From the Department of Biochemistry and Molecular Biology, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612
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ABSTRACT |
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ST6Gal-I ( The sialyltransferases are a large family of glycosyltransferases
that act to modify N-linked and O-linked
oligosaccharides and glycolipids as these molecules traverse the Golgi
apparatus of the cell. Their activity is required for the synthesis of
important sialylated oligosaccharide structures that modulate or
mediate a variety of interactions. These include selectin-leukocyte
interactions in inflammation and lymphocyte homing; virus, parasite,
and toxin binding to host cells; maintenance of glycoproteins in the
circulation; cell interactions in B cell maturation and activation; and
antiadhesive effects during metastasis and development (for review, see
Ref. 1).
Many of the glycosyltransferases have been precisely localized in the
cisternae of the Golgi of various cell types (for review, see Ref. 2).
From these localization studies, it appears that the
glycosyltransferases are organized throughout the Golgi cisternae in
roughly the same order in which they act to add sugar residues to the
growing oligosaccharide chains. It has been presumed that this
relatively strict localization pattern allows for efficient glycosylation by ensuring that enzymes are compartmentalized with their
glycoconjugate substrates and sugar nucleotide donors. In support of
this idea, recent studies have shown that differential compartmentalization of enzymes that compete for the same substrates does alter the types of oligosaccharide structures made by the cell (3,
4).
In addition to compartmentalization, other post-transcriptional or
post-translational events may control glycosyltransferase activity.
Glycosyltransferases have been found as soluble forms in a variety of
body fluids (5-11). Not surprisingly, many of these enzymes are
cleaved and secreted after expression in tissue culture cells (12-21).
This type of turnover restricts the residence time of the enzymes in
the Golgi and their function as glycosyltransferases. Recently, we have
found that there are two isoforms of ST6Gal-I (ST)1 which differ by a
single amino acid at position 123 in the catalytic domain (22). This
single amino acid difference leads to alterations in activity,
localization, and proteolytic processing and secretion. The higher
activity STTyr isoform is found in the Golgi and at low
levels on the cell surface, whereas the lower activity
STCys isoform is found exclusively in the Golgi and even in
the endoplasmic reticulum (ER) upon overexpression. Most notably, the
STTyr isoform is cleaved and secreted with a half-time of
3-6 h, whereas the STCys remains completely
cell-associated over long periods of time. These results suggest that
differences in the conformation of the STTyr and
STCys catalytic domains lead to differences in their
trafficking and that this results in differences in their proteolytic processing.
Phosphorylation of cytoplasmic sequences has also been implicated in
controlling the catalytic activity and trafficking of glycosyltransferases. For example, Scheideler and Dawson (23) showed
that activity of the UDP-N-acetylgalactosamine
GM3-N-acetylgalactosaminyltransferase is increased by a
cytoplasmic cyclic AMP-dependent phosphorylation event. Yu
and colleagues (24) demonstrated that incubation of purified
CMP-NeuAc:GM1 and CMP-NeuAc:LacCer sialyltransferases with protein
kinase C decreased the activities of these enzymes in a
time-dependent manner and that phosphatase treatment
reversed this inhibition. Because protein kinase C is a cytoplasmic
enzyme, it is unclear whether the sites phosphorylated in these
in vitro studies are phosphorylated in vivo. Work
done by Strous et al. (25) demonstrated that
Here we report that both ST6Gal-I isoforms are phosphorylated on Ser
and Thr residues in different cell types. The phosphorylation is
confined to the luminal sequences with the STTyr
phosphorylated on stem and catalytic domain sequences and the
STCys phosphorylated on catalytic domain sequences only.
Retention of the STTyr luminal sequences in the ER
abolishes nearly all enzyme phosphorylation, suggesting that
phosphorylation occurs in the Golgi or a post-Golgi compartment.
Treatment of cells with monensin blocks STTyr cleavage and
secretion but not STTyr phosphorylation, suggesting that
phosphorylation of the ST occurs in the cis-medial Golgi cisternae.
Materials
Tissue culture media and reagents, including Dulbecco's
modified Eagle's medium (DMEM), Opti-MEM, and Lipofectin, were
purchased from Life Technologies, Inc. Fetal bovine serum was obtained
from Atlanta Biologicals (Norcross, GA). A Sequenase version 2.0 DNA sequencing kit was obtained from U. S. Biochemical Corp. A QIAquick PCR purification kit and polyvinylidene difluoride (PVDF) membranes were purchased from Qiagen Inc. (Chatsworth, CA). Vent DNA polymerase and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). Protein A-Sepharose Fast Flow was purchased from Amersham Pharmacia Biotech. Protein molecular weight standards were purchased from Bio-Rad. 35S-Express protein labeling mix was
purchased from NEN Life Science Products. 35S-dATP for DNA
sequencing and [35S]methionine for stoichiometry
estimations were purchased from Amersham Pharmacia Biotech.
32P was purchased from ICN Biomedicals (Irvine, CA).
Oligonucleotides and restriction enzymes were purchased from Life
Technologies, Inc. Selecto Scientific flexible cellulose TLC plates
were purchased from Fisher Scientific. Fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG was purchased from EY
Laboratories (San Mateo, CA). All other chemicals, including monensin,
and phosphoamino acid standards were purchased from Sigma.
