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* Program of Signal Transduction,
Cell Analysis and Histology Facility, and
Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, CA 92037; and
Department of Clinical Chemistry, Uppsala University Hospital, Uppsala, Sweden
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 68-kDa PTP-MEG2 (11) is the sole mammalian
representative of a group of PTPases that contain a unique 250-aa
N-terminal region with homology to cellular retinaldehyde-binding
protein and Sec14p, a yeast protein with phosphatidylinositol transfer
activity. Two PTPases related to PTP-MEG2, termed PTPX1 and PTPX10,
have been cloned from Xenopus laevis (12). In
addition, Sec14p homology domains are also found in
-tocopherol-binding protein (13), a 45-kDa secretory
protein from olfactory epithelium (14), human Sec14p
homologs (15), and a large number of other proteins from
yeast and plants to humans (16). A common theme among
these proteins is binding of hydrophobic molecules and involvement in
secretion.
Very little is known about the biology of PTP-MEG2. The enzyme is expressed in many cell types (11), including at low levels in Jurkat T cells (17), and its expression is induced during monocyte/macrophage differentiation of U937 cells (18). We have recently found that PTP-MEG2 differed from 13 other tested intracellular PTPases in that its expression resulted in its accumulation in large intracellular vesicles of unknown nature (17). In this study, we demonstrate that these vesicles represent secretory vesicles, which fuse and swell in a manner that depends on the catalytic activity of PTP-MEG2. Based on our findings, we discuss a possible physiological function of PTP-MEG2 in the biogenesis of secretory vesicles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-influenza hemagglutinin tag epitope (HA) mAb 12CA5 conjugated to FITC or rhodamine isothiocyanate (TRITC) were from Roche Molecular Biochemicals (Indianapolis, IN). The 16B12 anti-HA from BAbCO (Richmond, CA) was used for immunoblotting. Anti-carboxypeptidase E was from Transduction Laboratories (Lexington, KY). FITC-conjugated rabbit anti-IL-2 was from Alexis (San Diego, CA). Goat anti-rabbit IgG (F(ab)2)-FITC was from Pierce (Rockford, IL). Anti-Lamp-1 (931B) antiserum, anti-Lamp-2 (932B), and anti-TGN46/51 polyclonal Abs have been described (19, 20). Anti-chromogranin antisera were from M. Stridsberg (21). A polyclonal Ab against PTP-MEG2 was generated against a GST N-terminal MEG2 fusion protein as described (22). Another antiserum was raised against a synthetic peptide corresponding to the last 30 aa of PTP-MEG2 conjugated to keyhole limpet hemocyanin. A third antiserum was raised against the internal peptide HSYRETRRKEGIVK, corresponding to aa 66–79 of human PTP-MEG2.
Plasmid construction
The cDNA for PTP-MEG2 was kindly provided by P. Majerus (Washington University, St. Louis, MO). It was subcloned in to the pEF/HA vector (23), which adds an HA to the N terminus of the cloned insert. The catalytically inactive mutant PTP-MEG2-C515S was generated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutation was verified by nucleotide sequencing.
Cells and transient transfection
PBLs were isolated from buffy coats from healthy volunteers by gradient centrifugation. The cells were cultured in RPMI medium with 10% heat-inactivated FCS, 10 µg/ml PHA, and 100 U/ml of IL-2 for 72 h before use. Rat basophilic leukemia (RBL), MCP5 mast cells, and Jurkat cells were kept at logarithmic growth in RPMI supplemented with 10% heat-inactivated FCS, L-glutamine, and antibiotics. Transient transfections were conducted by electroporation as described before (24, 25, 26, 27). Electroporation conditions typically contained 20 x 106 cells and a total of 1–10 µg of plasmid DNA, and in each transfection the DNA amount was kept constant by the addition of empty vector. Cells were used for experiments 48 h after transfection.
Immunoblots and immunoprecipitation
Immunoprecipitation was performed as before (24, 25, 26, 27, 28). Immunoblots were developed by the ECL technique (ECL kit; Amersham Pharmacia Biotech, Little Chalfont, U.K.) according to the manufacturer’s instructions.
