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Review article
PTEN:
The down side of PI 3-kinase signalling
Nick R. Leslie*, C. Peter Downes
Division of Cell Signalling, School of Life Sciences, Medical Sciences Institute, University of Dundee, Dundee, DD1 5EH Scotland, UK
Received 6 June 2001; accepted 14 August 2001
Abstract
The PTEN tumour suppressor protein is a phosphoinositide 3-phosphatase that, by metabolising phosphatidylinositol 3,4,5-trisphosphate
(PtdIns(3,4,5)P3), acts in direct antagonism to growth factor stimulated PI 3-kinases. Awealth of data has now illuminated pathways that can
be controlled by PTEN through PtdIns(3,4,5)P3, some of which, when deregulated, give a selective advantage to tumour cells. Early studies
of PTEN showed that its activity was able to promote cell cycle arrest and apoptosis and inhibit cell motility, but more recent data have
identified other functional consequences of PTEN action, such as effects on the regulation of angiogenesis. The structure of PTEN includes
several features not seen in related protein phosphatases, which adapt the enzyme to act efficiently as a lipid phosphatase, including a C2
domain tightly associated with the phosphatase domain, and a broader and deeper active site pocket. Several pieces of data indicate that
PTEN is a principal regulator of the cellular levels of PtdIns(3,4,5)P3, but work is only just beginning to uncover mechanisms by which the
cellular activity of PTEN can be controlled. There also remains the vexing question of whether any of PTEN’s cellular functions reflect its
evolutionary roots as a member of the protein tyrosine phosphatase superfamily. D 2002 Elsevier Science Inc. All rights reserved.
Keywords: Phosphoinositide; Tumour suppressor; Phosphatase; Lipid signalling
1. Introduction
1.1. Lipid signalling and the role of PtdIns(3,4,5)P3
Many different phosphorylated derivatives of the lipid,
phosphatidylinositol (PtdIns), exist in cells and play diverse
roles in cellular signalling. These inositol phospholipids
differ in both the number and distribution of phosphate
groups around the inositol ring, resulting in at least eight
naturally occurring phosphoinositide species. The versatility
of these molecules as cellular signals results from the ability
of lipid-binding protein domains (such as pleckstrin homo-
logy and FYVE zinc finger domains) specifically to recog-
nise particular configurations of the inositol phosphate
headgroup. Each inositol lipid signal molecule is thus able
to recruit and, in some cases, activate a range of protein-
binding partners. Moreover, since these lipids are likely to
be limited in their distribution to the membranes in which
they are made, both temporal and spatial changes in their
abundance are likely profoundly to influence the signalling
outcomes, (see Refs. [1–5] for recent reviews). The approx-
imate levels of the naturally occurring inositol phospholi-
pids and some proposed routes of production in a ‘model’
mammalian cell are indicated in Fig. 1.
Much attention has focused on PtdIns(3,4,5)P3 (Fig. 1) as
an intracellular second messenger produced rapidly in
response to many divergent cellular stimuli, including many
hormones and growth factors (reviewed by Refs. [4,6–8]).
It is one of the least abundant inositol phospholipids, being
present only at approximately 0.005% of the abundance of
PtdIns in most cell types analysed (this corresponds to a
cellular concentration in the tens of nanomolar range, but
presumably far higher in the membrane compartments
within which they are found), broadly similar to
PtdIns(3,4)P2 and PtdIns(3,5)P2 (Fig 1). The general mech-
anism for the production of PtdIns(3,4,5)P3 appears to be
through the regulated action ofClass I PI 3-kinase enzymes on
the relatively abundant phosphoinositide, PtdIns(4,5)P2. The
production of PtdIns(3,4,5)P3 stimulates many downstream
0898-6568/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S0898 -6568 (01 )00234 -0
* Corresponding author. Tel.: +44-1382-345156; fax: +44-1382-
201063.
E-mail addresses: [email protected] (N.R. Leslie),
[email protected] (C.P. Downes).
www.elsevier.com/locate/cellsig
Cellular Signalling 14 (2002) 285–295
cellular processes through its interactions with proteins that
contain modular ligand-binding domains (pleckstrin homo-
logy or PH domains) able to bind with some specificity to
PtdIns(3,4,5)P3 [3,9,10].
2. PTEN is a tumour suppressor gene that encodes
a PtdIns(3,4,5)P3 3-phosphatase
PTEN (Phosphatase and TENsin homologue deleted on
chromosome TEN) is one of the few human tumour sup-
pressor genes for which this status is supported by several
strong lines of evidence. PTEN, also called MMAC1 and
TEP1, was first identified in 1997 as a candidate tumour
suppressor locus found to be mutated in sporadic glioblas-
tomas, prostate and breast cancers and located at chro-
mosome 10q23, a region deleted at very high frequency in
many tumour types [11,12]. Since then it has become clear
that PTEN mutation is associated with many human tumour
types, but at particularly high frequency in endometrial
tumours and glioblastomas (reviewed in Refs. [13,14]).
Human germline mutations in PTEN are associated with
the rare autosomal dominant hamartomatous syndromes,
Cowden Disease and Bannayan–Riley–Ruvalcaba syn-
drome, the former including an increased risk of breast
and thyroid tumours. Mice homozygous for targeted dele-
tions within the PTEN gene (PTEN� /�) die during
embryonic development (between Days 6.5 and 9.5), while
heterozygous mice develop normally, but are prone to a
wide range of tumour types. Evidence supporting the
tumour suppressor status of PTEN has been reviewed
recently [15–17].