Methods
Construction of Iip33-ST and Other ST Mutants--
The
The experiments comparing the phosphorylation, localization, and
cleavage of the Iip33-ST to that of the STTyr also used a STTyr coding sequence cloned into the V5-pcDNA 3.1 vector. To clone the STTyr into this vector, the entire
STTyr coding sequence lacking the original stop codon was
obtained by PCR using STTyr-pSVL as the template, a sense
primer based on the pSVL sequences 5' to the ST insert (5'-GCT CTA AAC
CGG AT-3'), and the same antisense primer used for PCR of the ST-trunc
sequences for the Iip33-ST-V5-pcDNA 3.1 construct (shown above).
The PCR product was cut with BamHI (in pSVL polylinker) and
XbaI and ligated into these sites in the V5-pcDNA 3.1 vector polylinker resulting in the STTyr-V5-pcDNA 3.1 construct.
Transfection of COS-1 Cells--
COS-1 cells maintained in DMEM
and 10% fetal bovine serum were plated on 100-mm tissue culture dishes
and grown in a 37 °C, 5% CO2 incubator until 50-70%
confluent. Lipofectin transfections were performed according to
protocols provided by Life Technologies, Inc. Briefly, 30 µl of
Lipofectin was incubated with 1.5 ml of Opti-MEM for 40 min at room
temperature in a polystyrene tube. 20 µg of DNA was mixed with 1.5 ml
of Opti-MEM, added to the Lipofectin solution, and incubated at room
temperature for 15 min. Cells to be transfected were washed with
Opti-MEM, and the transfection mixture was added to the tissue culture
dishes. Cells were incubated with the DNA-Lipofectin transfection
solution (3 ml) for 6 h at 37 °C in a 5% CO2
incubator. After 6 h, 7 ml of DMEM and 10% fetal bovine serum
were added to the plates, and expression was allowed to continue for
16 h in the 37 °C, 5% CO2 incubator.
Metabolic Labeling of Cells and Immunoprecipitation of ST
Proteins--
Pulse-chase analysis and immunoprecipitation were
performed as described previously (22). For labeling of proteins with [35S]methionine/cysteine, transfected COS-1 cells (in
100-mm culture dishes) were incubated with methionine- and
cysteine-free DMEM for 1 h. The medium was removed and replaced
with 3 ml of fresh methionine-free DMEM containing 100 µCi/ml
35S-Express protein labeling mix, and the cells were
labeled in a 37 °C 5% CO2 incubator for 4 h. The
radioactive medium was then removed, the cells were washed extensively,
and 3 ml of DMEM and 10% fetal bovine serum was added, and the cells
were incubated in the CO2 incubator for a chase time of
2 h. Cell medium was collected at each time point, and the cells
were washed extensively with phosphate-buffered saline (PBS) and lysed
in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1%
SDS) containing protease inhibitors. For labeling of proteins with
32P, transfected COS-1 cells were incubated in DMEM without
sodium phosphate for 30 min in a 5% CO2 incubator at
37 °C. The medium was removed, and 3 ml of DMEM without sodium
phosphate containing 2 mCi of 32P was added to the cells.
Cells were incubated with label for 2-4 h at 37 °C. Cells were
rinsed once with 10 ml of PBS and either lysed in immunoprecipitation
buffer (described above) or chased with DMEM and 10% fetal bovine
serum for 2 h. In some experiments, the medium was also collected.
ST proteins were immunoprecipitated from both cell lysates and medium
using anti-ST antibody and protein A-Sepharose as described previously
(22). For treatment of cells with monensin, transfected cells were
incubated in phosphate- or methionine/cysteine-deficient DMEM
containing 10 µM monensin (Sigma) for 1 h in a 5%
CO2, 37 °C incubator. The same concentration of monensin
was maintained throughout the labeling and chase periods. Radiolabeled
cell lysate and medium fractions were processed and immunoprecipitated
as described above. Immunoprecipitated proteins were analyzed by
SDS-polyacrylamide gel electrophoresis and fluorography. Bio-Rad
prestained broad range gel standards were used to estimate molecular
mass: myosin, 203 kDa; Estimation of the Stoichiometry of Phosphorylation--
To
estimate roughly the number of mol of phosphate/mol of ST protein, we
performed a double labeling experiment using
[35S]methionine and 32P. 100-mm plates of
transfected COS-1 cells were incubated for 1 h in methionine- and
sodium phosphate-free DMEM specially prepared by Life Technologies,
Inc. Cells were then labeled for 3 h with 300 µCi of
[35S]methionine and 2 mCi of 32P in 3 ml of
fresh methionine- and sodium phosphate-free DMEM. Cells were washed
extensively with PBS and lysed in immunoprecipitation buffer containing
0.5 mM activated sodium orthovanadate (Sigma). ST proteins
were immunoprecipitated from cell lysates as described above and
immunoprecipitates counted in a Beckman LS 6500 liquid scintillation
counter using different windows for 35S (0-670) and
32P (670-1,000) radioactivity. Spillover of
32P radioactivity into the 35S window was
determined and taken into account when doing final calculations. To
eliminate contributions from nonspecifically immunoprecipitated
material, similar experiments were performed on nontransfected COS-1
cells, and these values were subtracted from those of transfected
cells. Calculations were made on the basis of specific activities
provided by the vendors (1,000 Ci/mmol, [35S]methionine
(Amersham Pharmacia Biotech) and 9,104 Ci/mmol, 32P (ICN)).