Confocal microscopy
Double immunofluorescence staining was done as before (17). Briefly, cells were washed in PBS and fixed in freshly made 3.7% formaldehyde. Fixed cells were permeabilized with 0.05% saponin, 0.5% BSA in PBS for 10 min at room temperature, and then incubated with primary and secondary Ab diluted in the same buffer for 1 h each at room temperature. After three washes with PBS, the cells were mounted onto glass slides and viewed under a confocal laser scanning microscopy MRC-1024 (Bio-Rad, Hercules, CA). A differential interference contrast image was also taken of most cells.
Electron microscopy
Cells were fixed in 3.7% paraformaldehyde and 0.2% glutaraldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES (pH 6.9), 10 mM EGTA, 2 mM Mg2SO4, and 0.02% NaN3) for 45 min at room temperature. Cells were washed three times with 0.002% glycine in PHEM buffer, and were postfixed in 1% (w/v) osmium tetroxide in 0.2 M cacodylate (pH 7.2), washed in distilled water, and dehydrated successively in ethanol. Dehydrated pellets were infused with propylene oxide and embedded in Epon 812. Ultrathin sections were obtained on a Reichert-Jung microtome, and double stained with uranyl acetate and lead citrate (29). Specimens were examined in a Hitachi H-600 electron microscope (Hitachi, Tokyo, Japan) at 75 kV with magnifications ranging from x3,000 to x100,000.
IL-2 secretion assay
A total of 20 x 106 Jurkat T cells cotransfected with PTP-MEG2 or empty vector and green fluorescent protein were cultured for 48 h, and then 2 x 106 green fluorescent cells were sorted in a BD Biosciences FACStar fluorescence-activated cell sorter with a 488-nm argon laser (BD Biosciences, Mountain View, CA) to >95% purity. A total of 0.33 x 106 sorted cells in triplicate were stimulated in 100 µl of medium with 50 ng/ml PMA plus 0.3 µg/ml ionomycin for 12 h, and 20 µl of the supernatant used for measurement of the amount of IL-2 using an ELISA kit (Roche Molecular Biochemicals). Results are given as picograms per milliliter of secreted IL-2 (by 66 x 103 cells).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A polyclonal antiserum was raised against the N terminus of human
PTP-MEG2. This antiserum immunoprecipitated PTP-MEG2 (Fig. 1
A) and reacted well with
transfected PTP-MEG2 in immunofluorescence staining (Fig. 2, A–D). Indirect
immunofluorescence staining of MCP5 (Fig. 1B) or RBL (Fig. 1F) mast cells gave rise to a granular cytoplasmic staining.
The preimmune serum did not react with these structures (Fig. 1C). Double immunofluorescence staining with Abs against
carboxypeptidase E, an enzyme typically located in secretory vesicles
(30) where it processes prohormones by cleaving C-terminal
arginine-containing motifs (31, 32), showed a great deal
of colocalization (Fig. 1, D and E), suggesting
that PTP-MEG2 is primarily located on secretory vesicles. In agreement
with this notion, the antiserum reacted weakly with resting Jurkat T
cells (Fig. 1G), which have very few secretory vesicles, but
considerably better with cells stimulated for 8 h with phorbol
ester and ionomycin (Fig. 1H). By immunoblotting, low levels
of PTP-MEG2 were detected in T cell lines and better levels in RBL mast
cells (Fig. 1I). Expression of PTP-MEG2 was also induced in
normal T lymphocytes by polyclonal activation with PHA and IL-2 (Fig. 1J).
