Sialyltransferase Isoforms Are Phosphorylated in the Cis-medial Golgi on Serine and Threonine Residues in Their Luminal Sequences*

Jiyan Ma, Miljan Simonovic, Rong Qian, and Karen J. ColleyDagger

From the Department of Biochemistry and Molecular Biology, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

ST6Gal-I (alpha 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

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 beta 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.

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.

    EXPERIMENTAL PROCEDURES

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 Delta Stem, Delta Tail, and Delta StemDelta 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.

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; beta -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.

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 -70 °C.

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 -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 alpha 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

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).


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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 Delta Stem, Delta Tail, or Delta TailDelta 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.

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, Delta Stem, Delta Tail, and Delta TailDelta 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, Delta Tail-STCys, and Delta Stem-STCys all incorporated equivalent amounts of 32P (Fig. 3, STCys), there was a significant difference in the labeling of the STTyr, Delta Stem-STTyr, and Delta TailDelta 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.

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.


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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.

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.

    DISCUSSION

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 beta -amyloid precursor protein to a post-Golgi compartment and the cell surface.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: ST, ST6Gal-I (alpha 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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