From the Departments of Molecular Oncology,
§ Protein Engineering, and ¶ Pathology, Genentech,
Inc., South San Francisco, California 94080
Received for publication, February 15, 2001, and in revised form, March 1, 2001
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ABSTRACT |
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The tumor suppressor PTEN is a
phosphatidylinositol phospholipid phosphatase, which indirectly
down-regulates the activity of the protein kinase B/Akt survival
kinases. Examination of sequence data bases revealed the existence of a
highly conserved homologue of PTEN. This homologue, termed PTEN 2, contained an extended amino-terminal domain having four potential
transmembrane motifs, a lipid phosphatase domain, and a potential
lipid-binding C2 domain. Transcript analysis demonstrated that PTEN 2 is expressed only in testis and specifically in secondary
spermatocytes. In contrast to PTEN, PTEN 2 was localized to the Golgi
apparatus via the amino-terminal membrane-spanning regions. Molecular
modeling suggested that PTEN 2 is a phospholipid phosphatase with
potential specificity for the phosphate at the 3 position of inositol
phosphates. Enzymatic analysis of PTEN 2 revealed substrate specificity
that is similar to PTEN, with a preference for the dephosphorylation of
the phosphatidylinositol 3,5-phosphate phospholipid, a known mediator
of vesicular trafficking. Together, these data suggest that PTEN 2 is a
Golgi-localized, testis-specific phospholipid phosphatase, which may
contribute to the terminal stages of spermatocyte differentiation.
The production of 3-phosphorylated phosphatidylinositol lipid
products by the PI3K1 pathway
is an important control point for the regulation of cell proliferation,
growth, survival, and vesicular trafficking (1). The activation of this
pathway by various growth factors, extracellular matrices, or oncogenic
events results in a diversity of signals, including the up-regulation
of the catalytic activity of the Akt/PKB kinases (2). These
kinases enhance cell survival by phosphorylation of a number of
substrates, including a subfamily of forkhead transcription factors
(3). A novel mechanism for the control of the Akt/PKB pathway was
identified when genetic evidence pointed to a tumor suppressor locus on
chromosome 10 at q23-25. Analysis of a candidate tumor suppressor gene
from this region demonstrated that the locus encoded a phosphatase,
which was termed PTEN (also called MMAC and TEP) (1, 4-6).
Further studies demonstrated that PTEN was mutated in a
large percentage of brain, endometrial, and prostate tumors as well as
a smaller percentage of other tumors (7-9). In addition, Cowden
disease and Bannayan-Zonana syndrome, which are both characterized by
increased susceptibility to breast and thyroid tumors, showed a range
of germline PTEN mutations, which were similar to those observed in
tumors (10). Enzymatic studies demonstrated that PTEN is a lipid
phosphatase, which down-regulates the PI3K pathway by removing the
3-phosphate from the phosphatidylinositol 3,4(and 3,4,5)-phosphate
phospholipids (PIP3,4 and PIP3,4,5)
(11). Importantly, many of the tumor-derived missense mutations
observed in PTEN resulted in a complete loss of phospholipid
phosphatase catalytic activity (12). The loss of the PTEN lipid
phosphates activity due to mutation was expected to result in increased
levels of PIP3,4 and PIP3,4,5 and the
up-regulation of the Akt/PKB cell proliferation/survival pathway, an
event that might induce tumor resistance to chemotherapy and radiation
(13-15). Data supporting this conjecture demonstrated that
glioblastoma cell lines mutated for the endogenous PTEN
locus suffered deleterious effects on cell cycle progression
(G1 arrest), proliferative capacity, and survival
when transfected with a wild type but not a catalytically inactive,
form of the phosphatase, suggesting that the enzymatic activity of the
enzyme was involved with the regulation of this phospholipid signaling
(6, 16, 17). These studies suggested that the loss of this phosphatase
in tumors induced the up-regulation of the Akt/PKB signaling pathway,
which resulted in cell cycle progression and inhibition of apoptosis.