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To better study the subcellular location of PTP-MEG2, we
transfected an epitope-tagged PTP-MEG2 expression plasmid into RBL mast
cells and into Jurkat T leukemia cells. Both of these cell types have a
regulated secretory apparatus typical of immune cells
(33), in the latter case induced by treatment with
anti-CD3 mAbs or calcium ionophore and phorbol ester. After fixing,
the cells were stained either with the polyclonal Ab followed by
FITC-conjugated anti-rabbit Ig (Fig. 2
, A, B,
and D) or a directly FITC-conjugated anti-HA mAb (Fig. 2, G–M), and viewed under a confocal microscope. In both
cell types and with both staining protocols, most of the fluorescence
was seen in large, and often solitary, vesicles in the cytoplasm of the
transfected cells. These bubble-like structures have been seen in >50
separate transfection experiments, and are consistently found in at
least 90% of cells expressing detectable exogenous PTP-MEG2. No
fluorescence above background was seen in cells incubated with
preimmune serum (Fig. 2, C and E). The effect of
PTP-MEG2 was dose-dependent (Fig. 2, K–M): transfection of
Jurkat cells with 0.2 µg of plasmid DNA resulted in relatively small
vesicles, while 2 µg of DNA was sufficient to increase the size of
the vesicles and to reduce their number. Although there was some
variation in size, the enlarged vesicles often had diameters of 1–2
µm, occasionally reaching 3 µm, and were usually found in the
broadest part of the cytoplasm, often reaching from the nucleus to the
plasma membrane. The enlarged vesicles were also seen by differential
interference contrast in many experiments (see
Figs. 5–7).
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Ultrastructure of PTP-MEG2-induced large secretory vesicles
Electron microscopy of Jurkat cells transfected with PTP-MEG2
showed that the enlarged vesicles were membrane-enclosed vesicles with
an amorphous content typical of secretory vesicles
(Figs. 3 and 4). In cells with very large vesicles,
the amorphous content was diluted and dispersed throughout the lumen,
while in smaller vesicles it remained more densely packed. For
comparison, Fig. 3B shows an electron micrograph of an
activated blood T lymphocyte with normal secretory vesicles with
diameters of 200–300 nm (Fig. 3C).
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E). In addition, many vesicles contained signs of recent
fusion with other vesicles in the form of rings inside the enclosing
membrane (Figs. 3, A, D, and F, and 4,
G and H). On closer inspection, these rings
consisted of the amorphous secretory vesicle contents (Fig. 4, G and H), not lipid bilayer. The enlarged
vesicles were often found adjacent to the trans-Golgi
cisternae (Fig. 3G). Taken together, these electron
microscopy studies suggest that PTP-MEG2 causes a striking increase in
fusion and/or lack of fission within the secretory vesicle compartment,
resulting in giant vesicles with diluted contents. Lack of markers for other organelles in the fused secretory vesicles
Although the electron microscopy studies suggested that PTP-MEG2
promotes homotypic fusion between secretory vesicles, we felt that it
was important to determine whether the enlarged vesicles represented
only fused secretory vesicles, or whether they included parts of other
organelles as well. This was conducted by confocal microscopy using
several different markers for subcellular compartments. Although the
PTP-MEG2-induced enlarged vesicles stained positive for the secretory
vesicle markers carboxypeptidase E and chromogranin C (Fig. 5, A–C), they were negative
for galactosyl transferase (Fig. 5, D and E), an
enzyme confined to the Golgi apparatus, for TGN46 (Fig. 5F),
a protein found in the trans-most Golgi and the adjacent
vesicles (20), and for catalase (Fig. 5, G and
H), 
COP, clathrin, dynamin, and caveolin (data not
shown). Thus, they appear not to contain parts of the Golgi apparatus,
the trans-Golgi network, peroxisomes, endoplasmic reticulum,
clathrin-coated vesicles, or caveolae. Interestingly, the lysosomal and
immature secretory vesicle marker Lamp-1 (33) was seen
inside most PTP-MEG2-containing vesicles as well as in separate, small,
and much more intensely stained dots (Fig. 5, I–K). The
latter represent bona fide lysosomes, in which Lamp-1 is enriched
(19), while the weaker staining of the lumen of the
enlarged vesicles is compatible with an immature secretory vesicle
nature of these vesicles. The localization of Lamp-1 and PTP-MEG2 in
the same vesicles was also readily detectable by coimmunoprecipitation
(Fig. 5L).
