1Department of Physiology, University College London, London, United Kingdom; and 2Department of Chemistry, National Taiwan University, Taipei, Taiwan
Submitted 18 January 2005 ; accepted in final form 12 April 2005
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ABSTRACT |
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vesicular trafficking; liver; genistein; pervanadate
One of the major physiological functions of the liver is the constitutive secretion of serum albumin, the principal blood plasma protein by one order of magnitude. Blood albumin is replaced at a rate of 2% per day (40) and so by protein mass this process is the largest internal constitutive secretory process in mammals. The primary amino acid sequences, structure, and function of albumins are highly conserved across species as are the processes of albumin synthesis and secretion (42). In all cases, translation produces the short-lived, primary product preproalbumin, which is rapidly co-translationally cleaved in ER lumen, at its NH2 terminus, to produce the stable precursor proalbumin (17). Proalbumin then passes to the Golgi and is converted to mature albumin by a second NH2 terminal cleavage (17). This process is easily monitored because proalbumin can be resolved experimentally from albumin. The cleavage occurs in a late Golgi compartment that is indistinguishable from the trans-Golgi network (TGN) and depends on Furin (38, 45). Furin and other members of the mammalian subtilisin/Kex2p-like proprotein convertase family are localized largely in the trans-Golgi network with small amounts in the plasma membrane and recycling endosomes (28, 35) pinpointing the TGN as the crucial compartment in albumin production from proalbumin. In the absence of efficient proteolysis, e.g., because of mutations in the cleavage signal, proalbumin is not trapped but is itself secreted from the cell (30) indicating that passage through the TGN is not dependent on cleavage but that it is a usual feature.
Some enzymes mediating reversible protein phosphorylation are known to play roles in secretory processes. Protein kinase(s) C (PKCs) were implicated in Golgi vesicle formation (9, 47, 51, 52, 61) and more recently, PKD has been shown to promote the Golgi budding induced by the -subunits of heterotrimeric G proteins (15, 22, 57). Tyrosine kinases have prominent roles in some forms of regulated secretion acting in signal transduction pathways that can modulate the final exocytotic step (36, 41, 48). Protein tyrosine kinases and phosphatases were not thought to influence the mechanisms of storage/secretory granule formation preceding exocytosis but studies with inhibitors (2) revealed the action of these enzymes in trafficking between the Golgi and the plasma membrane during regulated secretion. More recently, protein tyrosine phosphatase-MEG2 has been shown to be the protein tyrosine phosphatase associated with secretory storage granules, which allows maturation and fusion by regulating N-ethylmaleimide-sensitive fusion protein tyrosine phosphorylation (59, 63). In addition, studies (1, 18) of stimulated melanosome aggregation in cells from lower vertebrates indicate that protein tyrosine phosphorylation has been shown to be essential for nonsecretory vesicular trafficking.
Here we demonstrate that reversible protein tyrosine phosphorylation is an essential feature of the constitutive albumin secretory pathway in rat hepatocytes, the major natural protein transport system in these cells.
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MATERIALS AND METHODS |
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Hepatocyte primary cell culture.
Cells were prepared from the livers of male rats (200300 g body wt) by perfusion of the liver with Ringer's solution containing EDTA (2 mM). The cells were washed twice with HEPES-buffered Ringer's solution (HBR) containing 1 mM MgCl2, 1 mM CaCl2, 4 mg/ml bovine serum albumin, and 25 mM HEPES (pH 7.4), and then purified on a discontinuous Percoll gradient. The viable cells were suspended in Williams E medium [supplemented with 5% (vol/vol) FCS, 0.04 mg/ml gentamicin, and 2 mM glutathione] at 5 x 105 cells/ml. Insulin (1 mg/ml) and hydrocortisone (0.1 mM) were added to the suspension, of which 2 ml was placed in 3.5-cm-diameter collagen-coated culture dishes or 50 µl on similarly treated glass coverslips. The cultures were incubated overnight and replenished with fresh medium each day if maintenance is required or washed into HBR three times and then left in a final volume of 1 ml for experimentation. For studies of albumin secretion, 3.7 MBq (100 µCi) of [35S]-labeled methionine was added to the buffer ("pulse"). The cells were incubated for 10 min, after which they were washed with ice-cold saline containing 15 mM unlabeled methionine and cysteine ("chase") and the cells transferred to similar medium containing 10 µg of rat serum albumin, as a carrier, along with any inhibitors. After 30 min of incubation at 37°C, the medium was removed and kept for the analysis of secreted albumin (proalbumin is not released under these conditions). The secretion of albumin over this period is strictly linear with time. The cells were then washed once with fresh buffer and then lysed with the same solution containing 0.5% (wt/vol) Triton X-100. The sample was centrifuged briefly, and the supernatant containing the cellular albumin and proalbumin was kept for analysis.