The tumour suppressor gene, PTEN, encodes a 403
amino acid protein, PTEN, that is a member of the protein
Fig. 1. (A) Pathways of cellular inositol phospholipid synthesis. The inositol phospholipids identified in mammalian cells are shown, along with the proposed
pathways for their synthesis from PtdIns. We have indicated the relative abundances of these lipids in resting cells by the size of the represented box and text.
The data for these relative levels are based on the well-studied 1321N1 astrocytoma cell line, but are representative of many mammalian cell types and are
shown as the percentage abundance relative to PtdIns. PtdIns(4)P and PtdIns(4,5)P2 each comprise about 5% of PtdIns; PtdIns(3)P and PtdIns(5)P are about
0.25% of PtdIns; and PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3 are the least abundant of the identified phosphoinositides comprising about 0.005%
under basal conditions. Of the many pathways of phosphoinositide dephosphorylation, only those thought to be catalysed by PTEN are shown. (B) Structure of
PtdIns(3,4,5)P3. The structure of sn-1:2 phosphatidyl D-myo-inositol 3,4,5-trisphosphate [elsewhere abbreviated to PtdIns(3,4,5)P3] is shown. The fatty acid
side chains have been omitted. Most cellular PtdIns appears to be sn-1-stearoyl:2-arachidonyl PtdIns and it is assumed that other phosphoinositides derived
from PtdIns share this composition.
N.R. Leslie, C.P. Downes / Cellular Signalling 14 (2002) 285–295286
tyrosine phosphatase family. Soon after the identification of
PTEN, data were published making a strong connection
between the phosphatase activity of PTEN and its tumour
suppressor function and this conclusion has been supported
by extensive studies of tumour-derived PTEN mutants
[18–20]. It now appears that almost all pathological PTEN
mutations remove or impair phosphatase activity in vitro
[18,21,22]. This indicated that the tumour suppressor func-
tion of PTEN requires the dephosphorylation of a particular
substrate or substrates. The poor activity of PTEN against
phosphoproteins and peptide substrates, and its preference
for highly acidic substrates suggested that the physiological
substrates of PTEN might be acidic nonphosphoprotein
molecules, such as phospholipids [23,24]. This led to
the significant finding that PTEN dephosphorylates
PtdIns(3,4,5)P3 in vitro and in vivo [23–25]. Additionally,
the analysis of a tumour-derived PTEN mutant (PTEN
G129E) that retained activity against phosphotyrosine sub-
strates, but lacked detectable activity against phosphoinosi-
tides, established the link between the tumour suppressor
activity of PTEN and this lipid phosphatase activity
[24,26]. Many forms of evidence have now shown that
PtdIns(3,4,5)P3 is a physiological substrate of PTEN and
that PTEN functions to antagonise growth factor stimulated
PI 3-kinase signalling (see Fig. 2).
2.1. Biochemical analyses of substrate specificity
PTEN will dephosphorylate several phosphoinositide
signalling molecules in vitro, specifically removing phos-
phate from the D-3 position of the inositol ring in each case
[23,24]. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are the most
efficient substrates for PTEN in vitro, although it will also
dephosphorylate PtdIns(3)P and the soluble headgroup
of PtdIns(3,4,5)P3, inositol 1,3,4,5-tetrakisphosphate
(Ins(1,3,4,5)P4). There is also a report of activity against
PtdIns(3,5)P2 [27]. However, PtdIns(3)P and Ins(1,3,4,5)P4have been tested and are poorer substrates in vitro, and
overexpression of PTEN in cells has little effect on the
levels of PtdIns(3)P or PtdIns(3,5)P2 (McConnachie, Ben-
nett, Pass, Leslie and Downes, unpublished information).
Therefore, it is believed that the relevant physiological
substrates of PTEN are PtdIns(3,4,5)P3 and possibly
PtdIns(3,4)P2. Importantly, this phosphoinositide phospha-
tase activity of PTEN is not a feature of other related protein
tyrosine phosphatase family members, such as PTP1D,
VHR, Cdc25 and Cdc14 [23,24].
2.2. PTEN reduces cellular levels of PtdIns(3,4,5)P3 and
antagonises PI 3-kinase signalling
The effects of PTEN activity on cellular PtdIns(3,4,5)P3levels have been studied in a variety of cell types including
several PTEN null cell lines [23,25,28,29]. Murine fibro-
blasts and embryonic stem cells with targeted deletion of
both Pten alleles have greatly elevated unstimulated levels
of PtdIns(3,4,5)P3 relative to isogenic Pten wild-type cells
[25,29]. Similar results have also been obtained with human
tumour cell lines lacking PTEN, in which PtdIns(3,4,5)P3levels appear higher than other PTEN expressing cells and
are reduced dramatically upon reexpression of the phospha-
tase [28]. These experiments involving HPLC analysis of
labelled cellular phosphoinositides have also shown that
levels of PtdIns(3,4)P2 appear similarly high and are
reduced by PTEN reexpression [28]. In addition to this
direct evidence, a wealth of data has shown greatly
increased activity of the downstream PtdIns(3,4,5)P3 regu-
lated kinase, protein kinase B (PKB, also known as Akt), in
many PTEN null cell types, and that this activity is reduced
by expression of PTEN [24,25,29–36].