Phosphoamino Acid Analysis--
One-dimensional thin layer
electrophoresis was performed to identify phosphoamino acids according
to the method described in the protocol included with the HTLE (Hunter
thin layer electrophoresis) 7000 apparatus (C. B. S. Scientific Company, Inc. (Del Mar, CA). The 32P-labeled
immunoprecipitated ST proteins were separated by SDS-polyacrylamide gel
electrophoresis and transferred electrophoretically to PVDF membrane.
Proteins were hydrolyzed and amino acids eluted from the membrane
according to the method of Kamps and Sefton (28). Briefly, the membrane
was washed three times for 2 min each in deionized water with
continuous agitation. After washing, the membrane was wrapped in Saran
Wrap and exposed to x-ray film. According to the band pattern on the
x-ray film, the desired bands were excised from the PVDF membrane. The
excised membranes were wetted in methanol for 1 min followed by a 1-min
incubation in deionized water. Then the excised membranes were immersed
in 6 N HCl and incubated at 110 °C for 1 h. At the
end of the incubation, samples were centrifuged at 14,000 rpm for 5 min. The supernatant was transferred to another tube and lyophilized.
The sample was then dissolved in 10 µl of deionized water and spotted
on a flexible cellulose thin layer chromatography plate (100-µm
thickness). Phosphoamino acid standards were loaded on the same spot.
The plate was wetted using pH 3.5 electrophoresis buffer (0.87 M acetic acid, 0.5% pyridine, 0.5 mM EDTA) and
electrophoresed at 1.3 kV for 45 min on a HTLE 7000 apparatus. After
electrophoresis, the plate was dried in a 60 °C oven for 20 min. The
phosphoamino acid standards were visualized by spraying the plate with
ninhydrin and reheating it in the 60 °C oven for 10 min. To
visualize the labeled phosphoamino acids, the plate was exposed to
x-ray film at Immunofluorescence Microscopy--
Immunofluorescence microscopy
was performed as described previously (22). COS-1 cells were plated on
glass coverslips and transfected with the designated expression
vectors. After 16 h of expression, cells were fixed and
permeabilized using The ST6Gal-I Isoforms Are Phosphorylated--
Analysis of the
biosynthesis and processing of the ST6Gal-I suggested that the ST
undergoes a post-translational modification that is not related to
N-linked glycosylation. This modification results in a
slight molecular mass increase (data not shown). One possibility is
that the enzyme is phosphorylated either on its single cytoplasmic Thr
or on luminal Ser, Thr, or Tyr residues. To test this possibility, we
expressed both ST isoforms (STTyr and STCys) in
COS-1 cells and labeled these cells for 4 h with 32P
or 35S-Express protein labeling mix. Proteins were
immunoprecipitated from cell lysates using affinity-purified anti-ST
antibodies, and immunoprecipitated proteins were analyzed by
SDS-polyacrylamide gel electrophoresis (Fig.
1). In COS-1 cells, the ST proteins were
labeled with 32P, demonstrating that they are modified by
phosphate residues. We also observed 32P labeling of the
two isoforms expressed in Chinese hamster ovary cells and of the
endogenous ST proteins expressed in the rat hepatoma cell line FTO2B
(data not shown and Fig. 2). Comparison
of the levels of 35S-labeled and 32P-labeled
proteins in COS-1 cells suggested that the STTyr isoform appeared to be more highly phosphorylated than the STCys
isoform (also see Fig. 3).
To obtain an estimate of the stoichiometry of ST protein
phosphorylation, we performed double-labeling experiments using
[35S]methionine and 32P. Transfected COS-1
cells were labeled for 3 h with 300 µCi of [35S]methionine and 2 mCi of 32P. Cells were
washed and lysed, and ST proteins were immunoprecipitated from cell
lysates and analyzed for both 35S and 32P
radioactivity (for details, see "Methods"). Immunoprecipitations were also performed on cell lysates from untransfected control cells to
eliminate any contribution from nonspecifically immunoprecipitated proteins. We found that both STTyr and STCys
isoforms incorporated between 0.18 and 0.32 mol of phosphate/mol of
protein (average, 0.25 mol of phosphate/mol of protein) in duplicate
experiments. Experiments below suggest that these steady-state labeling
experiments are likely to yield a minimum estimate of stoichiometry
because phosphorylation of these proteins occurs primarily in the Golgi (see Fig. 5), and a significant proportion of the
35S-labeled protein could be in the ER and therefore not
phosphorylated. The results indicate that at least 25% of the total ST
molecules are phosphorylated, or alternatively, that a smaller
proportion of ST molecules possess multiple phosphorylated amino acids.
Work by Eipper and Mains (29) suggested that 41-63% of
adrenocorticoptropin hormone is phosphorylated in vivo, and
work by Liberti et al. (30) demonstrated that 17-33% of
ovine growth hormone is phosphorylated in vivo. The levels
of ST phosphorylation compare well with the levels of phosphorylation
of these other phosphoproteins that are likely to receive their
phosphate as they traverse the secretory pathway.
The ST6Gal-I Isoforms Are Phosphorylated on Ser and Thr
Residues--
Phosphoamino acid analyses of the
32P-labeled, immunoprecipitated STTyr and
STCys proteins expressed in COS-1 cells and the endogenous
enzyme expressed in FTO2B rat hepatoma cells demonstrated that Ser and
Thr residues are phosphorylated in both isoforms and in both cell lines
(Fig. 2). No phosphotyrosine residues were detected. Interestingly, the
levels of Ser and Thr phosphorylation differed in the two cell lines.