Analysis of albumin and proalbumin secretion. The albumin and proalbumin in each sample was precipitated by the addition of 110 µl of sheep anti-rat serum albumin, centrifuged, washed twice with 150 mM NaCl, and then taken up in dilute TCA. Redundant antibody was removed by the addition of ethanol. Albumin and proalbumin were reprecipitated from the acid with diethylether, air dried, redissolved in 20 µl of water, and analyzed by one-dimensional isoelectric focusing [5% polyacrylamide gels containing 6 M urea, 5% (vol/vol) glycerol, and ampholines]. The gels were then air dried and phosphorimaged for quantification.
Immunocytochemistry.
Cells on optical glass coverslips were treated with indicated compounds in HBR for 10 min at 37°C. After being washed with phosphate-buffered saline (PBS), the cells were fixed with 4% (wt/vol) paraformaldehyde, permeabilized with 0.1% (wt/vol) TX-100, and then blocked with 5% (vol/vol) goat serum. The coverslips were then individually soaked in rabbit anti-rat TGN38 serum (1/200 dilution), or mouse anti-GM130. After being washed to remove the primary antibodies, anti-rabbit IgG-Alexa 488 or anti-mouse IgG-Alexa 568 were added as appropriate and incubation continued for up to 1 h. After being washed, the cells were sealed in Mowiol on glass microscope slides. Routine confocal microscopy was carried out on a Bio-Rad µRadiance imaging system fitted with a Zeiss Axiovert 100TV inverted microscope. Alexa 488 was excited with the 488-nm line of an Argon laser and the emission collected with a 520-nm (40 nm bandwidth) band-pass filter. Alexa 546 was excited at 514 nm (shoulder on the excitation spectra) and collected with a 520-nm (40-nm bandwidth) band-pass filter. Identical results could be obtained with excitation at 568 nm on a Zeiss LSM510 microscope with a x40 (1.3 numerical aperture) oil-immersion lens. For data analysis, images of either small groups of hepatocytes (1020 members) or individual cells could be scrutinized using MetaMorph software with qualitatively identical results. Changes in the organized, patterned distributions of Golgi staining were quantified by normalizing the standard deviation of pixel intensity to average pixel intensity. The loss of a discrete staining pattern is revealed as a decrease in this index.
Preparation of rat liver Golgi membranes. In a variation of the surgical and perfusion procedures for the isolation of hepatocytes, the livers were cleared of blood and then removed and placed in ice-cold 0.5 M sucrose. The tissue was then processed up to the gradient centrifugation step described by Taylor et al. (54). All procedures were conducted on ice and all centrifuge rotors were of the swinging-bucket type unless otherwise stated. Briefly, the tissue was chopped and then minced with a stainless steel press before two successive steps of disruption in a Dounce homgenizer. After low-speed centrifugation (1,000 gmax) to remove large debris and nuclei, the microscopic particulate fraction was recovered from the supernantant by further centrifugation at higher speed (5,000 gmax). After resuspension of this pellet in 250 mM sucrose buffer, the particulate fraction was floated on a discontinuous density gradient consisting of 2, 1.3, 1.1, and 0.85 M sucrose and then ultracentrifuged (200,000 gav, 2 h). The Golgi-enriched fraction (which was clearly resolved from the ER-rich particles in the 1.3 M fraction) was recovered from the 0.85:1.1 M sucrose interface. This preparation could be snap frozen in liquid nitrogen and stored in aliquots at 80°C if required.
Probing Golgi membranes for tyrosine phosphatases, ATP-binding proteins, and phosphotyrosyl-containing proteins. For these experiments, snap-frozen Golgi preparations were thawed and the sucrose was diluted with an equal volume of water to allow the membranes to be sedimented by centrifugation (100,000 gav, 30 min, fixed angle rotor). Membranes were then resuspended in an assay buffer composed of (in mM) 20 HEPES, pH 7.5, 1 MnCl2, 1 MgCl2, and 1 DTT. We have already established that freeze-thaw, followed by centrifugation in fixed-angle rotors, removes the soluble contents from the lumen of rat liver Golgi (45) providing only the membranes for analysis. The Golgi membrane preparations in assay buffer were then treated with various agents. In one set of investigations membranes were treated with various concentrations of MgATP in the presence or absence of 100 µM pervanadate and a second set of membranes were treated with 1 mM LCL2 (diluted from a 50 mM stock in water). In both cases, after 1 h at 37°C, the membranes were recovered by brief ultracentrifugation (100,000 gav, 10 min), taken up in SDS-PAGE sample buffer, and heated (80°C for 5 min). Golgi membrane proteins were displayed on 10% SDS-PAGE gels and transferred to nitrocellulose. After a blocking step with bovine serum albumin or milk protein, as appropriate, the membranes were probed with either mouse monoclonal anti-phosphotyrosine IgG or streptavidin-HRP as needed. In membranes probed with anti-phosphotyrosine, a secondary goat anti-mouse HRP conjugate antibody was added after being washed. In each experiment, reactive bands were revealed by a chemiluminescent reaction. The apparent molecular weight of any reactive bands were estimated conventionally: we modeled the migration distance in relation to log10 molecular weight using either first-, or occasionally, second-order polynomial functions and regression interpolation.
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RESULTS |
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We also studied the effects of serum starvation to determine if surface receptor kinases or phosphatases or any of the many coupled downstream kinases might be loci for genistein's action. We found that serum can be withdrawn for up to 20 h (the longest time tested) without any measurable effect on albumin secretion. The subsequent readdition of serum did not produce any rebound effect. Many serum factors stimulate the synthesis of 3-phosphorylated polyphosphoinositides, but there was no effect of wortmannin on albumin secretion even at high, nonspecific, concentrations (110 µM). Similar tests with LY-294002, a complimentary phosphatidylinositol 3-kinase inhibitor (4), also failed to disrupt albumin secretion from hepatocytes (data not shown).
Once we obtained inhibition of secretion with broad-spectrum inhibitors of tyrosine kinases and phosphatases, we contrasted the effects of genistein and pervanadate with broad-spectrum inhibitors of other classes of protein kinases. Staurosporine caused a relatively small inhibition of albumin secretion from hepatocytes (20% of total). Staurosporine is known to inhibit various serine/threonine kinases, including isoforms of protein kinases C (PKCs) (62), cAMP-dependent protein kinase (PKA) (44), calmodulin kinase II (65), and myosin light chain kinase (32).
Finally to exclude the possibility that we have characterized a novel, upregulated de novo radioprotein synthesis rather than the normal constitutive albumin production, we examined the effects of rapamycin and found no significant inhibition.
Both genistein and pervanadate inhibit constitutive secretory pathway at the level of trans-Golgi. Because both genistein and pervanadate have a similar effect on the secretion of albumin, we can hypothesize that a cycle of protein phosphorylation and dephosphorylation might be important in the function of the constitutive secretory pathway. By analyzing the relationship between the observed inhibition of albumin secretion (%inhibition) and the distribution of albumin and proalbumin between the inside and the outside of the cells, we were able to establish important features of the inhibitory processes using two complimentary indexes. The first index reports the ratio of mature, cleaved albumin found inside and outside the hepatocytes (albumin in/albumin out, Fig. 3, A and C). The second index is the ratio of mature, cleaved albumin inside the cells to the total albumin-like protein inside the cells [the albumin-like protein is taken as the sum of albumin and proalbumin (Fig. 3, B and D)]. We also confirmed that albumin trapped inside the hepatocytes is not degraded by intracellular proteases. With the use of either index it is immediately apparent that both genistein and pervanadate cause similar changes in the distribution of the proteins between the inside and outside of the cells (compare Fig. 3A with 3C and 3B with 3D). Genistein and pervanadate increase the mass of unsecreted albumin with increasing inhibition of secretion indicating a blockade after the cleavage of proalbumin to albumin, a TGN-mediated process. Proalbumin also accumulates inside the cells showing that there is no alternative pathway that bypasses the blockaded TGN. This indicates that once the export of albumin is blocked a compartment proximal to the TGN accommodates the stalled precursor pool.