Expression of PTP-MEG2 disturbs secretion
The Jurkat T cell represents a somewhat immature, immortalized,
Th cell. Upon activation with phorbol ester plus calcium
ionophore, these cells synthesize and secrete IL-2. Therefore, we
tested whether the enlarged secretory vesicles induced by expression of
PTP-MEG2 would contain IL-2 upon stimulation of the cells. Double
immunofluorescence staining of cells for PTP-MEG2 and IL-2 showed that
IL-2 was undetectable in resting cells (Fig. 6, A and B), but
visible as discrete spots in control cells stimulated for 6 h with
phorbol ester plus ionomycin (Fig. 6C). In stimulated cells
expressing PTP-MEG2, IL-2 was found in the lumen of the large vesicle
(Fig. 6, D–F). These experiments again illustrate the size
difference between normal secretory vesicles and the fused secretory
compartment in cells transfected with PTP-MEG2. They also show that the
contents of the fused vesicle are not as concentrated as normally.
To measure the secretion of IL-2 by PTP-MEG2-expressing cells, we
cotransfected cells with green fluorescent protein and empty vector,
PTP-MEG2, or the catalytically inactive PTP-MEG2-C515S. Two days later,
fluorescent cells were sorted out by FACS, stimulated for 12 h
with phorbol ester plus calcium ionophore and the supernatants used for
quantitation of IL-2. As shown in Fig. 6G, expression of
PTP-MEG2 caused a substantial reduction in IL-2 in the medium. The same
result was obtained in triplicate also in a second independent
experiment. Because PTP-MEG2 had no effect on the transcriptional
activation of the IL-2 gene as measured by a luciferase reporter gene
driven by the IL-2 promoter (17), and there clearly was
intracellular IL-2 in the cells, we conclude that the reduced secretion
was likely due to a disturbance in the exocytosis of the content of
these vesicles. This was not unexpected, given the enlarged size of the
vesicles and their diluted contents. Nevertheless, we cannot exclude
the possibility that PTP-MEG2 has some effect on the exocytosis process
itself.
Secretory vesicle size is regulated by PTP-MEG2 catalytic activity
The dephosphorylation of substrates by PTPases depends on a
critical cysteine residue in the bottom of their catalytic center
(34). This residue is Cys-515 in PTP-MEG2. In contrast to
active PTP-MEG2, which caused secretory vesicle fusion in the same
experiments (Fig. 7, A–D and
G–J), expression of the catalytically inactive
PTP-MEG2-C515S mutant in either RBL or Jurkat cells did not lead to
secretory vesicle fusion (Fig. 7, E, F, and
K–N). Instead the staining was diffuse, but somewhat
granular, throughout the cytosol. In sharper images, it was clear that
the staining was predominantly in the form of very small vesicles. By
electron microscopy, the secretory vesicles were numerous and small
(Fig. 7O) in Jurkat cells expressing PTP-MEG2-C515S with an
average size of 196 ± 34 nm (n = 14), compared
with 288 ± 63 nm (n = 8) in vector-transfected
Jurkat T cells. Thus, it appears that inactive PTP-MEG2 causes
secretory vesicles to be somewhat smaller than normal. This may be a
dominant-negative effect by competition with endogenous PTP-MEG2.
To further test the notion that PTP-MEG2 must be catalytically active
to induce secretory vesicle fusion, we treated Jurkat cells expressing
active PTP-MEG2 with the general PTPase inhibitor pervanadate. This
resulted in a rapid shrinking or fragmentation of the fused secretory
vesicles, which became numerous and undetectably small in most cells
within minutes (Fig. 7, P and Q). A minority of
cells still contained medium-sized vesicles, but these were never >0.5
µm in diameter. Thus, it appears that secretory vesicle fusion
depends on the catalytic activity of PTP-MEG2 and probably the
dephosphorylation of one or several regulatory proteins.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-islet cells, proinsulin
synthesized in the endoplasmic reticulum and transported through the
Golgi apparatus is sorted in the trans-Golgi network into
small transport vesicles that fuse into condensing vacuoles or immature
secretory vesicles. These subsequently undergo an editing and
concentration process to become mature secretory vesicles
(35). The initial condensing vacuoles contain some
lysosomal proteins, such as Lamp-1, which are recycled back to the
Golgi during secretory vesicle maturation. By analogy with other
membrane-trafficking processes, recycling of Lamp-1 and other proteins
from the maturing secretory vesicle probably occurs by sorting of cargo
molecules into regions that subsequently bud off to become small
transport vesicles destined for the Golgi.