In COS-1 cells, 50-60% of the phosphate residues were found on Thr
and 40-50% on Ser, whereas in FTO2B cells 27% of the phosphate
residues were found on Thr and 73% on Ser. It is possible that either
differences in compartmentation and/or differences in resident kinases
can account for differences in the ratio of Ser to Thr phosphorylation
in COS-1 and FTO2B cells.
The ST6Gal-I Isoforms Are Phosphorylated on Luminal
Sequences--
Because two ST isoforms possess a single Thr residue in
their cytoplasmic tails which could be phosphorylated, the presence of
Ser phosphorylation and the differences in STTyr and
STCys phosphorylation levels in COS-1 cells argued that the
luminal sequences of the isoforms are phosphorylated. To investigate
where within the protein sequence phosphorylation occurs in these
proteins, the phosphorylation of the full-length enzymes, The ST6Gal-I Isoforms Are Phosphorylated in the Cis-medial
Golgi--
The differences in ST isoform phosphorylation levels could
be the result of differences in the trafficking of the two enzyme isoforms. Initial experiments suggest that the STTyr is
transported into very late Golgi and possibly post-Golgi compartments
where it is cleaved into a soluble form that is secreted from the cell (22). The STCys is not cleaved or secreted from COS-1
cells, suggesting that it is retained in an earlier Golgi compartment. Alternatively, the differences in STTyr and
STCys phosphorylation could be caused by differences in
their conformations which are suggested by the observed differences in
the catalytic activities of these proteins (22). One possibility is
that the differences in isoform conformation and phosphorylation may
control the protein trafficking through the secretory pathway and/or
their proteolytic cleavage.
Previous work has demonstrated kinase activities in the lumen of the ER
(31-34) and in the trans-Golgi (35, 36). For example, studies using
monensin and brefeldin A showed that secretory granule proteins
chromogranin B and secretogranin II and the osteopontin protein are
phosphorylated in the trans-Golgi and sulfated in the trans-Golgi
network (35, 36). To investigate whether phosphorylation of the ST
proteins was occurring in the ER or Golgi or both, we constructed and
expressed an ER retained/retrieved chimeric ST protein. This protein
consists of the class II major histocompatibility complex Iip33 protein
cytoplasmic tail fused to the transmembrane domain, stem, and catalytic
domain of the STTyr isoform. The cytoplasmic tail of the
Iip33 protein possesses the RRXX motif, which has been
demonstrated to act as an ER retention/retrieval signal (37, 38).
Proteins containing this sequence appear to be concentrated in the ER
in the steady state, but if they exit the ER, they can be retrieved
back from the intermediate compartment or the Golgi cisternae (39).
Based on these facts, we expected that if phosphorylation of the
STTyr occurred in the Golgi, then phosphorylation of the ER
retained/retrieved Iip33-ST chimeric protein would be eliminated or
severely decreased. We also predicted that the Iip33-ST protein would
demonstrate little to no cleavage and secretion because STTyr cleavage and secretion are believed to occur in the
late Golgi or even post-Golgi (22).
Indirect immunofluorescence microscopy of COS-1 cells expressing either
the STTyr or the Iip33-ST protein demonstrated that the
STTyr was localized in the Golgi apparatus, whereas the
Iip33-STTyr protein was localized in the ER, as expected
(Fig. 4). Metabolic labeling and
pulse-chase analysis followed by immunoprecipitation demonstrated that
Iip33-ST migrated with a higher molecular mass than wild type
STTyr. This was expected because we had replaced the
9-amino acid cytoplasmic tail of the STTyr with the
33-amino acid cytoplasmic tail of Iip33 (Fig.
5). We also observed that the Iip33-ST
protein was not cleaved in the stem region and secreted, whereas the
STTyr was cleaved and secreted as observed previously (22)
(Fig. 5, compare 35S-labeled ST and Iip33-ST medium
fractions). This was consistent with the immunofluorescence results
that suggested that the bulk of the Iip33-ST chimera was not
transported out of the ER. Comparison of the phosphorylation of the
STTyr and the Iip33-ST demonstrated that although the
proteins were expressed at relatively equal levels (Fig. 5,
35S-labeling, ST versus Iip33-ST), the
phosphorylation of the ER retained/retrieved Iip33-ST protein was
dramatically decreased relative to the STTyr protein (Fig.
5, 32P-labeling, ST versus Iip33-ST). Any
phosphorylation we did see could be explained either by the transport
of a small population of the Iip33-ST protein to the Golgi and its
retrieval back to the ER or by cytoplasmic phosphorylation of the Iip33
tail. In fact, there is one predicted casein kinase (CK) 2 site in the cytoplasmic tail of Iip33 (Ser-Asn-Asn-Glu) (27, 40). These results
suggested that the bulk of the Ser/Thr phosphorylation of the
STTyr occurs in the Golgi. Interestingly, further evidence for the phosphorylation of the luminal catalytic domain also came from
the presence of a phosphorylated, cleaved and secreted form of the
STTyr in the cell medium.