The conclusion that PTEN is a functional antagonist of
PI 3-kinase has been greatly strengthened by genetic ana-
lyses in nematodes (Caenorhabditis elegans), and insects
(Drosophila melanogaster). Previous results from these
organisms established that several components of the human
PI 3-kinase signalling pathway are conserved in both
sequence and function, including the activation of Class I
PI 3-kinases by growth factor receptors, the production of
PtdIns(3,4,5)P3, and the activation of PKB [37–39]. The
conservation of this signalling module has allowed genetic
analysis in flies and worms to demonstrate the role of
orthologues of PTEN as functional antagonists of PI
3-kinase upstream of PKB [40–46]. Stimulation of this
signalling pathway transduces rather different signals in
these organisms, however, inducing survival, proliferation,
and many of the metabolic actions of insulin in mammals;
Fig. 2. Model for the inhibition of PI 3-kinase signalling by PTEN and SHIP. PTEN is a phosphoinositide 3-phosphatase, able to remove phosphate from the 3
position of the inositol ring of several cellular signalling molecules including PtdIns(3,4,5)P3, converting it to PtdIns(4,5)P2. The reverse reaction is catalysed
by Type I PI 3-kinase enzymes activated by numerous cellular stimuli, including growth factors. SHIPs (two isoforms, SHIP1 and SHIP2 are currently known)
are 5-phosphatases that convert PtdIns(3,4,5)P3 to PtdIns(3,4)P2.
N.R. Leslie, C.P. Downes / Cellular Signalling 14 (2002) 285–295 287
increasing proliferation and cell size in flies; and inhibiting
dauer arrest, fat storage, and longevity in worms.
Final evidence for the mutually antagonistic functions of
PTEN and PI 3-kinase arises from the frequent finding that
many of the cellular effects of PTEN are mimicked by
pharmacological inhibitors of PI 3-kinase. Two structurally
unrelated inhibitors have been extensively used, the revers-
ible ATP-competitive, LY294002 and the irreversible fungal
metabolite, wortmannin [4,9,47]. While many cellular
effects of these inhibitors are shared with PTEN, in addition
to the Class I PI 3-kinases, these compounds are also able to
inhibit a number of other related enzymes, although often
only at higher concentrations. This can lead to inhibition, for
example, of processes mediated by Type III PI 3-kinases via
PI(3)P production, that would not be affected by PTEN. As
with all pharmacological agents care must be exercised both
in the dose of inhibitor employed and in the understanding
that specificity is rarely absolute.
2.3. Is PTEN the principal cellular regulator of basal
PtdIns(3,4,5)P3 levels?
Currently, there is little evidence that other enzymes or
mechanisms reduce basal cellular PtdIns(3,4,5)P3 levels in
most cells in the absence of PTEN. Several PTEN null cell
types from both tumours and knockout mice have been
demonstrated to have greatly elevated basal levels of
PtdIns(3,4,5)P3 [25,28,29] and PKB activity [25,29–31].
However, since elevated PtdIns(3,4,5)P3 is likely to confer a
selective advantage on cultured cells, it is possible that
analysis of both tumours and mouse embryos misses cell
types in which loss of PTEN does not lead to elevated levels
of PtdIns(3,4,5)P3 due to functional redundancy, and there-
fore does not confer a selective advantage. This is an
important point, as it may be a factor in determining the
occurrence of PTEN mutations in different tumour types.
2.4. Other PtdIns(3,4,5)P3 phosphatases— the SHIP
proteins and PTEN homologues
Other PtdIns(3,4,5)P3 phosphatases exist, for example,
the 5-phosphatases, SHIP and SHIP2 [48], and at least two
PTEN homologues, TPTE and PTEN2 [27,49]. However,
while there is strong evidence that the SHIP proteins do
metabolise PtdIns(3,4,5)P3 in several cell types (to produce
PtdIns(3,4)P2), they appear not to regulate basal
PtdIns(3,4,5)P3 levels, but rather may control the duration
and magnitude of stimulated increases in this lipid [50]. This
conclusion is derived from data showing that SHIP null
mice develop normally, and that bone marrow-derived mast
cells from wild-type and mutant mice appear to have similar
basal levels of cellular PtdIns(3,4,5)P3 and PKB activation.
However, upon IL-3 stimulation, the cells from SHIP null
mice gave a larger and more prolonged response than wild-
type cells in terms of both PtdIns(3,4,5)P3 levels and PKB
activation [50]. Mice lacking SHIP2, the more widely
expressed 5-phosphatase, also develop normally up to birth
but exhibit insulin hypersensitivity, supporting the idea that
these enzymes may not be regulators of basal PtdIns(3,4,5)P3levels in many cell types [51]. However, currently, no work
has been published investigating downstream signalling in
cells from these mice.
