibody for 1 h
and with the FITC-conjugated anti-mouse secondary antibody
(Jackson ImmunoResearch Laboratories) for 1 h. All of the
incubations were performed at room temperature. The cells were washed
twice in PBS between the incubations and four times for 5 min in PBS
containing 2 mg/ml gelatin after the incubations with the antibodies.
The cells were mounted in Vectashield (Vector Laboratories) and
pictures were taken with a Nikon immunofluorescence microscope coupled
to a charge-coupled device color camera.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 between the seventh and eighth
ß-strands; and (c) Cß1/2 between the first and second
ß-strands (Fig. 1)
. We have shown
previously that the mutations of the basic and hydrophobic residues at
the tip of the CBR3 and C2 loops affect the lipid membrane-binding
and the tumor-suppressor function of PTEN (15)
.
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. The expression of the
myristylated forms of both wild-type PTEN and M-CBR3 changed the cell
morphology and the cellular localization of the protein (Fig. 2A)
. The cells appeared flattened and, in contrast to the
nonmyristylated proteins that were localized mostly in the perinuclear
cytoplasm, the myristylated proteins were distributed all over the
cell, including the fine processes extending from the cell body. The
perinuclear granular staining of the myristylated forms may be derived
from Golgi and/or endosomal localization. The myristoylation signal
added to wild-type PTEN induced a drastic proliferation arrest,
increasing PTEN tumor-suppressor function probably by more efficient
membrane targeting (Fig. 2B)
. In contrast, its addition in
Myr-CBR3 altered moderately the growth effect of M-CBR3 (Fig. 2B and C)
. Although Myr-CBR3 suppressed the tumor
growth more efficiently than did M-CBR3 (Fig. 2C)
, the full
tumor-suppressor ability of wild-type PTEN was not rescued by membrane
targeting, suggesting that the CBR3 loop might function to orient
productively the active site toward the substrate.
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). The mutants PTEN-L186V, PTEN-P204S, PTEN-S227F, PTEN-G251C,
PTEN-K289E, and PTEN-D331G, which each contained a change in a
different loop connecting C2 ß-strands, were expressed stably in
U87-MG glioblastoma cells (Fig. 3A)
. They had decreased
expression levels compared with wild-type PTEN except for PTEN-K289E,
indicating that mutations situated in the loops of the C2 domain affect
the protein stability. To ascertain that mutations in the C2 domain
loops affect the protein stability by increasing the degradation rate
similarly to mutations in the C2 domain ß-strands (10)
,
pulse-chase analysis of protein turnover was performed for PTEN-P204S
and PTEN-G251C loop mutants (Fig. 3B)
. The rate of
degradation, represented by the slope of the curve, was considerably
higher for the loop mutants than for wild-type PTEN, showing that
protein degradation is the cause for the reduced protein stability of
the C2 domain mutants.
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and decreased the
tumor-suppressor activity of PTEN (not shown), indicating that
misfolding of both domains has a destabilizing effect on the entire
protein.
In contrast to the effects on the protein stability, the ability to
dephosphorylate a water-soluble PIP3 analogue was
preserved for most of the C2 loop mutants (Fig. 3D)
. Only
two of the loop mutants, PTEN-P204S and PTEN-G251C, which had also the
lowest expression level, completely lost phosphatase activity,
indicating profound structural changes of the whole protein.
The loop mutants showing phosphatase activity had a slightly higher
proliferation advantage in U87-MG cells relative to PTEN, but their
tumor growth in soft agar was rather similar to PTEN (Fig. 4)
. The two mutants lacking phosphatase
activity had impaired tumor-suppressor abilities. PTEN-G251C showed an
intermediate tumor growth phenotype, behaving similarly to mutants in
ß-strands (PTEN-L345Q) or to the lipid-binding deficient mutants
(M-C2 and M-CBR3; Fig. 4
). The PTEN-P204S mutant presented a
complete inability to suppress tumor growth (Figs. 2C
and 4)
, providing the first example of a total loss of function for a
C2-domain point mutant.
|
. We observed that
L186 is entirely solvent when exposed, and has no apparent
structure-stabilizing role consistent with its reduced destabilizing
effect (Fig. 3A)
. S227 is situated on the edge of the ß
sheet, and because its side chain is involved in stabilizing one of the
ß-strands, its mutation to phenylalanine would be expected to cause
local structural defects. The K289 residue belongs to a loosely
structured loop that was not included in the crystallization construct.
Although the K289E mutation reversed the charge of the amino acid, the
mutant protein had a high expression level, suggesting no alteration of
the structure. The D331G mutation significantly reduced the expression
level of the protein consistent with its structuring role by
stabilizing the turn at the end of the C2 helix (Fig. 3A)
. In the case of the G251C mutation, its localization
near the interface between the phosphatase and the C2 domain
(15)
most likely explains the destabilization of the
domains and the phosphatase activity loss (Fig. 3)
.
P204 is positioned in the ß1/2 loop that packs with both CBR3
and C2 loops. Because its side chain reaches into the hydrophobic
core, its replacement with a polar one in the P204S mutant would
disrupt the packing of the CBR3 and C2 loops and possibly more of
the structure. On the basis of these putative effects on the structure,
we attempted to explain the complete loss of tumor suppression for
PTEN-P204S as a consequence of two concurrent defects: one of protein
folding coupled to one of membrane binding. If this hypothesis were
correct, these defects which, taken apart, affect only partially the
tumor-suppressor function, when combined, would sum up and entirely
abrogate the tumor suppression. To demonstrate this, we introduced the
L345Q destabilizing mutation into the membrane binding-deficient
mutant M-CBR3 and analyzed the tumor growth of the double mutant
CBR3-L345Q. The presence of both defects totally inactivated the
tumor-suppression function of PTEN similarly to PTEN-P204S (Fig. 4)
.
Partial Recovery of Tumor Suppression for C2 Domain P204S Mutant
But Not for Phosphatase Domain Mutant in LNCaP Cells.
The C2 domain mutant P204S differed from the other C2 domain point
mutants by presenting a total loss of tumor suppression in the U87-MG
glioblastoma cells similarly to mutants in the phosphatase domain (Fig. 4)
. Surprisingly, when we expressed this mutant in LNCaP prostate
cancer cells, it presented partial tumor suppressor ability, inducing
cell death and a decrease in proliferation (Fig. 5)
. These results suggested that the
P204S mutation induced more or less severe structural defects depending
on the cellular context. The PTEN-H93A mutant in the phosphatase domain
maintained its loss of tumor suppression in LNCaP cells almost to the
level of the vector control consistent with the proposed role of H93A
as a catalytic site residue.
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ß7-strand mutant as well as in the
PTEN-G251C and PTEN-D331G loop mutants had no effect on their stability
(Fig. 6A)
.
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-351, G251C-351, and D331G-351 presented increased tumor
suppression compared with their nondeleted counterparts in both
proliferation and soft agar assays (Fig. 6A)
, suggesting a
structural role for the PEST region.
We have shown that the level of PKB/Akt activation correlates to the
tumor-suppressor phenotype of PTEN mutants in U87-MG cells
(10)
. For the PEST-deletion mutants, we also found
correlation between PKB/Akt activation and tumor-growth (Fig. 6B)
. Depending on the integrity of the C2 domain, the
deletion of the PEST sequences from wild-type PTEN or from C2-domain
mutants had opposite effects on the activation of PKB/Akt. For the
lipid-binding-deficient mutants M-C2 and M-CBR3, which had
phosphatase activity toward water-soluble PIP3
but lost the ability to suppress tumor growth, the activation of
PKB/Akt correlated to the growth phenotype (Fig. 6B)
,
indicating decreased in vivo lipid phosphatase activity.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(25)
. For membrane binding, in addition to the C2 domain,
PLC also has a PH domain that is flexibly connected to the rest of
the enzyme. The mechanism that was proposed for PLC membrane
attachment involved membrane tethering by the PH domain and then
protein fixation to the membrane by the C2 domain (25)
. In
the case of PTEN, a similar mechanism could operate, and we showed here
that the C2 domain does more than simple membrane recruitment and is
likely involved in the productive positioning of the active site in a
manner analogous to that proposed for the C2 domain of PLC. A role
for membrane tethering could be postulated for the PDZ-binding
motif, but additional evidence is needed to ascertain the cooperation
between the C2 domain and the PDZ motif in membrane association. The tumor-suppressor function of PTEN is impaired in tumors by mutations occurring in the phosphatase or in the C2 domains. To assess the importance of the membrane-binding function of PTEN C2 domain for tumor evolution we analyzed the biological effect of C2 tumor-derived missense mutations. These mutations fall in two categories: (a) mutations disrupting the hydrophobic ß-sheet core; and (b) mutations at the level of the loops connecting the ß-strands of the C2 domain. We have previously shown that the disruption of the C2 core by mutations in the ß-strands impairs the tumor-suppressor function by decreasing the phosphatase activity of the protein (10) . From a structural perspective, the mutations in the hydrophobic core of the C2 domain represent significant changes that could lead to the destabilization of the folded state of the C2 domain with consequences on the entire protein. On the other hand, most of the mutations in the loops are not expected to cause large-scale destabilization of the folded state of PTEN. Rather, it is likely that these mutations will create local structural defects involving small shifts in local elements or the local loss of structure. We introduced tumor-derived mutations in almost every loop of the C2 domain and found that they variably affect the tumor-suppressor function. Almost all of the changes also destabilized the protein to different extents, indicating an alteration of the folding. However, four of six examined loop mutations (L186V, S227F, K289E, and D331G) did not significantly change the PTEN growth-suppression in the U87-MG glioblastoma cell line. Only two of the loop mutations, G251C and P204S, impaired PTEN tumor-suppressor function.
The loss of the tumor-suppression for PTEN-G251C was partial and comparable with that induced by mutations affecting the C2 hydrophobic core. These mutants with intermediate tumor-growth phenotype lacked in vitro phosphatase activity toward PIP3 but also toward PI 3,4-bisphosphate and PI 3,5-bisphosphate (not shown). Because they appeared to suppress slightly the activation of PKB/Akt, it is likely that these mutants may have a residual phosphatase activity within the cells that cannot be detected in vitro. Unlike the other C2 mutants, PTEN-P204S mutant had a total loss of tumor suppression. On the basis of structural predictions, we assumed that both phosphatase activity impairment and membrane-binding loss are the cause. By concomitantly introducing distinct mutations ablating each of these functions, we observed that they confer an additive effect on tumor suppression. This double inactivation may also be the cause of the total loss of tumor suppression observed for C2 truncation mutations (10) , which are the most frequent genetic events affecting the C2 domain.
The evidence that a mutation in the C2 domain does not determine a fixed structural defect came from the P204S mutant that altered differently the cell growth in different cell types. The observed variations in phenotype could be caused by interactions between PTEN and cell-type specific factors resulting in structural changes affecting its stability and phosphatase activity. Very recently, two groups cloned two PDZ domain-containing proteins that, upon binding to PTEN, stabilized the protein and modulated its ability to inactivate PKB/Akt (27 , 28) . It will be interesting to investigate whether the interaction between PTEN and PDZ domain-containing proteins is the one responsible for the variations in phenotype that we observed.
The differences in tumor suppression with the same mutant protein (PTEN-P204S) in different cell lines suggested a possible explanation for the lack of a significant tumor suppression loss for four of six C2-loop mutants. With the exception of PTEN-P204S, which was detected in glioblastoma (29) , the other mutations were detected in endometrial carcinoma, D331G and S227F (30) , breast carcinoma, L186V (20) , lung cancer, G251C (31 , 32) , or Cowden’s disease, K289E (33) . Because all these mutants that developed in heterogeneous tissues were tested in a single cell line, it is reasonable to conceive that their real tumor-suppressor ability might be slightly different from the one we detected.
Another indication about the possibility to modulate the tumor-suppressor function for PTEN C2 domain mutants came from the analysis of the deletion of the last 53 amino acids of PTEN. This region contains two PEST sequences and a terminal three-residue PDZ-binding motif. The deletion of the PDZ-binding motif alone had no effect on the tumor growth in U87-MG cells, whereas the deletion of the whole region decreased slightly the tumor-suppressor function (10) . Surprisingly, the same deletion in three C2-domain mutants increased the tumor-suppressor function without increasing the expression level of the proteins. The mechanism appears to be related to an increase in the in vivo phosphatase activity of these mutants, as shown by the decrease in the PKB/Akt activation. While this paper was under review, a report showed that constitutive phosphorylation of three residues situated in the second PEST sequence decreases PTEN tumor-suppressor function (34) . Removal of these residues in the C2-domain mutants most likely accounts for the increased tumor suppression. It seems that the alleviation of the structural defects in PTEN C2-domain mutants by modifying the PEST tail might be a possible conformational intervention similar to the one undertaken for the p53 tumor suppressor (35) .
Our study showed that the PTEN C2 domain functions to tightly regulate the catalytic activity of the protein. This regulation depends upon structural constraints that are altered by tumor-derived mutations and modified in different cellular contexts.
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FOOTNOTES |
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1 M. M. G. was supported by fellowships from the
Medical Research Council of Canada and National Cancer Institute
CA09673, and N. P. acknowledges support from the Howard Hughes Medical
Institute and the NIH. 
2 To whom requests for reprints should be
addressed, at The Rockefeller University, Box 98, 1230 York Avenue, New
York, NY 10021. Phone: (212) 327-7323; Fax: (212) 327-7319; E-mail: georgem{at}rockvax.rockefeller.edu
3 The abbreviations used are: PIP3,
phosphatidylinositol 3,4,5-trisphosphate; PI-3 kinase,
phosphatidylinositol-3-OH kinase; PLC, phospholipase C; PKB/Akt,
protein kinase B; PEST sequences, sequences rich in proline, glutamic
acid, serine, and threonine; PDZ domains, domains present in
PSD-95/Dlg/Zo-1 proteins.
Received 6/ 5/00. Accepted 10/25/00.
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