To determine where in the Golgi phosphorylation of the
STTyr was occurring, we used the drug monensin to block
transport of proteins from the medial to the trans-Golgi (35, 36, 41). Previous work on Golgi phosphorylation has demonstrated that many phosphorylated proteins like chromogranin B, secretogranin II, and
osteopontin appear to be phosphorylated in the trans-cisternae of the
Golgi (35, 36). If this were the case for ST, then we would expect
monensin to eliminate phosphorylation as it has in these systems.
Treatment of COS-1 cells expressing STTyr with 10 µM monensin blocked the cleavage and secretion of the
STTyr but did not significantly decrease its level of
phosphorylation (Fig. 5, compare ST and ST + M, cell lysate and medium
fractions). These results suggest that, unlike other secretory proteins
that are phosphorylated in the ER, trans-Golgi, or later, the ST
proteins are phosphorylated in the cis- or medial Golgi. Notably, it
appears that no significant additional phosphorylation of the
STTyr occurs in the trans-Golgi or trans-Golgi network
because the monensin-treated and untreated samples appear to be
phosphorylated to the same degree. This suggests that the differences
in STTyr and STCys phosphorylation may be
related to conformational differences between the two isoforms and not
differences in their access to different kinases in different compartments.
We have found that the ST6Gal-I isoforms STTyr and
STCys are phosphorylated on luminal Ser and Thr residues in
their catalytic domain (STCys) or stem and catalytic domain
(STTyr) sequences (Figs. 1 and 3). They join a limited
family of secretory pathway proteins that are phosphorylated on
luminal/ectodomain sequences in the secretory pathway. These include
proteins of diverse functions such as progastrin (42), osteopontin (36,
43, 44), chromogranin B, and secretogranin II (35), fibronectin (45),
the vitellogenins (46), apolipoprotein B (47), prolactin (48),
grp94/endoplasmin (32), and grp78/BiP (31, 49). Most of these proteins
are phosphorylated primarily on Ser residues in vivo.
Notably, the ST6Gal-I isoforms exhibits significant levels of Thr
phosphorylation in addition to Ser phosphorylation.
In contrast to other secretory pathway phosphorylation events, we have
found that the phosphorylation of ST6Gal-I luminal sequences occurs in
the cis-medial Golgi cisternae (Fig. 5). The secretory pathway
phosphorylation of other proteins has been localized to the ER and the
trans-cisternae of the Golgi by a variety of techniques. Some kinases
involved in secretory pathway phosphorylation are presumed to be
ER-localized because the proteins that are phosphorylated, such as
grp94/endoplasmin and grp78/BiP, are localized in the ER in the steady
state (31, 32, 34). Early in vitro studies by Hirschberg and
colleagues (50, 51) identified a functional ATP transporter in the
Golgi membrane and several phosphorylated proteins associated with
Golgi membranes. Later in vitro reconstitution studies
showed that the phosphorylation of apolipoprotein B (47), preprogastrin
(42), and osteopontin (43, 44) occurred after incubation with purified
Golgi membranes.
Further dissection of the Golgi compartments containing Ser kinases was
achieved using reagents such as monensin and brefeldin A. An
elegant study by Rosa et al. (35) demonstrated that
phosphorylation of chromogranin B and secretogranin II was blocked by
monensin, a drug that blocks transport from the medial to the
trans-cisternae of the Golgi (41). In contrast, brefeldin A, a drug
that causes the cis-, medial, and trans-cisternae of the Golgi to flow
back into the ER while leaving the trans-Golgi network intact (52), blocked sulfation but not phosphorylation or galactosylation of these
proteins. From these experiments, they concluded that these secretory
granule proteins are phosphorylated in the trans-Golgi and sulfated in
the trans-Golgi network. A similar study by Ashkar et al.
(36) showed that osteopontin phosphorylation occurs in the
trans-cisternae of the Golgi of chicken osteoblasts. Brefeldin A was
used by Ridgway et al. (53) and Dockray and colleagues (54,
55) to demonstrate a trans-Golgi network or post-Golgi phosphorylation
of the oxysterol-binding protein and progastrin, respectively.
Interestingly, Walter et al. (56) have localized the
phosphorylation of The kinases involved in many of these secretory pathway phosphorylation
events may be related to the authentic casein kinase, a Ser kinase
originally found in the Golgi apparatus of mammary glands (57 and
references therein). Pinna and colleagues (40, 58) have done extensive
analyses of the authentic mammary gland Golgi-enriched fraction casein
kinase (GEF-CK), related kinases in the Golgi compartment of other
tissues (G-CK), and the CK1 and CK2 kinases that are functionally
unrelated to casein phosphorylation. Genuine casein kinase (GEF-CK) has
a specificity for Ser-Xaa-Glu/SerP sequences which is distinct from the
CK2 specificity for Ser/Thr-Xaa-Xaa-Glu/Asp/SerP/TyrP sequences and CK1
specificity for SerP-Xaa-Xaa-Ser/Thr-X (40). In addition, Lasa et
al. (58) have shown that the GEF-CK authentic casein kinase is
found in the Golgi, whereas the CK1 and CK2 enzymes are found in low
levels in this compartment and are present predominantly at other
locations in the cell. These researchers also demonstrated that an
activity similar to the authentic casein kinase was found in the Golgi
membranes of rat liver and called this kinase G-CK. This Golgi kinase
has the same consensus sequence for phosphorylation as does the GEF-CK
kinase and may function to phosphorylate some of the Ser residues in
the ST6Gal-I sequences and in many of the phosphorylated secretory
pathway proteins described above.