A number of animal model studies supported an important role for PTEN
in the control of proliferation, survival, and cell size. Importantly,
mice with homozygous null mutations in the PTEN locus showed
early embryonic lethality due to an apparent hyperproliferative effect,
whereas heterozygous animals developed tumors postnatally with apparent
loss of heterozygosity at the PTEN locus (18, 19). This
latter result suggested that the loss of PTEN expression was an
advantageous event, which allowed tumors to grow in a more unregulated
manner after the accumulation of other oncogenic mutations. Cell lines
derived from PTEN null embryonic mice demonstrated higher levels of
PIP3 phospholipids and enhanced activation of the Akt/PKB
kinase (19). These cells also showed significant resistance to a
diversity of apoptotic stimuli, further endorsing a role for this
phosphatase in the regulation of cell survival through the PI3K
pathway. Additional evidence in both Caenorhabditis elegans
and Drosophila supported a role for the PTEN phosphatase in
the regulation of cell growth and survival. C. elegans
contains an insulin-like pathway, including an insulin-like receptor
tyrosine kinase, a PI3K, PDK, and Akt/PKB kinases and a forkhead-like
transcription factor, which is involved with dauer formation, a
developmental stage where worms undergo a quiescent state. Genetic
analysis of this pathway demonstrated that a worm homologue of
PTEN, termed DAF 18, could suppress upstream mutations in
either the insulin-like receptor or PI3K, completely consistent with
results found in the mammalian PI3K pathway (20). The involvement of
this pathway in the regulation cell size was further suggested by
studies in Drosophila. These data demonstrated that the fly
homologue of PTEN was involved with the determination of cell size,
consistent with other studies, which established the importance of
several components of the PI3K pathway, such as Akt/PKB (21, 22).
Together, these three separate animal models provided strong proof for
the relevance of the PTEN lipid phosphatase in the regulation of
various aspects of the PI3K pathway.
X-ray crystallographic analysis of PTEN structure revealed that this
phosphatase contains a novel substrate recognition pocket with
positively charged residues potentially involved with the association
of the phosphates on the inositol ring substrate (12). Positioning of
many tumor-derived mutations known to disrupt catalytic activity to the
active site in part served to explain the mechanism of action of the
phosphatase. The mechanism by which PTEN appears to be associated with
its phospholipid substrates appears to be quite complex. Structural
analysis revealed a functional C2 lipid binding domain in the
carboxyl-terminal region of the protein, which was proposed to serve as
a lipid association motif (12). Many tumor-derived mutations have been
mapped to the carboxyl terminus, and a fraction of these are involved
with the formation of an interface between the phosphatase domain and
the C2 domain. These latter results helped to explain why mutations in
the carboxyl-terminal region appeared to affect catalytic activity.
Human, mouse, and Drosophila PTEN all contain a PDZ binding
motif ((S/T)XV) at their carboxyl termini, and yeast
two-hybrid analyses established that the PTEN phosphatase binds to PDZ
domains of a family of membrane-associated guanylate kinases (MAGUKs),
peripheral membrane-associated proteins with multiple protein
interaction domains, which function to juxtapose signaling molecules
and position them near the plasma membrane (23). Interestingly, some
tumor-derived mutations are found at the extreme carboxyl terminus of
PTEN, and these mutations would be expected to disrupt PDZ domain
binding interactions, consistent with an important functional role for
PDZ domain binding in PTEN tumor suppression. Because these MAGUKs are
localized specifically to intercellular tight junction regions, these
studies also suggested mechanisms for the positioning of the PTEN
phosphatase to the lipid domains of subcellular regions such as the
epithelial tight junction, a site known to be involved with the
regulation of cell survival (24).
In this paper we describe a novel homologue of PTEN, termed PTEN 2, which has been identified using data base searches. Interestingly, this
novel phosphatase is expressed uniquely in testis, and specifically in
the secondary spermatocytes. In contrast to PTEN, PTEN 2 contains several potential transmembrane domains, which appear to target the
phosphatase to the Golgi apparatus. Molecular modeling suggests that
PTEN 2 is a lipid phosphatase with many active site residues conserved
with PTEN, and enzymatic analysis demonstrates that the novel
phosphatase actively dephosphorylates PIP3,5 and
PIP3,4,5 in vitro. Together, these data suggest
that PTEN 2 is a phospholipid phosphatase, which may play a role in the
terminal stages of spermatocyte maturation by regulating intracellular
levels of phosphatidylinositol 3-phosphate phospholipids.
PTEN DNA Constructs--
A human expressed sequence tag related
to PTEN was initially identified from searches of public and private
data bases. By using various protocols, including screening a testis
cDNA library and PCR methods, we obtained a full-length sequence of
human PTEN 2. A fragment of human PTEN 2 cDNA was used as a probe
to isolate a full-length murine cDNA from a murine testis cDNA
library. GST-PTEN 2 (amino acids 378-683) were subcloned into an
expression vector containing a cytomegalovirus promoter. A
catalytically inactive form of murine GST-PTEN 2 was constructed by
changing Cys458 to Ser. To determine the
localization of PTEN 2 in mammalian cells, a myc tag was placed at the
carboxyl terminus of the gene. GFP-PTEN 2 was constructed by subcloning
either the full-length cDNA or the amino or carboxyl terminus into
CLONTECH's pEGFPN3 vector. The PTEN 2 amino
terminus includes amino acids 1-377, whereas the PTEN 2 carboxyl
terminus includes amino acids 378-683. Fluorescence in situ
hybridization mapping of mouse murine PTEN 2 was performed by
SeeDNA Biotech Inc. The probe was an 8-kb BamHI genomic DNA
containing the first exon of PTEN 2.