It appears that PTP-MEG2 both promotes the fusion of
trans-Golgi-derived vesicles into condensing vacuoles and
prevents the recycling and concentration process. As a consequence,
immature secretory vesicles fuse into larger structures than normal and
retain membrane components that normally leave by retrograde transport.
As a result, the vesicles swell enormously. Conversely, the rapid
shrinking of these large vesicles upon inhibition of the catalytic
activity of PTP-MEG2 is likely to represent the reversal of the block
in recycling, as well as a halt in fusion. Because the catalytic
activity of PTP-MEG2 is required, we suggest that PTP-MEG2
dephosphorylates one or several proteins involved in fusion and
retrograde transport and that the effect of PTP-MEG2 expression
represents the result of excessive dephosphorylation of these proteins
due to more PTP-MEG2 protein than normal in the transfected cells.
Conversely, cells expressing the inactive PTP-MEG2-C515S had secretory
vesicles of a somewhat smaller size than normal. This may also explain
the somewhat lower level of IL-2 secreted by these cells (Fig. 6G)
We propose that the physiological function of PTP-MEG2 may be to
regulate the formation of secretory vesicles of a defined and cell
type-specific size. It appears that mast cells, which contain
relatively large granules (400–600 nm), express higher levels of
PTP-MEG2 than lymphocytes, in which secretory granules are smaller
(200–300 nm). The induction of PTP-MEG2 expression during phorbol
ester-induced maturation of HL-60 cells (18), or in
phorbol ester- and ionomycin-treated Jurkat cells (Fig. 1H),
also parallel the formation of secretory granules. Thus, PTP-MEG2 may
be a molecule whose expression controls the extent of the secretory
apparatus of hematopoietic cells.
Although the finding that a PTPase is specifically localized to secretory vesicles is novel, a role for tyrosine phosphorylation in the regulation of secretory vesicle formation has been suggested before, based on effects of kinase and phosphatase inhibitors (36). However, none of the enzymes involved in this regulation have previously been identified. A direct role for tyrosine phosphorylation is perhaps not unexpected, given the central role of secretion in cell-cell communication and in the regulation of cell growth and differentiation. These cellular functions are typically regulated by reversible tyrosine phosphorylation.
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Acknowledgments |
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Footnotes |
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2 Current address: Gene Therapy Inc., San Diego, CA 92121.
3 Current address: Department of Medical Microbiology, Malmö University Hospital, Lund University, SE-205 02 Malmö, Sweden.
4 Address correspondence and reprint requests to Dr. Tomas Mustelin, Program of Signal Transduction, Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: tmustelin{at}burnham-inst.org
5 Abbreviations used in this paper: PTPase, protein tyrosine phosphatase; HA, hemagglutinin tag epitope; RBL, rat basophilic leukemia; TRITC, rhodamine isothiocyanate.