CK2 is the only kinase found in the secretory pathway that is
documented to possess Thr kinase activity. Pinna and colleagues (58)
have localized the majority of CK2 activity to the ER/mitochondria and
cytoplasmic fractions, with only a small amount of this enzyme activity
found in the Golgi. Other studies by Wu et al. (43) have
localized substantial amounts of CK2 activity in both the ER and Golgi
of osteoblast-like cells. It is clear from our studies of ST6Gal-I
phosphorylation that there is a significant amount of Thr
phosphorylation, and it is likely that this is occurring in the Golgi.
Consequently, it is possible that the phosphorylation of ST6Gal-I
isoform Thr residues is catalyzed by CK2 or another unidentified Thr
kinase. Analysis of the ST6Gal-I sequence reveals that there are
potential CK2 phosphorylation sites and potential GEF/GF-CK
phosphorylation sites.
The data in Fig. 3 suggest that the STTyr isoform is
phosphorylated in the stem and catalytic domain, whereas the
STCys isoform is phosphorylated primarily in the catalytic
domain. These two isoforms differ in their catalytic activity, and we
wondered whether differences in their phosphorylation patterns resulted
in these differences in activity. However, initial analyses using
alkaline phosphatase to dephosphorylate the partially purified
STTyr enzyme did not reveal any alteration of enzyme
activity after amino acid dephosphorylation.2
The most striking observations concerning the two ST6Gal-I isoforms
relate to their differences in cellular localization and processing.
The STTyr isoform is found in the Golgi, at low levels on
the cell surface, and is cleaved and secreted from cells with a
half-time of 3-6 h (22). In contrast, the STCys is found
in the Golgi and is retained intracellularly for long periods of time.
This isoform is never observed at the cell surface and is never cleaved
and secreted from COS-1 or HeLa cells (22). Analysis of the
N-linked oligosaccharide structures of the STTyr
and STCys isoforms expressed in COS-1 cells demonstrated
that both isoforms possess galactosylated oligosaccharides and suggests
that both reside in or have passed through the trans-Golgi cisternae
(22). Because we have localized the bulk of STTyr
phosphorylation to the cis-medial Golgi, it is unlikely that
differences in the phosphorylation patterns of the two isoforms are a
result of differences in their transport, as both isoforms would
traverse these compartments on their way to the trans-Golgi,
trans-Golgi network, or post-Golgi compartments. A more likely
possibility is that conformation differences in the luminal sequences
of the two isoforms lead to differences in not only in transport but
also in phosphorylation.
We originally hypothesized that the STTyr isoform was
cleaved because it was not retained as efficiently in the Golgi as the STCys isoform. In fact, blocking transport of the
STTyr protein out of the trans-Golgi network using a
20 °C block also blocked cleavage and secretion, suggesting that the
cleavage event might occur in a post-Golgi compartment (22). Another
possibility is that phosphorylation of the STTyr stem
region controls its trafficking to and/or cleavage within the late
Golgi or a post-Golgi compartment. A role for Ser phosphorylation in
controlling the maturation (cleavage) of progastrin has also been
hypothesized (55). Interestingly, potential Ser phosphorylation sites
are found near ST6Gal-I stem cleavage sites identified previously (21).3 Could differences in
expressed kinases and phosphorylation control the site of ST stem
cleavage and/or whether the ST is cleaved at all? Investigation of
these and other questions concerning the role of phosphorylation in
ST6Gal-I function and processing require the mapping of the ST6Gal-I
isoform phosphorylation sites. Our laboratory will pursue these studies.
2,6-sialyltransferase) is expressed
as two isoforms, STTyr and STCys, which
exhibit differences in catalytic activity, trafficking through the
secretory pathway, and proteolytic processing and secretion. We have
found that the ST6Gal-I isoforms are phosphorylated on luminal Ser and
Thr residues. Immunoprecipitation of 35S- and
32P-labeled proteins expressed in COS-1 cells suggests that
the STTyr isoform is phosphorylated to a greater extent
than the STCys isoform. Analysis of domain deletion mutants
revealed that STTyr is phosphorylated on stem and catalytic
domain amino acids, whereas STCys is phosphorylated on
catalytic domain amino acids. An endoplasmic reticulum
retained/retrieved chimeric Iip33-ST protein demonstrates drastically
lower phosphorylation than does the wild type STTyr isoform. This suggests that the bulk of the ST6Gal-I phosphorylation is
occurring in the Golgi. Treatment of cells with the ionophore monensin
does not significantly block phosphorylation of the STTyr isoform, suggesting that phosphorylation is occurring in the cis-medial Golgi prior to the monensin block. This study demonstrates the presence
of kinase activities in the cis-medial Golgi and the substantial
phosphorylation of the luminal sequences of a glycosyltransferase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
1,4-galactosyltransferase is phosphorylated on Ser residues. Their
results suggest that the majority of phosphoserine is found in the
cytoplasmic sequences of the enzyme, and they suggest a potential role
for phosphorylation in the trafficking and targeting of the
galactosyltransferase. However, to our knowledge, substantial
phosphorylation of glycosyltransferase luminal sequences has not been
reported previously.