Molecular Modeling--
For the molecular modeling, a sequence
alignment was obtained by using ClustalW and a threading approach. The
HOMOLOGY/MODELLER module from the Insight II package (version 98.0, MSI, San Diego, CA) was used for molecular construction and display.
Docking of inositol (1,3,4,5)-tetrakisphosphate in the active site of
PTEN 2 was manually performed as previously described for PTEN by Lee and co-workers (12).
PTEN 2 Expression Pattern--
In addition to Northern analysis,
we also determined the tissue distribution pattern of PTEN 2 using the
PCR method. Mouse Multiple Tissue cDNA panels were purchased from
CLONTECH. After 35 cycles, the mouse PTEN 2 gene
was only detected in testis.
In Situ Hybridization--
PCR primers (upper
5'-GAACTGGAACCATGGTG and lower 5'-TAGGAAGATTCGGAGAGAG) were designed to
amplify a 423-bp fragment of PTEN 2. Primers included extensions
encoding T7 and T3 RNA polymerase initiation sites to allow in
vitro transcription of sense and antisense probes, respectively,
from the amplified products. The hybridization was performed on 5-µm
paraffin sections of formalin-fixed tissues. Prior to hybridization the
sections were deparaffinized and treated with proteinase K for 15 min
at 37 °C. [33P]UTP-labeled sense and antisense probes
were hybridized to the sections at 55 °C overnight. Unbound probe
was removed by treatment with RNase A for 30 min at 37 °C, followed
by a high stringency wash (0.1× SSC for 2 h at 55 °C) and
dehydrated in 70, 95, and 100% ethanol, respectively. The slides were
dipped in NBT2 nuclear track emulsion (Eastman Kodak), exposed for 4 weeks at 4 °C, developed, and counterstained with hematoxylin and eosin.
Intracellular Localization of PTEN 2--
The
intracellular localization of PTEN 2 gene was done in COS7 cells.
36 h after transfection, the cells were fixed using formaldehyde
and stained using an anti-myc monoclonal antibody. The
YFP-Golgi marker was purchased from CLONTECH.
Brefeldin was purchased from Sigma Chemical Co., and the transfected
cells were treated for 40 min before fixation.
Preparation of Catalytically Active PTEN
Proteins--
Approximately 5 × 108 293 transfected
cells were collected and resuspended in 100 ml of 0.5% Triton X-100,
50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol,
2 mM DTT, and protease inhibitors (Roche Molecular
Biochemicals, 1836145). After sitting on ice for 15 min, the lysates
were centrifuged at 10,000 × g and the supernatant was
collected. The lysate was applied to a 2-ml reduced
glutathione-Sepharose column and recirculated several times. The column
was washed in 10 column volumes of 0.03% Brij35, 50 mM
Tris, pH 7.5, 0.5 M NaCl, 10% glycerol, and 2 mM DTT. The GST fusion protein was eluted with 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM
DTT, 30% glycerol, and 10 mM reduced glutathione. The
protein is stored in aliquots of 30 µl at Phosphoinositide Phosphatase Assay--
The lipid-based
phosphatase reactions were performed essentially as described
previously (25). The reactions (50 µl) contained 100 mM
Tris (pH8.0), 10 mM dithiothreitol, 100 µM
phosphatidylinositol phosphate substrates (Echelon), 1.0 mM
phosphatidylserine (Avanti, 830052), and 50 µg/ml PTEN 2 or 10 µg/ml PTEN. Reactions were run at 37 °C for 3 h and
centrifuged at 20,000 × g for 15 min. The supernatants
were treated with malachite green (Biomol Green, AK-111), and
absorbance was measured at A650.
Sequence Characterization of a PTEN Homologue--
Perusal of
human and murine DNA sequence data bases demonstrated a closely related
homologue of PTEN in both species. Cloning of cDNAs encoding both
the human and murine forms of the homologue revealed that, whereas a
diversity of sequences were expressed in the human, a single species
was found in the mouse. While this work was being completed, a human
homologue of the murine sequence was reported, and chromosomal mapping
suggested that there were a number of different
PTEN-homologous genes encoded in the human genome, some of
which appeared to be pseudogenes (26). The sequence of the 664-amino
acid (molecular mass 76,719 Da) mouse protein is illustrated in
Fig. 1 and compared with the reported
human PTEN 2 homologue and with PTEN. This figure illustrates that both the human and murine PTEN homologues are extended at their amino termini, and hydropathy analysis (Fig. 1) reveals that, in contrast to
PTEN, the murine and human homologues appear to contain four high
probability transmembrane domains followed by the catalytic domain.