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DISCUSSION |
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We examined the effects of genistein and pervanadate on the cleavage of proalbumin to albumin. Because cleavage occurs in the hepatocyte TGN, the sensitivity of this step can then discriminate between blockade upstream or downstream of this compartment. We find that both agents prevent the transfer of the newly formed mature albumin from the TGN to the outside of the cell and that proalbumin is trapped in a proximal compartment (for examples, see Figs. 1 and 3). The common factor with both genistein and pervanadate is failure of the secretory pathway at the level of the trans-Golgi.
Hepatocytes account for 93% of liver volume, and Golgi represents
2% of hepatocellular membrane (5), making the rat liver one the best-described tissues for the biochemical study of Golgi function. We isolated Golgi membranes free of luminal contents (including albumin and presumably any soluble protein kinases, phosphatases, or substrates that might be in transit) from Golgi preparations proven to authentically catalyze the cleavage of proalbumin (45). To test our case in general (before proceeding to the specific) and to avoid bias in searches for specific proteins, we screened these membranes with nonspecific probes, particularly broad specificity antiphosphotyrosine antibodies to detect tyrosine kinases and endogenous substrates and with an activity probe for tyrosine phosphatases. In principle, at least one component of a cycle for protein tyrosine phosphorylation (one of either the family of tyrosine kinases, phosphatases, or suitable substrates) must be resident in the Golgi to anchor a cycle in this compartment. The absence of all three elements would allow the hypothetical cycle to be immediately dismissed. We found that isolated membranes do not contain a pool of phosphotyrosyl proteins that survives postmortem subcellular fractionation. However, because we could cause phosphotyrosyl proteins to be generated by ATP in vitro but only in the presence of pervanadate then Golgi membranes must contain active systems for both the rapid generation and removal of resident phosphotyrosyl proteins. Phosphoprotein synthesis showed apparent saturation kinetics with ATP over the 0100 µM range (not shown) which is significant because virtually all well-described protein tyrosine kinases have micromolar Kms for ATP. The apparent molecular weights of some phosphoproteins are roughly in accord with elements already described by Austin and Shields (2) in regulated secretion. A probe of these same membranes for active tyrosine phosphatases revealed several candidate Golgi tyrosine phosphatases and consequently it is established that all of the machinery for reversible protein tyrosine phosphorylation is present in rat liver Golgi membranes.
Correct Golgi function is associated with the structural and organisational integrity of this dynamic compartment. We found that pervanadate but not genistein caused major changes in the distribution of the trans-Golgi marker TGN38 and that neither compound induced any change in the localization of the cis-Golgi marker GM130. A simple hypothesis is that the accumulation of one or more unphosphorylated proteins stabilizes the TGN structure and blocks cargo exit (and also import from distal compartments), whereas the accumulation of the corresponding phosphotyrosyl protein(s) permits outward traffic but prevents final exit from the cell.
Gross disruptions to either actin filaments or microtubule networks might explain the effects of genistein or tyrosine phosphatase inhibitors. However, we discount genistein and pervanadate sensitivity in the actin cytoskeleton because this can be entirely dispersed with latrunculin B without significant effect on albumin secretion. This is surprising given the proposed role of myosins and actin in the organization and function of Golgi during protein secretion from cultured cell lines (56, 60). Microtubules are essential for the operation of the secretory pathway providing tracks for migrating transport intermediates. Colchicine and nocodazole disrupt hepatocellular microtubules and block albumin secretion (10, 14, 49), but we find no gross perturbation of microtubules with either genistein or pervanadate. This is not surprising because there is little evidence for an obligatory role for any tyrosine kinase or phosphatase in the organization of microtubules except possibly effects mediated through MAP (serine/threonine) kinases, which are discussed above.
In conclusion, rat liver Golgi membranes contain all of the elements for the endogenous cyclic generation of resident phosphotyrosyl proteins. Inhibition of this cycle at either one of two different points (phosphorylation or dephosphorylation) causes an identical trafficking lesion at the level of the trans-Golgi and in the case of phosphatase blockade can cause the redistribution of an established TGN marker. Despite the absence for precise targets for the effective kinase and phosphatase inhibitors our results indicate a previously unacknowledged, permissive role for protein tyrosine phosphorylation at the level of the TGN during the constitutive secretion of serum albumin from the liver. Because this new role for reversible tyrosine phosphorylation is established for a large-scale, constitutive secretory process in primary cells then this mechanism is highly likely to be universal.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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