EXPERIMENTAL PROCEDURES
Stem,
Tail, and
Stem
Tail mutants of the STCys protein
were constructed as described previously (26). The STTyr
forms of these mutants were constructed by converting the
STCys versions to the STTyr form by replacing
the BglII fragment from the STCys forms with
that of the STTyr form. This 608-base pair fragment consists of nucleotides 322-930 and contains the codon for amino acid
123, the amino acid that differs in the two isoforms. The full-length
Iip33 coding sequence in pCMV IV was obtained from Dr. William W. Young, University of Louisville. A fragment containing the coding
sequence of the amino-terminal Iip33 cytoplasmic tail plus upstream
untranslated sequences from the pCMV IV vector which include a
BamHI site was obtained by PCR using Vent DNA polymerase, the sense primer 5'-AAG TCT AGA ATA AAC GCT CAA CTT TGG-3' (based on
sequences in the pCMV IV vector), and the antisense primer 5'-CGG AAT
TCG CGG CTG CAC-3' (based on sequences encoding the carboxyl-terminal
end of the Iip33 tail). To facilitate cloning, the antisense primer was
engineered to incorporate an EcoRI site at the 3'-end of the
Iip33 tail fragment. A fragment containing the STTyr
transmembrane region, stem region, and catalytic domain (ST-trunc) was
obtained by PCR using STTyr-pSVL as a template, the sense
primer 5'-ACC GAA TTC AAG AAA AAG TTC AGC-3', and the antisense primer
5'-GCT CTA GAC AAC GAA TGT TCC G-3'. To facilitate cloning, the primers
incorporated an EcoRI site at the 5'-end of the ST-trunc
fragment and a XbaI site at the 3' end of the ST-trunc
fragment. The antisense primer also abolished the existing stop codon
in the ST sequence. The ST-trunc fragment was first cloned into the
V5-pcDNA 3.1 vector (the EpiTag vector from Invitrogen) using
existing EcoRI and XbaI sites in the vector's
polylinker. This cloning step fused the V5 epitope tag and 6His
sequences to the carboxyl terminus of the ST-trunc and reintroduced a
stop codon following these tagging sequences. This new construct
(ST-trunc-V5-pcDNA 3.1) was digested with BamHI (in
vector polylinker) and EcoRI (site engineered into ST-trunc
sequence) and the BamHI/EcoRI cleavage product of
the Iip33 fragment ligated into the vector using T4 DNA ligase (New
England Bio:labs). The new Iip33-ST-V5-pcDNA 3.1 construct was
verified by restriction enzyme digestions and DNA sequencing. The
resulting Iip33-ST construct includes the entire Iip33 cytoplasmic tail
(Met-Asp-Asp-Gln-Arg-Asp-Leu-Ile-Ser-Asn-Asn-Glu-Gln-Leu-Pro-Met-Leu-Gly-Arg-Arg-Pro-Gly-Ala-Pro-Glu-Ser-Lys-Cys-Ser-Arg) (27), containing its ER retention/retrieval signal (Arg-Arg), followed by Glu and Leu and then the ST transmembrane region, stem
region, and catalytic domain fused to the V5 epitope tag and 6His sequence.
-galactosidase, 118 kDa; bovine serum
albumin, 82 kDa; ovalbumin, 49.2 kDa; carbonic anhydrase, 34.8 kDa;
soybean trypsin inhibitor, 29.4 kDa; lysozyme, 19.2 kDa; aprotinin, 7.5 kDa.
70 °C.
20 °C methanol. After washing, cells were
subjected to 1-h incubations with blocking buffer (5% normal goat
serum, PBS), a 1:100 dilution of affinity purified rabbit anti-rat
2,6-ST antibody in blocking buffer, and a 1:100 dilution of
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary
antibody in blocking buffer. Cells were washed 4 × 5 min in PBS
after primary and secondary antibody incubations. After the final wash,
coverslips were mounted on glass slides and cells visualized and
photographed using a Nikon Axiophot microscope equipped with
epifluorescence illumination and a 60 × oil immersion Plan
Apochromat objective.
RESULTS
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Fig. 1.
STTyr and STCys are
phosphorylated when expressed in COS-1 cells. COS-1 cells
transiently expressing either STTyr-pSVL or
STCys-pSVL were labeled with ~660 µCi/ml
32P or 100 µCi/ml 35S-Express protein
labeling mix for 4 h in a 37 °C, 5% CO2 incubator.
Sialyltransferase proteins were immunoprecipitated from cell lysates
and immunoprecipitates analyzed by SDS-polyacrylamide gel
electrophoresis and fluorography as described under "Methods."
Protein molecular mass markers: 49.2 kDa, ovalbumin; 34.8 kDa, carbonic
anhydrase; 29.4 kDa, soybean trypsin inhibitor.
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Fig. 2.
ST isoforms are phosphorylated on Ser and Thr
residues when expressed in COS-1 cells or FTO2B rat hepatoma
cells. One-dimensional thin layer electrophoresis was performed to
identify phosphoamino acids in the ST proteins. The
32P-labeled immunoprecipitated ST proteins were separated
by SDS-polyacrylamide gel electrophoresis and transferred
electrophoretically to PVDF membrane. 32P-Labeled protein
bands were hydrolyzed directly on the PVDF membrane by incubation with
6 N HCl at 110 °C for 1 h (28). Hydrolyzed samples
and phosphoamino acid standards were electrophoresed on a flexible
cellulose thin layer chromatography plates at 1.3 kV for 45 min using
an HTLE 7000 apparatus. Phosphoamino acid standards were visualized
using ninhydrin, and their positions are indicated in the figure.