Another functional difference between PTEN and the
SHIP proteins is that whereas PTEN converts PtdIns(3,4,5)P3to the abundant PtdIns(4,5)P2, the SHIP proteins convert
PtdIns(3,4,5)P3 to PtdIns(3,4)P2 (see Fig. 2). Because
PtdIns(3,4)P2 appears to be of similar abundance to
PtdIns(3,4,5)P3 in most cell types, this reaction probably
has a significant effect on the cellular abundance of
PtdIns(3,4)P2. The recent identification of PH domain con-
taining proteins able to bind specifically to PtdIns(3,4)P2 has
strengthened the argument that PtdIns(3,4)P2 is a second
messenger with roles independent from PtdIns(3,4,5)P3[52]. Therefore, the production of PtdIns(3,4)P2 from
PtdIns(3,4,5)P3 by SHIP proteins may convert one signal to
another, whereas PTEN action appears to mediate a simple
inactivation of the PtdIns(3,4,5)P3 signal.
There are several genes in the human genome very
similar to PTEN, and there is significant evidence that at
least two should express functional proteins and at least one
is expected to be able to metabolise PtdIns(3,4,5)P3 (Refs.
[27,49], and Walker, Leslie, and Downes, unpublished).
However, current data suggest that the tissue distribution
of these proteins may be tightly restricted, possibly being
expressed only in the testis.
2.5. Is all PtdIns(3,4,5)P3 functionally equivalent?
Current data suggest that in many cell types PTENmay be
a principal negative regulator of PtdIns(3,4,5)P3 levels. How-
ever, phospholipids are clearly not uniformly distributed
throughout the cell, and are probably not even distributed
uniformly throughout topologically continuous membrane
compartments. For example, PtdIns(4,5)P2 is found in many
different membranes within the cell and even associated with
nonmembranous structures within the nucleus [1,5,53]. It
seems likely that the cellular distribution of PtdIns(3,4,5)P3within the plasma membrane is often not uniform, depending
upon the type and environment of the cell, and that the effects
of this nonuniformity on downstream processes may be
significant. Strong evidence, particularly from studies of
cellular chemotaxis supports this idea [54,55]. In both the
slime mold Dictyostelium discoideum and in mammalian
neutrophil-differentiated HL-60 cells, PtdIns(3,4,5)P3and/or PtdIns(3,4)P2 accumulates at the leading edge of the
migrating cell as indicated by the localised recruitment of a
fluorescently tagged PH domain [56,57].
It seems quite possible, therefore, that precise targeting of
PTEN to particular cellular pools of PtdIns(3,4,5)P3 could
have significant functional consequences. Several cellular
proteins have been identified that interact with PTEN through
PDZdomains and it has been shown that interferingwith these
N.R. Leslie, C.P. Downes / Cellular Signalling 14 (2002) 285–295288
interactions affects some functions of PTEN, but not others.
This will be discussed in more detail below in the Section 5.
2.6. Is the protein phosphatase activity of PTEN
functionally significant?
In addition to its lipid phosphatase activity, human PTEN
also has significant protein tyrosine phosphatase activity in
vitro [19,58]. While strong evidence connects the lipid
phosphatase activity of PTEN with most of its identified
downstream functions, including tumour suppressor activity,
it remains possible that there are also physiological protein
substrates for PTEN. PTEN is a dual-specificity protein
tyrosine phosphatase in vitro, as it has detectable activity
against phosphoserine- and phosphothreonine-containing
substrates in addition to phosphotyrosine. However, the best
peptide substrates that have been described for PTEN in
vitro are acidic phosphotyrosine-containing polypeptides
[19]. The signalling proteins, focal adhesion kinase (FAK)
and Shc, are substrates for PTEN in vitro that have been
proposed as potential cellular targets of the enzyme. It has
also been suggested that dephosphorylation of these sub-
strates could mediate the inhibition of cell motility caused
by PTEN [59,60]. However, two pieces of evidence argue
against this. Firstly, neither phosphorylation of FAK, nor
cell motility, appear to be affected by expression of a PTEN
mutant that lacks lipid phosphatase activity, but retains
activity against phosphotyrosine substrates in vitro (PTEN
G129E) [61]. Secondly, evidence has been presented iden-
tifying signalling pathways leading from PtdIns(3,4,5)P3,
the known lipid substrate of PTEN, to phosphorylation of
both FAK and Shc, that could explain the effects of PTEN
on the phosphorylation states of these proteins [62,63].
More recent data, however, have implicated the protein
phosphatase activity of PTEN in reducing the cellular levels
of cyclin D1 in the breast cancer cell line MCF7 [64]. In the
latter experiments, PTEN G129E failed to inhibit PKB
activity, but appeared fully capable of down-regulating
cyclin D1. It will be interesting to see whether these effects
are found in other cell types, and to identify specific
substrates upon which the protein phosphatase activity of
PTEN might be acting. In this regard, a mutation in PTEN
that mirrors the effect of the G129E mutation, by blocking
protein phosphatase activity while retaining activity against
lipid substrates, would be most useful. Such a mutation has
not yet been reported.
3. The PTEN protein
3.1. Linking structure and function
Initial analysis of its amino acid sequence showed that
PTEN has an N-terminal phosphatase domain with homo-
logy to both the protein tyrosine phosphatase family and the
nonphosphatases tensin and auxilin [11,12] (Fig. 3). The
extreme C-terminus of PTEN was first predicted, then
shown experimentally, to contain a binding motif for a class
of PDZ protein–protein interaction domains [65–67]. How-
ever, significant understanding of the possible functions of
the remainder of the molecule was provided only by the
solution of its crystal structure [21].