Interestingly, all four potential transmembrane domains contain charged
residues embedded within the hydrophobic potential membrane-spanning
sequence (Fig. 1). Using a structural algorithm, which predicts
membrane topology,2 we find
that the murine homologue of PTEN may have a membrane-spanning structure, which is reminiscent of ion channels (Fig. 1). This sequence
analysis suggests that the murine and human PTEN homologues appear to
have extended amino termini, which may be involved with intracellular
membrane association. Fluorescence in situ hybridization mapping
revealed that the murine homologue mapped to a single locus on
chromosome 8 between A3 and A4 (data not shown).
The murine homologue appears to have a number of residues throughout
the catalytic domain, which are conserved with PTEN (Fig. 1).
Importantly, many of these residues are likely to be involved with
substrate recognition and catalytic activity (Fig.
2) (12). For example, residues
Asp426, His427, Cys458,
Lys459, Gly461, Arg462, and
Gln510, which are identical between the two proteins, were
all proposed to be involved with substrate recognition in the
structural analysis of PTEN. In addition, Fig. 2 also illustrates
that the vast majority of the tumor-associated PTEN mutations, many of
which are known to disrupt catalytic activity, occur at residues that
are also either identical or conserved between the PTEN and the PTEN
homologue. These comparative data suggest that the murine and human
homologues are likely to have similar substrate specificity as PTEN,
and we have therefore termed the new protein PTEN 2.
The structure of PTEN has been recently solved by x-ray crystallography
at 2.1-Å resolution (12). This analysis revealed a molecule consisting
of an amino-terminal phosphatase domain and a carboxyl-terminal C2
lipid binding motif, which were tightly packing against each other
through a large interface. The phosphatase active site is similar to
that observed in protein tyrosine phosphatases, including the essential
catalytic residues, but is enlarged to allow for the binding of the
larger phosphoinositide substrate. C2 domains have been shown to
mediate membrane lipid association. Based on the 39% sequence identity
between PTEN 2 and PTEN, a three-dimensional model of the phosphatase
and C2 domains of PTEN 2 has been built by homology modeling and
threading techniques using the PTEN structure as template (Fig. 2). The
existence of a C2 domain in PTEN 2 (PTEN 2-C2) was established by using
threading analysis and the high conservation of residues forming the
phosphatase domain/C2 interface (Fig. 2). This domain presents a 23%
sequence identity with the PTEN C2 domain (PTEN-C2). In PTEN 2 there is a five-residue insertion in the "T1 loop" that could allow this loop to establish more extensive contacts with the C2 domain as compared with PTEN (Fig. 2). The catalytic residues essential for the
activity in all protein tyrosine phosphatases are conserved in PTEN 2 (Asp426, Cys458, and Arg464) and
occupy the same position in both, PTEN and PTEN 2 (Fig. 2). The high
conservation of residues forming the substrate binding site in PTEN and
PTEN 2 has structural implications for substrate recognition. In
particular, the positively charged residues that have been proposed to
interact with the negatively charged phosphate groups of the
phospholipid substrate in PTEN are conserved in PTEN 2 (His427, Lys459, and Lys462).
Manual docking of inositol (1,3,4,5)-tetrakisphosphate in the active
site of PTEN 2 was performed as described by Lee and collaborators (12)
and indicated that this phospholipid might bind to PTEN 2 in the same
binding mode as that proposed for PTEN (Fig.
3). Like the PTEN-C2, PTEN 2-C2 lacks the
residues present in other Ca2+-dependent
C2-containing proteins that coordinate Ca2+ and regulate
membrane binding. A patch of five lysines or "CBR3 loop" has been
proposed by Lee and collaborators (12) to mimic the Ca2+
charge in PTEN. In PTEN 2, none of the five lysine residues in the CBR3
loop of PTEN is conserved (Pro607, Pro610,
Tyr613, Asp614, and Cys616). A
second "positive patch" in helix c2 (Lys644,
Lys646, and Lys649) is conserved in both PTEN
and PTEN 2. This region is similar to a helix in phospholipase
A2 that has been shown to contribute to membrane binding.
Together, these analyses suggest that many of the functional
characteristics of the PTEN structure are conserved in PTEN 2.
