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Redistribution of Rab27-specific effector Slac2-c, but not Slp4-a, after isoproterenol-stimulation in rat parotid acinar cells
archives of oral biology 54 (2009) 361–368
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Redistribution of Rab27-specific effector Slac2-c, but not Slp4-a, after isoproterenol-stimulation in rat parotid acinar cells Akane Imai a,*, Mitsunori Fukuda c, Sumio Yoshie b, Tomoko Nashida a, Hiromi Shimomura a a
Department of Biochemistry, School of Life Dentistry at Niigata, The Nippon Dental University, 1-8, Hamaura-cho, Chuo-ku, Niigata 951-8580, Japan b Department of Histology, School of Life Dentistry at Niigata, The Nippon Dental University, 1-8, Hamaura-cho, Chuo-ku, Niigata 951-8580, Japan c Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan
article info
abstract
Article history:
Small GTPase Rab27 has been implicated in the regulation of different types of membrane
Accepted 31 December 2008
trafficking, including melanosome transport and various regulated secretion events. We have previously shown that Rab27 and its effectors, Slac2-c/MyRIP and Slp4-a/granuphilin-a, are
Keywords:
involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar
Parotid acinar cells
cells. The ability of Rab to interact with the specific effectors is important. However, little is
Slac2-c
known about the fate of these effectors after b-adrenergic stimulation in parotid acinar cells.
Slp4-a
The present study investigated changes in intracellular redistribution of Slac2-c and Slp4-a in
Isoproterenol-stimulation
parotid acinar cells after IPR treatment. Subcellular fractionation studies detected Slac2-c and
Redistribution
Slp4-a in the apical plasma membrane (APM) and secretory granules under resting conditions. After 5 min of IPR treatment, Slac2-c was rapidly recruited to the luminal site, but after 30 min, the amount of Slac2-c in the APM fraction was reduced by approximately 80% compared to the increased level after 5 min of IPR treatment. Such reductions in Slac2-c are likely caused by the translocation of Slac2-c from the APM to the cytosol. In addition, we found that Slac2-c in the cytosolic fraction, but not other fractions, disappeared in the presence of Ca2+. Since Slac2-c contains multiple PEST-like sequences (i.e., potential signals for rapid protein degradation), we suggest that Slac2-c is Ca2+-dependently proteolyzed in the cytosol after exocytosis. In contrast, intracellular localization and expression levels of Slp4-a in parotid acinar cells were unaltered even after b-stimulation, indicating completely different fates for the two Rab27 effectors after b-stimulation. Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Parotid gland acinar cells secrete serous saliva containing amylase. These cells contain numerous secretory granules
holding proteins such as amylase, and exocytosis of these proteins is dramatically induced by isoproterenol (IPR), a b-adrenergic stimulant.1,2 Amylase release requires several steps: budding of secretory granules at the
* Corresponding author. Tel.: +81 25 267 1500; fax: +81 25 267 1134. E-mail address: [email protected] (A. Imai). 0003–9969/$ – see front matter . Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2008.12.007
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endoplasmic reticulum (ER) and cis-Golgi; maturation; transport to the apical surface; docking; priming; and fusion between the secretory granule and apical plasma membrane.2,3 Many Rab proteins are implicated in these steps. Rabs are small GTP-binding proteins like Ras, and display GTPase activity. More than 60 distinct Rab proteins have been identified in mice and humans,4–10 forming a large family of putative membrane trafficking proteins. In addition, Rabs interact with specific effectors, and the complex regulates different steps (or types) of membrane trafficking in cells.4,6,7 Several Rabs (Rab3D, Rab4, Rab8, Rab11, Rab26 and Rab27) are reportedly involved in exo- and endocytosis of secretory granules in parotid acinar cells.11–19 We have previously provided evidence that Rab27 and its effectors, synaptotagmin-like protein (Slp)4-a)/granuphilin-a, and Slp homologue lacking C2 domains (Slac2)-c/MyRIP, are involved in the control of IPR-induced amylase release.19– 21 The Slac2-c/Rab27 complex and binding of Slac2-c to actin regulate IPR-induced amylase secretion.19 Slp4-a controls amylase release through interactions between Slp4-a and syntaxin 2 or 3, target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs), depending on Munc18-2.20 We have recently reported the redistribution of Rab27 in rat parotid acinar cells after IPR stimulation.22 Rab27B on secretory granules translocates to the apical plasma membrane after 5 min of IPR treatment, then diffuses into the cytosol due to the function of GDP dissociation inhibitor (GDI) after 30 min. In preparation for the next round of exocytosis, GDP-Rab27 is converted to the GTP-bound form by a specific guanine nucleotide exchange factor (GEF), then is recruited to secretory granules. In contrast, however, nothing is known about the recycling pathway of Rab27 effectors, which are also required for amylase release. We thus considered elucidation of the behaviours of Rab27 effector proteins in parotid acinar cells after b-stimulation as important. This study investigated the intracellular distribution of Slac2-c and Slp4-a in parotid acinar cells after IPR treatment.
2.2. Preparation of the homogenate and membrane fraction from parotid acinar cells All animal protocols were approved by the Laboratory Animal Committee of The Nippon Dental University School of Life Dentistry at Niigata. Parotid acinar cells and subcellular fractions were prepared as described previously.23 Acinar cells were homogenized in buffer containing 5 mM Hepes– NaOH (pH 7.5), 50 mM mannitol, 0.25 mM MgCl2, 25 mM bmercaptoethanol, 0.1 mM ethylene glycol-bis (2-aminoethylether) tetraacetic acid (EGTA), 2 mM leupeptin, 2.5 mg/ml trypsin inhibitor, 0.1 mM 4-amidinophenylmethanesulfonyl fluoride hydrochloride (PMSF), 5 mM benzamidine and 2 mg/ml aprotinin, using a glass homogenizer and Teflon pestle. The supernatant fraction prepared by centrifugation at 300 g for 10 min at 4 8C was used as homogenate sample, and subjected to subcellular fractionation. Five subcellular fractions (basolateral plasma membrane, BLM; apical plasma membrane, APM; intracellular membrane, ICM; cytosol; and secretory granules, SG) were obtained. Secretory granule membrane (SGM) was prepared by treating SG with hypotonic medium (10 mM Tris–HCl, pH 7.2, containing protease inhibitors as described above). Protein assay was performed using a protein assay kit (Bio-Rad Laboratories, Inc., Alfred Nobel Drive Hercules, CA, USA).
2.3.
Western blotting
SDS–PAGE and immunoblot analysis were performed as described previously.24 Samples were separated by 7.5 or 10% SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis). Proteins in the gel were transferred to Immun-blot PVDF (polyvinylidine difluoride) membranes (Bio-Rad) using a semi-dry blotter. Membranes were probed with anti-Rab27B polyclonal antibody (1:5000), anti-Slp4-a polyclonal antibody (1:2000) or anti-Slac2-c polyclonal antibody (1:2000). Membranes were washed, then incubated with HPO-conjugated secondary antibody, followed again by washing. Immunoreactive bands were visualized using an ECL or ECLplus detection system. Intensity of immunoreactive bands on film was quantified using Image Gauze LAS-1000 software (Fuji Film, Tokyo, Japan).
2.
Materials and methods
2.4.
2.1.
Materials
Three aliquots of parotid acinar cells or slices under 1 mm3 were preincubated at 37 8C for 10 min in 5 ml of Hanks’ balanced salt solution (HBSS) containing 0.5% bovine serum albumin (BSA), then further stimulated with 1 mM IPR for 0, 5 and 30 min, respectively. After incubation, the medium was immediately removed by centrifugation at 500 g for 1 min at 4 8C. Acinar cells were washed twice with ice-cold homogenization buffer and subjected to membrane preparation. Parotid slices were used in immunohistochemical observation.
Anti-Rab27B, anti-Slp4-a-C2B domain (C2B), and anti-Slac2-cSlp homology domain (SHD) rabbit polyclonal antibodies were prepared as described previously.19 Trypsin and collagenase (CLSPA) were obtained from Sigma (St. Louis, MO, USA) and Worthington (Lakewood, NJ, USA), respectively. Horseradishperoxidase (HPO)-conjugated secondary antibody, anti-rabbit immunoglobulin (Ig)G and anti-mouse IgG + A + M were from Sigma and Zymed (San Francisco, CA, USA), respectively. ECL and ECLplus detection systems were obtained from GE Healthcare UK Ltd. (Buckinghamshire, England). Dynabeads1 M-280 sheep anti-rabbit IgG was obtained from Dynal Biotech (Oslo, Norway). All other chemicals were commercially obtained at analytical grade.
2.5.
IPR-stimulation of parotid acinar cells
Immunofluorescence microscopy
IPR-stimulated parotid slices were fixed with 4% paraformaldehyde in 0.07 M phosphate buffer (pH 7.3) and for 8 h at room
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temperature. Fixed specimens were then rapidly frozen in isopentane precooled to 35 8C. Frozen sections were cut at 6 mm using a cryostat, mounted on APS-coated glass slides and processed for the following immunostaining procedures using the indirect fluorescence method. Individual sections were incubated overnight at 4 8C with a polyclonal antibody against, either Slac2-c (1/100 dilution) or Slp4-a (1/200 dilution). After incubation, sections were exposed to Alexa Fluor 488-labeled anti-rabbit IgG (1/100 dilution) for 2 h at room temperature. Immunostained sections were further stained with Alexa Fluor 594-labeled phalloidin (Invitrogen, Carlsbad, CA, USA), which binds specifically to F-actin. Stained sections were examined and photographed under a confocal laser scanning microscope (LSM 510; Zeiss, Germany).
2.6. Limited proteolysis of Slac2-c by trypsin and mcalpain Recombinant T7-tagged Slac2-c protein was expressed in COS7 cells and purified with the anti-T7 tag antibody-conjugated agarose (Merck Biosciences Novagen, Darmstadt, Germany), but all procedures were performed in the absence of any protease inhibitors.25 Purified proteins were incubated at 25 8C for 30 min (or 30 8C for 15 min) with various concentrations of trypsin (or m-calpain in the presence or absence of 750 mM Ca2+), as described previously.25 Digested proteins were then analyzed by 10% SDS–PAGE, followed by immunoblotting with anti-Slac2-c-SHD antibody, as described above.
2.7. Effect of calcium ions on degradation of Slac2-c and Slp4-a Homogenate of parotid acinar cells in the buffer without protease inhibitors was divided into two tubes in the presence or absence of 1 mM CaCl2 or 2 mM EGTA (Fig. 5A), or in the presence of protease inhibitors and 750 mM CaCl2 or 2 mM EGTA (Fig. 5B and C), and then incubated at 37 8C for 30 min. Subcellular fractionation was then performed as described above.
3.
Results
3.1. Subcellular distribution of Slac2-c and Slp4-a in rat parotid acinar cells before IPR stimulation At first, to investigate subcellular distributions of Slac2-c and Slp4-a before IPR stimulation in parotid acinar cells, homogenates were separated into five fractions (BLM, APM, ICM, SGM and cytosol) by centrifugation, and Western blotting was performed using specific antibodies. Slac2-c and Slp4-a were mainly detected in SGM and APM fractions before stimulation (Fig. 1). Although Slac2-c was found in all fractions, both Slac2c and Slp4-a were highly localized in the APM fraction. In contrast, we previously reported that Rab27B, a specific binding partner of Slac2-c and Slp4-a in parotid acinar cells, was detected mostly in the SGM fraction and was also found in the APM fraction to a lesser extent.21 To further confirm localization of Slac2-c and Slp4-a in rat parotid glands at an immunohistochemical level, we stained endogenous Slac2-c
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Fig. 1 – Subcellular distribution of Slac2-c and Slp4-a in rat parotid acinar cells. Lanes are: BLM, basolateral plasma membrane; APM, apical plasma membrane; ICM, intracellular membrane; SGM, secretory granule membrane; and Cyto, cytosol. Each fraction (5 mg protein/ lane) was separated by 10% SDS–PAGE and Western blotted. Positions of molecular mass markers (kDa) are shown on the left.
and Slp4-a in acini using specific antibodies. Consistent with the results of the subcellular fractionation study described above, both proteins were predominantly localized in the subapical regions of acinar cells (Fig. 2A and J), where secretory granules were enriched. Colocalization of these Rab27 effectors with known secretory granule makers, amylase and VAMP-2, has already been shown by immunohistochemistry.19
3.2. Effect of IPR stimulation on intracellular distribution of Slac2-c and Slp4-a in acinar cells We next investigated changes in the subcellular localization of Slac2-c and Slp4-a in parotid acinar cells after IPR stimulation for 5 or 30 min. It has been reported that IPR treatment remarkably induces amylase release for 5 min and the release increases up to near maximum at 30 min.26–28 Then almost all granules have disappeared from parotid acinar cells29. Immunofluorescence micrographs demonstrated that Slac2c assembled into the luminal site after IPR stimulation for 5 min, and that Slac2-c had disappeared from the cytoplasm by 30 min (Fig. 2A–I). Slp4-a was not particularly altered by IPR treatment (Fig. 2J–R). The APM is thought to be the most important component for amylase release in parotid acinar cells, as the membrane of the secretory granule fuses with the APM in the final step of exocytosis.22 We thus further investigated the translocation of Slac2-c and Slp4-a to the APM or cytosol after 5 and 30 min of IPR stimulation using Western blotting (Fig. 3A and B). Total Slac2-c and Slp4-a in the acinar cells were shown to confirm translocation (Fig. 3C). Fig. 3A (top panel) and Fig. 3D showed that Slac2-c was increased in the APM by 5-min stimulation. After 30 min, Slac2-c was reduced by approximately 80% of the increased amount after 5 min of IPR treatment. Notably, Slac2c in the cytosol had disappeared by 30-min stimulation in Fig. 3B (top panel) and Fig. 3E. Though total Slac2-c was not changed by stimulation for 5 min, approximately 30% of Slac2c after IPR treatment for 5 min was disappeared from the acinar cells at 30 min of IPR treatment (Fig. 3C and F).
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Fig. 2 – Redistribution of Slac2-c, but not Slp4-a in parotid acinar cells after IPR stimulation. Gland slices were incubated with medium containing 1 mM IPR for 0 min (A–C and J–L), 5 min (D–F and M–O) or 30 min (G–I and P–R). Each section immunostained (green) for Slac2-c (A, D and G) and Slp4-a (J, M and P) was concomitantly stained with Alexa Fluor 594 phalloidin (red; B, E, H, K, N and Q) to confirm the luminal site of acini. Merged images are shown in C, F, I, L, O and R. Asterisks represent lumen. Bar, 7 mm.
Conversely, Slp4-a showed no marked change in the APM fraction, cytosol or total homogenate by semi-quantitative Western blotting (Fig. 3A–C). This result supported the results of immunohistochemistry shown in Fig. 2 (J, M and P).
3.3. Slac2-c in cytosol is digested through Ca2+-dependent proteolysis Why did Slac2-c fade away in the cytosol as shown in Figs. 2 and 3? We have previously found that Slac2-a, a closely related
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Fig. 3 – Effects of IPR treatment on distribution of Slac2-c and Slp4-a in the APM fraction, cytosol fraction and total homogenate. After parotid acinar cells were stimulated with IPR for 5 or 30 min, the APM fraction and cytosol were prepared as described in Section 2. (A–C) Each fraction [APM panels (A), 2 mg protein/lane; Cyto Slac2-c panel (B upper), 5 mg protein/ lane; Cyto Slp4-a panel (B lower), 10 mg protein/lane; homogenate panels (C), 5 mg protein/lane] was separated by 7.5% SDS– PAGE and Western blotted. Typical data from three independent experiments are shown. (D–F) The intensity of Slac2-cimmunoreactive bands in A–C was quantified using LAS-1000 Image Gauze software (Fuji Film) and normalized by values in the absence of IPR (0 min), respectively. Bars indicate mean W standard error for three independent experiments. *p < 0.01 (Student’s unpaired t test).
isoform of Slac2-c in melanocytes,30 contains multiple PESTlike sequences31 in the middle region and that Slac2-a is highly sensitive to proteases such as calpains both in vitro and in vivo.25 Inspection of the amino acid sequence of Slac2-c clearly indicated that Slac2-c also contains putative PEST sequences in the middle region (Fig. 4A). Since recombinant Slac2-c molecule was actually degraded by mild concentrations of trypsin and Ca2+-dependent m-calpain in the middle region (Fig. 4B), we expected that endogenous Slac2-c in the cytosol is digested through Ca2+-dependent proteolysis. To test this possibility, parotid homogenate was incubated with or without 1 mM of CaCl2, fractionated by centrifugation and investigated by Western blotting (Fig. 5A). The intracellular free calcium concentration in rat parotid acinar cell is maximally up to near 1 mM by muscarinic and adrenergic agonists.32 As expected, Slac2-c in the cytosol disappeared in the presence of Ca2+ (Fig. 5A, top lanes 5 and 6), whereas Slac2-c bands in the PM and SGM fractions were unaltered, irrespective of the presence of Ca2+ (Fig. 5A, top lanes 1–4). Total Slac2-c in the cells was markedly decreased in the presence of Ca2+ (Fig. 5A, top lane 8). Since degradation of Salc2-c was recovered in the presence of protease inhibitor cocktails (Fig. 5B) or a calpain inhibitor, calpastatin peptide (Fig. 5C), we concluded that Ca2+-dependent proteases, including calpains, in parotid acinar cells digest Slac2-c that is free from the membranes. By contrast, no change was observed for Slp4-a bands irrespective of the presence of Ca2+ (Fig. 5A, bottom panels), consistent with the fact that Slp4-a lacks PEST-like sequences and is highly resistant to limited proteolysis.33
4.
Discussion
Mammalian Rab27 consists of two isoforms, Rab27A and Rab27B. These isoforms are closely related to each other in terms of function and structure.34 Both Rab27A and Rab27B are localized on the SGM in parotid acinar cells, and regulate IPRinduced amylase secretion by binding to the effectors Slac2-c, Slp4-a and Noc2.19–22 In addition, we have reported the movement of Rab27B and Noc2 after IPR stimulation in parotid acinar cells.21,22 However, the fates of Slac2-c and Slp4-a after stimulation (retrieval or recycling) in parotid acinar cells or even in the well-characterized secretory cells (e.g., pancreatic b-cells and PC12 cells) have never been elucidated. The present study demonstrated that both Slac2-c and Slp4-a are present in the fractions of APM and amylasecontaining secretory granules close to the apical region (Fig. 1 , Fig. 2A and J). Nevertheless, both proteins behaved in a completely different manner after stimulation. Although intracellular distribution of Slp4-a was virtually unaltered by IPR stimulation, Slac2-c was assembled to the apical region at 5 min after IPR treatment (Fig. 2 and Fig. 3A). Thereafter, Slac2-c in the APM fraction was reduced to pre-stimulation levels by stimulation for 30 min. The most unexpected, surprising finding in this study was that Slac2-c almost fades away from the cytoplasm after IPRstimulation for 30 min (Fig. 2G) and that Slac2-c disappeared from the cytosol (Fig. 3B, top panel, E). Disappearance of Slac2c in the cytosol is most likely mediated by proteolysis, due to the following observations. First, Slac2-c is a member of the Slac2 family35 and Slac2 members contain PEST-like sequences in the middle of the molecule that are known to
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Fig. 4 – Limited proteolysis of recombinant Slac2-c by trypsin and m-calpain. (A) Schematic representation of three functional domains of mouse Slac2-c and content of 4 amino acids (Pro, Glu, Ser and Thr) in each domain. The SHD comprises 2 potential a-helical regions (SHD1 and SHD2; black boxes) separated by two zinc finger motifs (indicated by Zn2+) and functions as a specific Rab27A/Bbinding domain. The myosin VIIa-binding domain (MBD) and actin-binding domain (ABD) are indicated by shaded and hatched boxes, respectively30. Note that content of the 4 amino acids (PEST) is very high in the MBD, which is highly sensitive to proteolysis (see B). Putative proteolysis-sensitive sites are indicated by open arrowheads. (B) After incubation of T7-Slac2-c with trypsin (lanes 1–6) at 25 8C for 30 min or calpain (lanes 7–12) at 30 8C for 15 min, reactions were stopped by addition of SDS sample buffer and boiling for 3 min. Samples were then analyzed by 10% SDS–PAGE and immunoblotting with anti-Slac2-c antibody. Note that 3 major proteolytic fragments were detected with the anti-Slac2-c antibody (open arrowheads, sites 1-3). Positions of molecular mass markers (kDa) are shown on the left.
be sensitive to proteases such as calpains25,31 (Fig. 4A). Second, Slac2-c protein was easily digested by m-calpain or trypsin in vitro (Fig. 4B). Third, disappearance of Slac2-c was inhibited in the presence of protease inhibitors (Fig. 5B) or calpastatin peptide (Fig. 5C). Fourth, expression of some proteases has been reported in parotid acinar cells.36–38 Currently, however, whether proteolysis of Slac2-c is involved in the control of amylase release remains unknown. One possible role of Slac2c degradation is to efficiently terminate amylase release, as has been proposed for the role of Slac2-a in terminating actinbased melanosome transport in melanocytes.25 Although some researchers reported that IPR (1 mM) stimulation dose not cause any change in cytosolic Ca2+ concentration for a short period (<10 min) in rat parotid acinar cells,39,40 nothing is
Fig. 5 – (A) Proteolysis of Slac2-c, but not Slp4-a, by endogenous Ca2+-dependent proteases. After parotid homogenate was incubated with or without 1 mM CaCl2 and 2 mM EGTA, plasma membrane (PM), secretory granule membrane (SGM), cytosol (Cyto) fractions and total homogenate were prepared and determined by Western blot analysis as described in Section 2. Each lane received application of 2 mg of protein. (B) Inhibition of Slac2-c degradation in parotid homogenate by protease inhibitor (PI) cocktails, including 0.1 mM phenylmethylsulfonyl fluoride, 10 mM leupeptin and 10 mM pepstatin A. (C) Inhibition of Slac2-c degradation in parotid homogenate by 10 mM calpastatin peptide. Parotid homogenate in (B) and (C) was treated as described in Section 2. Positions of molecular mass markers (kDa) are shown on the left.
known about the intracellular Ca2+ level in parotid acinar cells after IPR-stimulation for more than 30 min. However, since it is well known that intracellular cAMP level is increased by IPRstimulation for 30 min, and cAMP has been shown to promote a concentration-dependent increase in Ca2+ mobilization from saponin-permeabilized rat parotid acinar cells,41 alteration of Ca2+ level may occur in the cells after 30 min. Further study is necessary to elucidate the exact mechanism of how Ca2+dependent proteolysis of Slac2-c is activated in parotid acinar cells after 30 min IPR stimulation. In contrast to Slac2-c, no marked changes in Slp4-a were observed in either the APM fraction or the cytosol (Fig. 2J–R and Fig. 3A). Since Slp4-a contains C-terminal tandem C2 domains (Ca2+ and/or phospholipid-binding motifs), unlike Slac2 members,35 Slp4-a may continue binding to phospholipids in the APM after IPR-induced granule secretion. Since intracellular localization of Slp4-a does not change during amylase release, action of Slp4-a may be regulated by conformational change and/or post-translational modifications. Future study is necessary to elucidate this issue. In summary, we have demonstrated the rapid redistribution of Slac2-c, but not Slp4-a, in parotid acinar cells after IPR-
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stimulation. The present results, together with our recent findings of IPR-dependent redistribution of Rab27B,22 indicate that three proteins, Rab27, Slac2-c, and Slp4-a, that are essential for amylase release behave differently in exocytotic and recycling pathways. Rapid translocation of Rab27 and Slac2-c to the luminal site mediates IPR-stimulated amylase release. Rab27 is then extracted to the cytosol by binding to GDI22 and Slac2-c is likely to be degraded by Ca2+-dependent proteases in the cytosol. Slp4-a remains in the APM after secretory granule exocytosis. Further study is necessary to clarify the particular mechanisms of Slac2-c degradation in parotid acinar cells after IPR-induced amylase release.
Acknowledgments We thank Eiko Kanno for technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, by a Research Promotion Grant (NDUF-07-10) from The Nippon Dental University, by the Naito Foundation, by the Takeda Science Foundation, by the Gushinkai Foundation and by the Uehara Memorial Foundation.
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14. Ngyen D, Jones A, Ojakian GK, Raffaniello RD. Rab3D redistribution and function in rat parotid acini. J Cell Physiol 2003;197:400–8. 15. Nashida T, Yoshie S, Imai A, Shimomura H. Co-localization of Rab4 with endocytosis-related proteins in the rat parotid glands. Arch Histol Cytol 2003;66:45–52. 16. Castle AM, Huang AY, Castle JD. The minor regulated pathway, a rapid component of salivary secretion, may provide docking/fusion sites for granule exocytosis at the apical surface of acinar cells. J Cell Sci 2002;115: 2963–73. 17. Yoshie S, Imai A, Nashida T, Shimomura H. Expression, characterization, and localization of Rab26, a low molecular weight GTP-binding protein, in the rat parotid gland. Histochem Cell Biol 2000;113:259–63. 18. Nashida T, Imai A, Shimomura H. Relation of Rab26 to the amylase release from rat parotid acinar cells. Arch Oral Biol 2006;51:89–95. 19. Imai A, Yoshie S, Nashida T, Shimomura H, Fukuda M. The small GTPase Rab27B regulates amylase release from rat parotid acinar cells. J Cell Sci 2004;117:1945–53. 20. Fukuda M, Imai A, Nashida T, Shimomura H. Slp4-a/ granuphilin-a interacts with syntaxin-2/3 in a Munc18-2dependent manner. J Biol Chem 2005;280:39175–84. 21. Imai A, Yoshie S, Nashida T, Shimomura H, Fukuda M. Functional involvement of Noc2, Rab27 effector, in rat parotid acinar cells. Arch Biochem Biophys 2006;455:127–35. 22. Imai A, Yoshie S, Fukuda M, Nashida T, Shimomura H. Redistribution of small GTP-binding protein Rab27B after isoproterenol-stimulation in rat parotid acinar cells. Eur J Oral Sci; submitted for publication. 23. Imai A, Nashida T, Yoshie S, Shimomura H. Intracellular localization of SNARE proteins in rat parotid acinar cells: SNARE complexes on the apical plasma membrane. Arch Oral Biol 2003;48:597–604. 24. Imai A, Nashida T, Shimomura H. Roles of Munc18-3 in amylase release from rat parotid acinar cells. Arch Biochem Biophys 2004;422:175–82. 25. Fukuda M, Itoh T. Slac2-a/melanophilin contains multiple PEST-like sequences that are highly sensitive to proteolysis. J Biol Chem 2004;279:22314–21. 26. D’Silva NJ, Jacobson KL, Ott SM, Watson EL. Beta-adrenergicinduced cytosolic redistribution of Rap1 in rat parotid acini: role in secretion. Am J Physiol 1998;274:C1667–73. 27. Benhar M, Boschwitz H, Linial M. Differential expression pattern of Rab-GDI isoforms during the parotid gland secretion cycle. Exp Cell Res 1997;233:207–15. 28. Nashida T, Imai A, Yoshie S, Shimomura H, Yokosuka H, Kumakura M. Unstimulated amylase secretion is proteoglycan-dependent in rat parotid acinar cells. Arch Biochem Biophys 2008;469:165–73. 29. Nashida T, Yoshie S, Imai A, Shimomura H. Presence of cytoskeleton proteins in parotid glands and their roles during secretion. Arch Oral Biol 2004;49:975–82. 30. Fukuda M, Kuroda TS. Slac2-c (synaptotagmin-like protein homologue lacking C2 domains-c), a novel linker protein that interacts with Rab27, myosin Va/VIIa, and actin. J Biol Chem 2002;277:43096–103. 31. Rechsteiner M, Rogers SW. PEST sequences and regulation by proteolysis. Trends Biochem Sci 1996;21:267–71. 32. Nezu A, Tanimura A, Morita T, Irie K, Yajima T, Tojyo Y. Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells. Biochem J 2002;363:59–66. 33. Holt O, Kanno E, Bossi G, Booth S, Daniele T, Santoro A, Arico M, Saegusa C, Fukuda M, Griffiths GM. Slp1 and Slp2-a localize to the plasma membrane of CTL and contribute to secretion from the immunological synapse. Traffic 2008;9:446–57.
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34. Barral DC, Ramalho JS, Anders R, Hume AN, