PTEN 2, a Golgi-associated Testis-specific Homologue of the PTEN Tumor Suppressor Lipid Phosphatase*

Yan WuDagger , Donald DowbenkoDagger , M. Teresa Pisabarro§, Lisa Dillard-Telm, Hartmut Koeppen, and Laurence A. LaskyDagger ||

From the Departments of Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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. 


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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.

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. 


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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.

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.


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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).

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 beta 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 beta 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.

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 right-arrow Ser) was also produced. Full-length wild type PTEN and active site-mutated (Cys124 right-arrow 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 right-arrow 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

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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