The determination of the structure of PTEN has
identified several characteristics that are not shared by
other PTP enzymes and which direct specificity towards
inositol phospholipid substrates [21]. Most enzymes that
metabolise membrane lipids have other membrane-binding
motifs in addition to the active site, allowing the enzyme
to remain associated with a membrane during the com-
pletion of multiple catalytic cycles [68]. In addition to its
N-terminal phosphatase domain, PTEN has a calcium-
independent C2 domain that has been shown to bind to
lipid vesicles in vitro [21]. In other proteins, C2 domains
have been shown to mediate lipid binding and membrane
localisation [2,69]. The functional significance of the
PTEN C2 domain has also been demonstrated, as muta-
tions that reduce the lipid binding of the domain, without
interfering with in vitro phosphatase activity against a
soluble substrate, dramatically to impair all aspects of
downstream function investigated in cells, including the
reduction of cellular PtdIns(3,4,5)P3 levels [21,70,71].
Recent results also show that these C2 domain mutations
are only partially rescued by artificial membrane targeting
of the phosphatase [70]. It therefore seems possible that
this C2 domain may not only allow transient association
of PTEN with cellular membranes, but may play a role in
ensuring the correct productive orientation of the active
site [21,70]. This idea is also supported by the extensive
tight interface between the phosphatase domain and the
Fig. 3. The PTEN protein. The 403 amino acid PTEN protein is represented. The N-terminal phosphatase domain (amino acids 7–185), including the catalytic
cysteine (C) and the C2 domain (186–351), Are hatched and both required for enzymatic activity. The extreme C-terminal PDZ-binding sequence is also
shown and although it is represented as a small region, the extent of further sequences required for optimal specificity and affinity of binding is not known. The
existence of probable phosphorylation sites in the C-terminal tail is represented by a circled letter P. The amino acid numbering of domains is derived from the
crystal structure of the protein [21].
N.R. Leslie, C.P. Downes / Cellular Signalling 14 (2002) 285–295 289
C2 domain, that is one of the most conserved regions of
the protein between divergent species, and is also a
frequent target of mutation in tumours. Extensive muta-
tion studies seem to show that together, the phosphatase
and C2 domains form a minimal catalytic unit, as
truncation mutations removing only a few residues from
the C-terminus of the C2 domain ablate all detectable
phosphatase activity, even against soluble substrates. On
the other hand, mutations in the C-terminal tail, beyond
the C2 domain, seem to have little or no effect upon
enzyme activity [18,21,22,72,73].
A further structural adaptation of PTEN, contrasting with
other PTPases, is the size of the active site pocket. Phos-
photyrosine-specific classical PTPases for which structures
have been determined, such as PTP1B, have a deep narrow
active site cleft, whereas the dual-specificity phosphatases
(phosphotyrosine and phosphoserine/phosphothreonine-spe-
cific), such as VHR have a more shallow active site [74].
The active site pocket of PTEN, however, is deep, but also
very broad, allowing access to the bulky phosphorylated
inositol headgroup. It is also highly basic, consistent with
the preference for acidic substrates [21].
Unfortunately, in order to obtain a crystal structure, the
C-terminal 49 amino acids of PTEN needed to be deleted (as
well as the N-terminal 7 amino acids and a 24 residue C2
domain internal loop) [21]. While this C-terminal region is
not required for catalytic activity, or for most cellular
functions analysed, evidence suggests that it may play an
important regulatory role. The C-terminal sequence, beyond
the end of the C2 domain, has now been shown to contain
several phosphorylation sites, a PDZ domain-binding
sequence and two possible PEST sequences (Fig. 3). The
mechanisms and functional significance of these regulatory
mechanisms remain rather unclear, and will be discussed in
greater detail below, but greatly influence the stability of the
protein. It should be noted that the significance of the
homology between PTEN and the nonphosphatase mole-
cules, tensin and auxilin, is not yet clear.
3.2. Downstream of PtdIns(3,4,5)P3—cellular effects
of PTEN
Conserved signalling pathways can be used to transduce
very different information in different organisms and in
different cells in the same organism [75,76]. Therefore,
the cellular effects of PTEN action, and possibly any
selective advantage of PTEN loss in a tumour, will depend
on the predominant functions for which the PI 3-kinase
signalling pathway is used in any particular cell type.
However, it has been known for some years that in many
and possibly most mammalian cell types, PI 3-kinase is
activated by many growth factors and acts as a strong
survival and proliferative signal. We will not rigorously
review all the identified signalling events and biological
endpoints downstream of PI 3K as these have been reviewed
recently by others [6,7,77,78] or those shown to be regu-
lated by PTEN [15,79]. However, we will give a brief
account of some recent work that has shed significant light
on the mechanism of tumour suppression by PTEN.
Currently, several biological processes have been iden-
tified that can be controlled by PtdIns(3,4,5)P3 signalling,
many of which can be down-regulated by PTEN and are
likely to be pathologically significant in tumours with PTEN
mutations. As previously mentioned, PTEN has been shown
to inhibit cell cycle progression and survival [25,29,80], but
can also inhibit cell spreading and motility [60,61] and the
release of angiogenic factors [81–84] (Fig. 4). Additionally,
many other cellular processes have some requirement for
Type I PI 3-kinase signalling, such as endocytic pathways
[85–87], regulation of translation [88,89], and the activation
of certain transcription factors. While the signalling path-
ways linking PtdIns(3,4,5)P3 with many of these complex
biological processes are still rather poorly understood,
considerable progress has been made in the identification
of direct effectors of PtdIns(3,4,5)P3 signals, particularly
those containing PH domains shown to bind directly to this
signalling lipid (reviewed in Refs. [3,7,9,90]).