PTEN 2 Is Expressed in a Specific Stage of Sperm
Development--
Northern blot analysis (Fig. 3) of various murine
tissues using a PTEN 2 probe revealed a discrete ~2.7-kb transcript,
which was specifically expressed in testis, in agreement with results for the human PTEN 2 sequence (26). The specificity of testis expression is emphasized by the observation that PCR analysis of
multiple murine tissues revealed a signal only in testis RNA, even
after a large number of PCR cycles (Fig. 3). Because testis contains a
diversity of cell types, some of which (spermatocytes) pass through a
number of developmental stages (27), we decided to analyze this tissue
using in situ hybridization. Isotopic in situ
hybridization was performed on adult testis, as well as on testes
representing various stages of adolescence. In the adult testis a
positive signal was observed in germ cells within seminiferous tubules,
whereas cells in the interstitium of the testis were negative. The
positive signal within the seminiferous tubules showed an uneven
distribution with some tubules displaying a strong signal, whereas
others were completely negative (Fig. 4).
A positive in situ hybridization signal appeared to
correlate with the presence of a specific cell population. The
positively reacting cells reside in the adluminal portion of the
tubule, are of small to medium size, have a round nucleus with evenly
distributed, finely granular chromatin, and a distinct nucleolus.
Based on morphological criteria, these cells are most consistent with
secondary spermatocytes and/or very early spermatids. The abundance of
the positive signal, together with the short half-life of secondary
spermatocytes, makes it likely that both cell types express PTEN 2. Sertoli cells, spermatogonia, primary spermatocytes, or mature
spermatids did not express PTEN 2 RNA. Expression in these cell types
was ruled out on the basis of their location within the seminiferous
tubules, size and shape of cell body and nucleus, and chromatin
pattern. Expression of PTEN 2 during testicular maturation is not
detected until day 19 of postnatal development (Fig. 4). In a time
course experiment we were unable to demonstrate PTEN 2 RNA in testes
removed on days 3, 7, 10, and 16. The expression of PTEN 2 RNA
therefore slightly precedes sexual maturity in the male mouse,
consistent with an association of PTEN 2 with late events of
spermatogenesis.
PTEN 2 Is Associated with the Golgi Apparatus--
Examination of
the PTEN 2 sequence suggested that four potential transmembrane domains
are found in the protein, consistent with the possibility that PTEN 2 is a membrane-associated molecule that passes through the secretory
pathway. To examine the subcellular localization of PTEN 2, a plasmid
encoding a form of the protein with a carboxyl-terminal myc
epitope tag was transfected together with a plasmid encoding a
yellow fluorescence protein-trans/medial Golgi marker (encoding the
first 81 amino acids of
To determine if the amino-terminal transmembrane-containing
region was involved with Golgi localization, truncated forms of the
protein encoding either the amino-terminal transmembrane motifs or the
carboxyl-terminal phosphatase and C2 domains were also expressed in COS
cells. Fig. 5 reveals that the carboxyl-terminal fragment of the
protein appeared as a diffusely stained signal throughout the cytoplasm
and in the nucleus, whereas the amino-terminal fragment containing the
transmembrane motifs was found to colocalize with the perinuclear Golgi
apparatus in a manner that was similar to the full-length protein,
consistent with the suggestion that the subcellular localization of the
protein is mediated by these hydrophobic domains. These results suggest
that, in contrast to PTEN, which appears to be cytoplasmically
localized in transfected cells, PTEN 2 appears to be localized to the
Golgi apparatus via an interaction that is likely to be mediated by one
or more of the amino-terminal transmembrane motifs.
Enzymatic Activity of PTEN 2--
Homology modeling (Fig. 2)
strongly suggested that PTEN 2 might be a lipid phosphatase with
specificity for the 3-phosphorylated phosphatidylinositols that are
products of a PI3K reaction. To examine the lipid phosphatase activity
of PTEN 2, a truncated form of the protein, including the catalytic
region and the potential C2 domain was produced in and isolated from
transfected mammalian cells. As a control, a truncated form of PTEN 2 with a mutation at the critical active site cysteine
(Cys458 The regulation of phosphatidyl inositol levels is a critical
component of a diversity of cellular functions ranging from cell survival to membrane trafficking (1). The levels of these various phospholipids are determined by a number of factors, including the
activities of various lipid kinases and phosphatases. One lipid
phosphatase, termed PTEN, is especially interesting because of its
association with a survival pathway in cells as well as its apparent
tumor suppressor activity. Because PTEN is the first phosphatase, which
appears to dephosphorylate phospholipids at the 3 position, the
identification of other phosphatases with similar activity is of great
interest. The data described here report a novel relative of PTEN,
termed PTEN 2, which has many of the enzymatic characteristics of PTEN,
but which shows significant differences with the original enzyme,
including a unique cell-type specificity and an association with the
Golgi secretory compartment.