Received for publication August 30, 2001. Accepted for publication February 27, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopherol transfer protein from rat liver: homology with cellular retinaldehyde-binding protein. J. Biol. Chem. 268:17705.-granules. J. Biol. Chem. 269:20838.This article has been cited by other articles:
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  K. Saito, S. Williams, A. Bulankina, S. Honing, and T. Mustelin Association of Protein-tyrosine Phosphatase MEG2 via Its Sec14p Homology Domain with Vesicle-trafficking Proteins J. Biol. Chem., May 18, 2007; 282(20): 15170 - 15178. [Abstract] [Full Text] [PDF]
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 Y. Wang, E. Vachon, J. Zhang, V. Cherepanov, J. Kruger, J. Li, K. Saito, P. Shannon, N. Bottini, H. Huynh, et al. Tyrosine phosphatase MEG2 modulates murine development and platelet and lymphocyte activation through secretory vesicle function J. Exp. Med., December 5, 2005; 202(11): 1587 - 1597. [Abstract] [Full Text] [PDF]
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 R. J. Webb, J. D. Judah, L.-C. Lo, and G. M. H. Thomas Constitutive secretion of serum albumin requires reversible protein tyrosine phosphorylation events in trans-Golgi Am J Physiol Cell Physiol, September 1, 2005; 289(3): C748 - C756. [Abstract] [Full Text] [PDF]
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 S. Rahmouni, T. Vang, A. Alonso, S. Williams, M. van Stipdonk, C. Soncini, M. Moutschen, S. P. Schoenberger, and T. Mustelin Removal of C-Terminal Src Kinase from the Immune Synapse by a New Binding Protein Mol. Cell. Biol., March 15, 2005; 25(6): 2227 - 2241. [Abstract] [Full Text] [PDF]
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 V. Labrador, C. Brun, S. Konig, A. Roatti, and A. J. Baertschi Peptidyl-Glycine {alpha}-Amidating Monooxygenase Targeting and Shaping of Atrial Secretory Vesicles: Inhibition by Mutated N-Terminal ProANP and PBA Circ. Res., December 10, 2004; 95(12): e98 - e109. [Abstract] [Full Text] [PDF]
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A. Alonso, S. Burkhalter, J. Sasin, L. Tautz, J. Bogetz, H. Huynh, M. C. D. Bremer, L. J. Holsinger, A. Godzik, and T. Mustelin The Minimal Essential Core of a Cysteine-based Protein-tyrosine Phosphatase Revealed by a Novel 16-kDa VH1-like Phosphatase, VHZ J. Biol. Chem., August 20, 2004; 279(34): 35768 - 35774. [Abstract] [Full Text] [PDF]
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A. Alonso, S. Narisawa, J. Bogetz, L. Tautz, R. Hadzic, H. Huynh, S. Williams, A. Gjorloff-Wingren, M. C. D. Bremer, L. J. Holsinger, et al. VHY, a Novel Myristoylated Testis-restricted Dual Specificity Protein Phosphatase Related to VHX J. Biol. Chem., July 30, 2004; 279(31): 32586 - 32591. [Abstract] [Full Text] [PDF]
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A. Alonso, N. Bottini, S. Bruckner, S. Rahmouni, S. Williams, S. P. Schoenberger, and T. Mustelin Lck Dephosphorylation at Tyr-394 and Inhibition of T Cell Antigen Receptor Signaling by Yersinia Phosphatase YopH J. Biol. Chem., February 6, 2004; 279(6): 4922 - 4928. [Abstract] [Full Text] [PDF]
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 H. Huynh, X. Wang, W. Li, N. Bottini, S. Williams, K. Nika, H. Ishihara, A. Godzik, and T. Mustelin Homotypic Secretory Vesicle Fusion Induced by the Protein Tyrosine Phosphatase MEG2 Depends on Polyphosphoinositides in T Cells J. Immunol., December 15, 2003; 171(12): 6661 - 6671. [Abstract] [Full Text] [PDF]
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 M.-j. Xu, X. Sui, R. Zhao, C. Dai, S. B. Krantz, and Z. J. Zhao PTP-MEG2 is activated in polycythemia vera erythroid progenitor cells and is required for growth and expansion of erythroid cells Blood, December 15, 2003; 102(13): 4354 - 4360. [Abstract] [Full Text] [PDF]
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Y. Yang, P. Villain, T. Mustelin, and C. Couture Critical Role of Ser-520 Phosphorylation for Membrane Recruitment and Activation of the ZAP-70 Tyrosine Kinase in T Cells Mol. Cell. Biol., November 1, 2003; 23(21): 7667 - 7677. [Abstract] [Full Text] [PDF]
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R. Zhao, X. Fu, Q. Li, S. B. Krantz, and Z. J. Zhao Specific Interaction of Protein Tyrosine Phosphatase-MEG2 with Phosphatidylserine J. Biol. Chem., June 13, 2003; 278(25): 22609 - 22614. [Abstract] [Full Text] [PDF]
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