Much attention has focused on the serine/threonine kinase
PKB (also known as Akt) as a downstream target of PTEN.
This is largely because the roles of PtdIns(3,4,5)P3 and
PtdIns(3,4)P2 in the activation of this enzyme are well
established and because, as a proto-oncogene, PKB has been
shown to play an important role in the transduction of
survival and proliferative signals in mammalian cells
(reviewed in Refs. [78,79,91,92]). However, it is also critical
that elevated levels of cellular PtdIns(3,4,5)P3 seem suf-
ficient for the activation of PKB in the absence of other
stimuli, as several PTEN null cell types have been shown to
Fig. 4. Signalling pathways downstream of PtdIns(3,4,5)P3. Some of the
known signalling pathways downstream of the second messenger,
PtdIns(3,4,5)P3, are shown. The synthesis of this lipid is stimulated by
many extracellular stimuli, including growth factors, hormones, and
extracellular matrix components. PtdIns(3,4,5)P3 can bind to PH domains
within many target proteins, of which the best understood are shown, and
plays a role in the activation of the indicated kinases (PKB, some PKC
isoforms, S6K1, SGK, and the Tec family), PLCg, and exchange factors for
the GTPase, Rac. A guide to some of the biological endpoints regulated by
these pathways is also shown.
N.R. Leslie, C.P. Downes / Cellular Signalling 14 (2002) 285–295290
have almost maximal PKB activity in the absence of acute
growth factor stimulation [24,25,30]. A number of potential
cellular PKB substrates have been identified that may be
responsible for the survival and proliferative effects of PKB
activity. The most convincing at present are the proapoptotic
regulatory protein, Bad, and transcription factors of the
Forkhead family [77,78,92]. More substrates of PKB sig-
nificant in the transduction of these signals probably remain
to be identified.
Recent work studying the downstream pathways
through which PTEN induces cell cycle arrest and pro-
motes apoptosis has highlighted the importance of Fork-
head transcription factors and the cyclin-dependent kinase
inhibitor p27Kip1. PKB-mediated phosphorylation of some
Forkhead transcription factors has been shown to inhibit
their transcriptional activity through cytoplasmic localisa-
tion and to be constitutive in cells lacking PTEN. Sig-
nificantly, expression of a Forkhead protein that could not
be phosphorylated by PKB was able to induce cell cycle
arrest and apoptosis with similar efficiency compared with
the effects of PTEN expression, suggesting that the func-
tion of the Forkhead proteins could be sufficient to
mediate many of the downstream effects of PTEN [80].
One of the significant targets for transcriptional regulation
by these factors is the cyclin-dependent kinase inhibitor
p27Kip1 and evidence suggests that increases in p27 may
be required for the induction of a G1 cell cycle arrest and
tumour suppression by PTEN [93–95]. PTEN appears able
to affect both the transcription and protein stability of p27
so that it may exert a two-pronged approach to regulating
the cellular amounts of this key cell cycle regulator
[80,96–98].
4. Regulation of PTEN
Given the critical role of PTEN in antagonising PI 3-
kinase signalling pathways, it might be anticipated that the
enzyme would be the target of complex control mecha-
nisms. Until recently, few studies had addressed this issue,
but evidence is now emerging for functional regulation of
PTEN through effects on protein stability, localisation, and
transcription of the PTEN gene. The regulation of stability
and localisation appears to be achieved through the C-
terminal tail of PTEN beyond the C2 domain, a region not
required for either enzymatic activity or reduction of the
major pool of cellular PtdIns(3,4,5)P3. Interestingly,
although mutations do occur in this region of the protein
in tumours, they do so at lower frequency than other regions
[13,14]. The evidence suggests that this is because most
tumour-derived mutations within the phosphatase domain or
the C2 domain (up to amino acid 351) remove all detectable
PTEN phosphatase activity [18,72]. Mutations in the C-
terminal tail appear only partially to interfere with PTEN
function, possibly through effects on protein stability/
abundance or localisation [22,67,71,99] and in tumours
appear not to lead to the dramatic increase in PKB activity
seen with null mutations [71]. It is therefore interesting that
mutations in the C-terminal tail have never been described
in either Cowden disease and other inherited conditions, or
in endometrial tumours, where PTEN mutation appears to
be a very early event in tumour progression [13,14,100]. By
contrast, such mutations have been found rather frequently
in gliomas, where PTEN mutation is associated with late-
stage aggressive tumours (Table 1).
4.1. Phosphorylation of PTEN
It has recently become clear through the work of two
research groups that PTEN exists in cells as a phosphopro-
tein [22,99]. When cells are labelled with radioactive
phosphate, both endogenous and expressed PTEN proteins
incorporate label, predominantly into phosphoserine, but
also phosphothroenine residues. Although indirect, the data
can be interpreted to suggest that most cellular PTEN is
constitutively phosphorylated. Deletion and mutational
studies indicate that phosphorylation sites are probably
located in a cluster in the C-terminal tail of PTEN, beyond
the end of the C2 domain, possibly at amino acids S370,
S380, T382, T383 and S385. Casein kinase 2 (CK2) has
been proposed as the responsible kinase, based largely on in
vitro data [99], but this needs confirmation by more tech-
nically demanding in vivo labelling approaches.