The three-dimensional structure of PTEN provided a number of insights
into the potential mechanisms by which this phosphatases recognizes and
dephosphorylates its 3-phosphate-containing phospholipid substrates
(12). Because of the high degree of sequence homology between PTEN and
the novel PTEN 2 described here, we were able to produce a model for
the novel enzyme by homology modeling. This model predicted a number of
interesting aspects about the novel enzyme. First the model suggested
that the novel enzyme should have lipid phosphatase activity specific
for the 3 position of the phospholipid, and this prediction was
confirmed in our enzymatic assays. In addition, the model predicted
that the novel phosphatase contains a carboxyl-terminal C2 domain,
which has the potential for interacting with membrane lipids. Perhaps
more interesting is the conservation in the interface between the
phosphatase and C2 domain in both PTEN and PTEN 2. This conservation
suggests that this region may have functional importance, and analysis of tumor-derived mutations in this region of PTEN suggested that this
interface is important for the maintenance of enzyme activity (12).
Interestingly, enzymatic analysis suggests that PTEN 2 has a greater
degree of specificity for phospholipids containing phosphate at both
the 3 and 5 positions. It will be important to examine the differences
between the PTEN and PTEN 2 phosphatase domains to determine the
mechanism by which this specificity is attained. The current model of
PTEN 2, together with the crystal structure of PTEN, should prove
useful for structural predictions that can be tested in enzymatic assays.
A major difference between PTEN and PTEN 2 is the extended amino
terminus, which contains four potential transmembrane domains. These
domains appear to be involved with the localization of PTEN 2 to the
Golgi apparatus, as demonstrated by colocalization with a known Golgi
marker protein as well as mislocalization of both PTEN 2 and the marker
protein in the presence of the Golgi-disrupting agent brefeldin. PTEN
was previously suggested to localize to the cell surface-restricted
phospholipid substrate, and particularly to the tight junctions of
epithelial cells, through a PDZ domain-mediated interaction with a
family of tight junction MAGUK proteins termed the MAGIs (23). PTEN 2 appears to have solved the membrane localization dilemma by
incorporating the hydrophobic amino-terminal region. However, this
hydrophobic region appears to have more complex functions than mere
localization to the Golgi apparatus. For example, the multiple
transmembrane domains in the amino terminus of PTEN 2 are reminiscent
of ion channels, and homology analysis of this region of PTEN 2 suggests a distant relationship with sodium channels (data not shown).
The role that PTEN 2 plays in the regulation of the Golgi apparatus
remains to be determined. However, a diversity of studies has suggested
that 3 phospholipids are involved with membrane trafficking and,
particularly, with the formation of multivesicular bodies (28-33). For
example, yeast mutants in phospholipid kinases, which produce either
PIP3 or PIP3,5, are found to be defective in
the formation of the multivesicular body (30). It should be noted that
PTEN 2, which has specificity for the PIP3,5 phospholipid,
might thus be involved with the regulation of this structure in
mammalian cells. Further work will be required to determine the exact
role of PTEN 2 in membrane trafficking, but its localization to the
Golgi, an important mediator of vesicular trafficking, together with
its specificity for PIP3,5, a known mediator of membrane
trafficking, jointly suggest that this enzyme may play a role in
regulating some aspect of vesicular localization in the cell.
Finally, the highly specific expression of the PTEN 2 phospholipid
phosphatase in a specific subset of developing sperm cells suggests
that it might play an important role in the terminal differentiation of
sperm. The cell type, which shows predominant expression of PTEN 2, was
the secondary spermatocyte, a stage of spermatogenesis that occurs just
before the large morphological changes that accompany the production of
mature sperm (27). A potential role for PTEN 2 in terminal sperm
differentiation is also supported by our finding that the transcript
for the enzyme appears simultaneously with the development of mature
sperm and sexual maturity (Fig. 4). Interestingly, the Golgi of the
late spermatocyte undergoes a profound morphological change to become the acrosome of the mature sperm. The possibility that PTEN 2 may be
involved with this morphological change is appealing for a number of
reasons. First, of course, is the temporal expression of the mRNA
encoding this phosphatase at a time when this profound morphological
change occurs. Second is the subcellular localization of the
phosphatase in the Golgi apparatus of transfected cells. Finally, and
perhaps most interestingly, is the notion that modulation of
phospholipid levels in subcellular compartments might be involved with
aspects of vesicular trafficking, as mentioned above. Together, these
results suggest that the specific expression of PTEN 2 in the Golgi of
secondary spermatocytes might be required for at least one of the
important developmental changes, which sperm progenitors undergo as
they differentiate to become mature sperm.
In summary, the results reported here suggest that a closely related
homologue of PTEN, termed PTEN 2, appears to be a Golgi-associated lipid phosphatase, which is expressed, in the terminal stages of sperm
development. Although the exact function of this phosphatase remains to
be determined, the data suggest a potential role in membrane
trafficking regulated by the Golgi apparatus. It will be of significant
interest to produce mice that are null mutants of this enzyme to
determine its role in spermatogenesis, and we are currently analyzing
mice with mutations in this phosphatase. In addition, further studies
into the exact specificity of the phosphatase in vivo, as
well as the effects of the expression of this enzyme on vesicular
trafficking, should provide insights into its function. Finally, the
mechanisms by which this phosphatase specifically degrades
3,5-containing phospholipids will require a further understanding of
the structure-function relationships of the catalytic site.
Together, these data may highlight a novel role for this phosphatase in
a critical aspect of mammalian reproduction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C. Full-length
human PTEN cDNA was cloned into the baculovirus expression vector,
PH.hif, as a carboxyl-terminal HIS-tag fusion. The PCR primers
used for this were: 5'-CATCGCGATCGCATGACAGCCATCATCAAAGAG-3' and
5'-CTACGCGGCCGCTCAGACTTTTGTAATTTGTGTATGC-3'. Insect "Hi-Five" cells (Expression Systems) at 7.5 × 105/ml were
infected with a multiplicity of 1.0 for 48 h and then harvested.
Pelleted cells were resuspended in 100 ml of 50 mM Tris, pH
7.5, 300 mM NaCl, 250 mM sucrose, 1 mM DTT, and 5 mM imidazole. The suspension was
sonicated for 1 min on ice and centrifuged at 10,000 × g for 15 min. The supernatant was collected and recirculated over a 2-ml column of nickel-nitrilotriacetic acid Superflow (Qiagen, 1004493). The column was washed with 10 column volumes of lysis buffer
and eluted with 5 × 1-ml steps of 50 mM Tris, pH 7.5, 250 mM sucrose, 150 mM NaCl, 2 mM
DTT, and 250 mM imidazole. Enzyme was stored at
70 °C
in 20-µl aliquots.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence, hydropathy, and domain model of
PTEN 2. A, the sequence comparison between murine and
human (26) PTEN 2 and human PTEN. The localization of putative
transmembrane domains as predicted by a structural algorithm (T. Wu,
unpublished data) as well as the predicted phosphatase domain are
overlined. B, hydropathy analysis of the murine
PTEN 2 sequence illustrated in A reveals the localization of
the four potential transmembrane domains. The localization of the
phosphatase domain is also illustrated. The human PTEN 2 sequence
showed a similar pattern (data not shown). C, a domain model
of PTEN 2 illustrating the membrane association mediated by the four
potential amino-terminal transmembrane domains and the phosphatase
domain.
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Fig. 2.
Modeling of PTEN 2. A,
sequence alignment of PTEN and PTEN 2 used for the molecular
modeling. Hydrophilic and charged residues are displayed in
red, and aromatic/hydrophobic residues are shown in
black. The consensus is shown below the alignment, with
conserved hydrophilic/charged and aromatic/hydrophobic residues marked as
red and black squares, respectively. Residues
forming the active site of the phosphatase and the phosphatase/C2
interface are boxed pink and green, respectively.
Cancer-related mutations are highlighted with blue
asterisks. Charged residues in the C2 loop involved in contacts
with the lipid membrane are marked with red asterisks.
B, ribbon diagram of the superimposition of the x-ray
structure of PTEN (in blue), and the modeled structure of
PTEN 2 (in white). Alpha helices are shown as
cylinders, and beta strands as arrows (root mean
square deviation, C = 0.33 Å). a, enlarged view of
the active site showing binding mode of a phosphatidylinositol
4-phosphate molecule as well as tartrate, a substrate mimic (12). Key
residues for substrate recognition are shown and labeled in
blue for PTEN and white for PTEN 2. b,
enlarged view of the PTP/C2 interface highlighting residue conservation
between PTEN and PTEN 2.
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Fig. 3.
Transcript distribution of PTEN 2. Northern blot (A) and PCR (B) analysis of PTEN 2 transcript reveals that the message is only found in testis.
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Fig. 4.
In situ hybridization analysis of
PTEN 2 expression in the testis. Brightfield (A) and
darkfield (B) image (200× magnification) of adult testis
showing expression in three of four testicular tubules. C,
mPTEN 2 RNA expression in subpopulation of germ cells
(arrow; 400× magnification). These cells demonstrate the
morphological features of secondary spermatocytes and early spermatids
(see text). D-I, dark- and brightfield images of testes at
day 10 (D and G), 16 (E and
H), and 19 (F and I) of postnatal life. Day 19, the time
when sexual maturity is reached, is the earliest time point with mPTEN
2 expression in testicular germ cells (200× magnification).
1,4-galactosyltransferase) into COS cells,
and confocal microscopy was performed. Fig.
5 shows that examination of permeabilized
cells revealed that PTEN 2 clearly colocalized with the
Golgi-associated marker in perinuclear, vesicular structures,
consistent with the suggestion that PTEN 2 was found in the secretory
pathway of the cell. This localization was further emphasized by
examining transfected cells treated with the Golgi-disrupting agent,
brefeldin. The vesicular, perinuclear localization of both PTEN 2 as
well as the YFP Golgi marker was disrupted in these cells (Fig. 5),
consistent with the localization of both of these proteins to the Golgi
apparatus.
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Fig. 5.
Subcellular localization of PTEN 2. The
top panel illustrates confocal analysis of COS cells
transfected with a PTEN 2 construct with a carboxyl-terminal
myc tag (red), a marker for the Golgi consisting
of the first 81 amino acids of 1,4-galactosyltransferase, and the
yellow fluorescence protein (green) and the overlapping
image (yellow). Note the perinuclear, vesicular morphology
of the co-stained subcellular region, which is consistent with the
Golgi apparatus. The middle panel shows that cells
transfected as in the top panel show delocalization of the
two markers when they are treated with the Golgi-disrupting agent,
brefeldin. The bottom panels illustrate the subcellular
distribution of various forms of PTEN 2. Note that the full-length and
amino-terminal, potential transmembrane containing, forms associate
with the perinuclear Golgi region, whereas the carboxyl-terminal form
shows a diffuse staining over the whole cell, including the
nucleus.
Ser) was also produced. Full-length wild type
PTEN and active site-mutated (Cys124
Ser) were also
produced as positive controls using the baculovirus system. Each of
these isolated proteins was tested for dephosphorylation of a variety
of phosphatidyl inositol substrates in an in vitro malachite
green enzyme assay. Fig. 6 illustrates
that the wild type version of PTEN was able to release phosphate from a
diversity of substrates, including those containing phosphate at the 3 position. This activity was completely abolished in the
Cys124
Ser active site mutant (data not shown).
Although this apparent lack of substrate specificity is at odds with
the accepted activity of PTEN toward only the 3 phosphate position, it
should be remembered that this assay is performed under artificial
conditions in vitro. Importantly, the activity of the PTEN 2 phosphatase domain appeared to be directed toward substrates containing
phosphates at both the 3 and 5 positions. Thus, although the enzyme
showed strong activity against PIP3,5 and
PIP3,4,5 substrates, there was lower activity against
PIP3 and PIP3,4. Because molecular modeling
predicts that this enzyme is a 3 phosphatase, together with the
complete lack of homology between PTEN 2 and phosphatases with
specificity for the 5 position of phosphatidyl inositols, we propose
that PTEN 2 removes the 3 phosphate from the PIP3,5 and
PIP3,4,5 substrates. However, further analysis of the
structures of the dephosphorylated substrates will be required to
delineate the exact specificity of this enzyme. In summary, as
predicted from molecular modeling, PTEN 2 appears to be a lipid
phosphatase with specificity for a subset of 3-phosphorylated
phosphatidylinositols.
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Fig. 6.
Phospholipid phosphatase activity of PTEN
2. The enzymatic activity of a GST fusion of PTEN 2 produced in
mammalian 293 cells and containing the phosphatase and C2 domains was
assayed using the illustrated phospholipids as substrates. Released
phosphate was assayed using the malachite green reagent. The enzymatic
activity of PTEN, including the phosphatase and C2 domains, produced in
baculovirus-infected cells was assayed as described for PTEN 2. In both
cases, analysis of mutants where the active site cysteine was converted
to serine showed absolutely no activity (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Dept. of Molecular
Oncology, Genentech, Inc., 460 Pt. San Bruno Blvd., South San Francisco, CA 94080. Tel.: 650-225-1123; Fax: 650-225-6127; E-mail: lal@gene.com.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M101480200
2 T. Wu, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PIP3, phosphatidylinositol 3-phosphate phospholipid; PIP3, 4, phosphatidylinositol 3,4-phosphate phospholipid; PIP3, 4,5, phosphatidylinositol 3,4,5-phosphate phospholipid; MAGUK, membrane-associated guanylate kinase; GST, glutathione S-transferase; GFP, green fluorescence protein; kb, kilobase(s); bp, base pair(s); DTT, dithiothreitol; PDZ, PSD95, discs large, Z01 domain; PTEN, phosphatase with tensin homology..
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REFERENCES |
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