Labeled phosphoamino acids were visualized by exposing the plate to
x-ray film at 80 °C.
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Fig. 3.
STTyr is phosphorylated in the
stem and catalytic domains, whereas STCys is phosphorylated
only in the catalytic domain. COS-1 cells transiently expressing
unaltered STTyr and STCys and Stem,
Tail,
or
Tail
Stem mutants of both isoforms were labeled with ~660
µCi/ml 32P for 2 h in a 37 °C, 5%
CO2 incubator. ST proteins were immunoprecipitated from
cell lysates and immunoprecipitates analyzed by SDS-polyacrylamide gel
electrophoresis and fluorography as described under "Methods."
[35S]Methionine/cysteine labeling and immunoprecipitation
revealed that relatively equivalent levels of these proteins were
expressed in identical transfection experiments (data not shown).
Protein molecular mass marker: 49.2 kDa, ovalbumin.
Stem,
Tail, and
Tail
Stem mutants were analyzed. Cells expressing the
different forms of each ST isoform were labeled for 4 h with
32P or 35S-Express protein labeling mix, the ST
proteins were immunoprecipitated from cell lysates, and
immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis. All proteins were expressed at comparable levels as
determined by 35S labeling and immunoprecipitation (data
not shown). Although the STCys,
Tail-STCys,
and
Stem-STCys all incorporated equivalent amounts of
32P (Fig. 3, STCys), there was a significant
difference in the labeling of the STTyr,
Stem-STTyr, and
Tail
Stem-STTyr (Fig.
3, STTyr). Removal of the STTyr stem region
caused a significant decrease in 32P incorporation. Further
deletion of the tail region did not cause any more decrease in
32P incorporation. These results suggest that the
STTyr is phosphorylated in the stem region and catalytic
domain, whereas the STCys is phosphorylated primarily in
the catalytic domain. These results also suggest that phosphorylation
of the single Thr residue in the cytoplasmic tail of the isoform does
not contribute significantly to the overall phosphorylation level of
either isoform.
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Fig. 4.
STTyr is localized in the Golgi,
whereas Iip33-STTyr is localized in the ER in the steady
state. COS-1 cells transiently expressing either
STTyr-V5-pcDNA 3.1 or
Iip33-STTyr-V5-pcDNA 3.1 were prepared for
immunofluorescence microscopy as described under "Methods." After
16 h of expression, cells were fixed and permeabilized with
20 °C methanol before incubations with anti-ST primary antibody
and fluorescein isothiocyanate-conjugated rabbit anti-rat secondary
antibody. Immunofluorescence was visualized using a Nikon Axiophot
fluorescence microscope and a 60 × oil immersion Plan Apochromat
objective. Magnification, × 750.
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Fig. 5.
Phosphorylation of the ST protein occurs in
the cis-medial cisternae of the Golgi. COS-1 cells expressing
STTyr-V5-pCDNA3.1 (ST) or
Iip33-STTyr-V5-pcDNA3.1 (Iip33-ST) labeled
for 4 h with 35S-Express protein labeling mix
(35S) or 32P. In lanes marked
ST + M, COS-1 cells expressing
STTyr-V5-pCDNA3.1 were incubated with 10 µM monensin throughout the 1-h preincubation,
4-h labeling, and 2-h chase periods. ST proteins were
immunoprecipitated from cell lysates (C) and medium
fractions (M), and immunoprecipitates analyzed by
SDS-polyacrylamide gel electrophoresis and fluorography. Protein
molecular mass markers: 82 kDa, bovine serum albumin; 49.2 kDa,
ovalbumin; 34.8 kDa, carbonic anhydrase.
DISCUSSION
-amyloid precursor protein to a post-Golgi compartment and the cell surface.
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ACKNOWLEDGEMENTS |
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We thank Dr. William Young for the generous gift of the Iip33 coding sequence and Dr. Pradip Raychaudhuri for the use of the HTLE 7000 apparatus and for reagents and helpful advice. We also thank Brett Close for a critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Research Grant GM48134 (to K. C.).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.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Dept. of Biochemistry and
Molecular Biology, University of Illinois at Chicago College of
Medicine, 1819 W. Polk St. M/C 536, Chicago, IL 60612. Tel.: 312-996-7756; Fax: 312-413-0364; E-mail: karenc{at}uic.edu.
2 K. Colley, unpublished results.
3 S. Kitazume-Kawaguchi, S. Tsuji, and K. Colley, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
ST, ST6Gal-I
(2,6-sialyltransferase);
ER, endoplasmic reticulum;
GM3, Il3NewAc-LacCer;
GM1, Il3NeuAc-GgOse4Cer;
DMEM, Dulbecco's modified
Eagle's medium;
PCR, polymerase chain reaction;
PVDF, polyvinylidene
difluoride;
PBS, phosphate-buffered saline;
HTLE, Hunter thin layer
electrophoresis;
CK, casein kinase (not equivalent to the authentic
casein kinase);
GEF-CK, Golgi enriched fraction-casein kinase (the
authentic casein kinase from mammary glands);
G-CK, Golgi-casein kinase
(a rat liver Golgi kinase with the same specificity as GEF-CK from
mammary glands).
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