Analysis of the functional consequences of phosphory-
lation revealed that wild-type PTEN or mutant PTEN
proteins with phosphorylation sites replaced by acidic
residues were found to be more stable in cells than proteins
in which phosphorylation sites had been replaced by alanine
residues, or where the whole C-terminus had been deleted
[22,99]. This agrees well with previous work, noted above,
demonstrating a role for the C-terminal tail in controlling
PTEN stability [72]. Evidence also suggests that PTEN may
be degraded by a proteosome-mediated pathway [99]. Phos-
phorylation appears to reduce the biological activity of trans-
fected PTEN as measured by the relative activities of
phosphorylation site mutants in the induction of growth arrest
and in a Forkhead transcriptional activation assay [22]. This
raises the interesting possibility that phosphorylation of
Table 1
Mutations in the C-terminal tail of PTEN
Total
mutations analysed
Mutations
in C-terminus
Glioblastomas 159 6
116 7
Endometrial carcinoma
and precancerous lesions
187
136
0
0
Germline 110 0
The frequency of occurrence of mutations in the whole coding region and
specifically the C-terminal tail (beyond the C2 domain, i.e., aa 352–
Stop404) of PTEN is shown. Two estimates for data from glioblastomas
and for endometrial tumours are shown from two reviews of PTEN
mutational data [13,14].
N.R. Leslie, C.P. Downes / Cellular Signalling 14 (2002) 285–295 291
PTEN in the C-terminal tail is able to stabilise but reduce
the activity of the protein. At present, there is no evidence of
conditions that regulate the degree of phosphorylation of
PTEN, and hence, the functional significance of these
observations remains unclear. It seems likely that phosphor-
ylation may not greatly affect the in vitro activity of the
enzyme against soluble substrates, since deletion of the
C-terminal phosphorylation sites appeared not to greatly
affect the activity of protein immunoprecipitated from
human 293T cells or expressed and purified from insect
cells [21,72]. Taken together with the activity of bacterially
expressed PTEN, this suggests that the protein has constitu-
tive phosphatase activity, but raises interest in the mech-
anism by which phosphorylation could be inhibiting the
biological actions of PTEN.
4.2. Regulation through localisation—PTEN
targeting proteins
It appears that in most cell types analysed, PTEN is
largely cytosolic [20,58,60]. Since its principal substrate is a
membrane phospholipid, and artificial membrane targeting
enhances effects on downstream signalling [66,70,71], sig-
nificant effort has been invested in investigating mecha-
nisms by which PTEN might become membrane associated
and/or incorporated in signalling complexes assembled at
membrane loci.
The extreme C-terminus of PTEN can mediate interac-
tions with PDZ domains in several proteins [65–67]. These
include the membrane associated guanylate kinase with
inverted orientation (MAGI) proteins 1, 2, and 3, the related
protein, discs large (DLG/SAP97), itself a tumour sup-
pressor in flies, and MAST205 (microtubule-associated
serine/threonine kinase of 205 kDa). An interaction between
endogenous proteins, however, has thus far only been
demonstrated between PTEN and MAGI-2 [67]. Unfortu-
nately, little is currently known about other binding partners
of these PDZ domain proteins and the signalling complexes
they are presumed to coordinate.
Two groups found that overexpression of MAGI proteins
enhanced PTEN’s ability to block PKB activation [66,67],
while others failed to observe any effect on PKB activity of
deleting the PDZ-binding sequence from PTEN [71,73]. On
the other hand, the C-terminal PDZ-binding sequence is
required for efficient inhibition of membrane ruffling and
cell spreading [71,101]. Since it was found that deletion of
the PDZ-binding sequence did not measurably affect the
ability of PTEN to reduce cellular levels of PtdIns(3,4,5)P3,
it seems that PDZ domain-mediated targeting of PTEN may
play a role in the regulation of processes that are highly
sensitive to small changes in the levels of PtdIns(3,4,5)P3(which include cell spreading) [71]. This in turn may
suggest that a small localised pool of PtdIns(3,4,5)P3 is
dedicated to regulating lamellipodia formation, but not the
activation of PKB.
Since the PDZ-binding sequence is tightly conserved
within vertebrate PTEN orthologues (from Mammals,
Zebrafish, and Xenopus), but absent in those from flies
and worms, it seems likely that these regulatory mecha-
nisms might have evolved for the ‘fine tuning’ of
PtdIns(3,4,5)P3 signalling in higher organisms. This could
include, for example, the signalling complexes on mem-
brane subdomains in which PDZ scaffolds have been
proposed to play roles in polarised epithelial cells and
synaptic membranes [102–105]. In other proteins, it has
been proposed that phosphorylation of a serine or threonine
residue within the C-terminal sequence bound by some PDZ
domains can modulate these protein–protein interactions.
Although it has been shown that phosphorylation of Thr401
can alter the pattern of PDZ proteins bound by PTEN-
derived peptides, there is currently no evidence for phos-
phorylation of this residue of PTEN in cells [65]. Human
tumours have been described with missense mutations in the
PTEN PDZ-binding motif, in the stop codon and in threo-
nine 401. These mutations interfere with the interaction of
PTEN with PDZ domain containing proteins and this would
argue that functions requiring PDZ targeting of PTEN may
be important in the development of these tumours [71].
However, it is also possible that these mutations have other
additional effects, such as influencing the stability of the
enzyme [67].
It should be noted that in some cell types PTEN appears
to be present in the nucleus [106–108]. Since there is no
strong evidence yet for the existence of PtdIns(3,4,5)P3 in
the nucleus, the role of nuclear PTEN remains unclear. It is
possible that it has functions other than PtdIns(3,4,5)P3metabolism, or that the protein is simply functionally
inactivated by sequestration in the nucleus away from
its substrates.
4.3. Regulation of expression
Although PTEN was initially named TEP1 by Li and Sun
[58] because of a dramatic decrease in its expression that
occurs in human keratinocytes treated with TGFb, relativelylittle is still known about mechanisms that regulate PTEN
expression. Evidence from breast, prostate, thyroid, pancre-
atic, and haematopoietic tumours suggests that PTEN
expression can be lost or greatly reduced in some tumours
without mutation of the coding sequence of the PTEN gene
[106,107,109–111]. These tumours may have mutation or
methylation of noncoding regions of PTEN required for
expression, particularly since the PTEN promoter has not
yet been identified. It is also possible that there is an
alteration in these tumours of mechanisms further upstream
regulating the expression of PTEN, and information is just
emerging concerning the normal regulation of PTEN
expression. It has been shown that PTEN expression is
greatly increased during either granulocytic or monocytic
differentiation of myeloid leukaemic cells [112]. Very
recently, it has been reported that PTEN expression is
N.R. Leslie, C.P. Downes / Cellular Signalling 14 (2002) 285–295292
profoundly regulated by agonists acting on the peroxisome
proliferator-activated receptor g (PPARg) in both macro-
phages and Caco-2 tumour cells [113]. PPARg appears
directly to regulate transcription of the PTEN gene leading
to increased mRNA, PTEN protein, PtdIns(3,4,5)P3 3-phos-
phatase activity and a concomitant reduction in the activity
state of PKB. These results suggest the exciting possibility
that up-regulation of PTEN might be one of the mechanisms
through which PPARg agonists exert their recently
described anti-inflammatory and anticancer actions [113].
They appear to present a paradox, however, since PPARg
agonists are currently proving effective in the treatment of
Type II diabetes, where, by antagonising the PI 3-kinase
pathway, up-regulation of PTEN would be expected to be
harmful. Critically, however, PTEN appears to up-regulate
expression of insulin receptor substrate (IRS)-2, which is
one of four related adaptor proteins through which insulin
and certain other stimuli activate PI 3-kinase [114]. This
may be a reflection of a feedback pathway in which a
persistently low basal level of PtdIns(3,4,5)P3 leads to up-
regulation of IRS-2 and an enhanced ability to respond to
insulin via IRS-2. Since transgenic mice lacking IRS-2, but
not those lacking IRS-1, exhibit many features of Type II
diabetes, it is likely that up-regulation of IRS-2 will be
beneficial in this disease. The idea that PTEN can be
regulated through changes in its level of expression, either
via transcriptional control pathways or by controlling the
turnover of the expressed protein, is in tune with an
important feature of the enzyme in that it appears to be a
constitutively active phosphatase. Currently, there are only
hints that PTEN may be acutely regulated through covalent
modifications or translocation to signalling complexes
(unlike the SHIP proteins, which are membrane recruited
upon many cellular stimuli).
5. Future perspectives
Changes in levels of expression of PTEN, such as in
response to pharmacological agents, stimuli such as TGFb,or in heterozygotes with only one functional PTEN allele,
appear to directly influence basal levels of PI 3-kinase-
dependent lipid signals. Bearing in mind that the up-regu-
lation of PTEN expression induced by PPARg agonists may
explain the therapeutic effects of these compounds not just
in cancer, but also in inflammatory conditions and non-
insulin-dependent diabetes, the identification of the PTEN
promoter must be a key step in future work. Further analysis
of the transcriptional regulation of PTEN may also go some
way to explaining the types of tumours in which PTEN
mutations occur. Recent work has highlighted the range of
processes that PI 3-kinase signalling can influence, and the
many relevant processes deregulated in tumours carrying
PTEN mutations. It will be interesting to see this list
expand, and to know whether the regulation of other
processes that require some input from PI 3-kinase signal-
ling, such as vesicle trafficking and translation, play roles in
PTEN-related tumorigenesis.
Another area of future interest will be the interplay
between PtdIns(3,4,5)P3 metabolism via PTEN and other
enzymes that fulfil a similar role. There are already hints of
PTEN homologues and it will be important to confirm not
only that these proteins possess lipid phosphatase activity,
but also to examine their functional roles in cells. Perhaps
more significant is the existence of a distinct pathway for
PtdIns(3,4,5)P3 metabolism via selective removal of the 5-
phosphate, catalysed by SHIP proteins. Since these enzymes
generate a product, PtdIns(3,4)P2, that is considered to be a
second messenger, metabolism via SHIP or PTEN should
have functionally distinct outcomes with implications for
these enzymes as targets for novel therapies.
Acknowledgments
We would like to thank Ian Batty and Ian Fleming for
helpful discussions and critical reviews of the manu-
script. Work in the CPD laboratory is supported by the
Medical Research Council and the Division of Signal
Transduction Therapy.
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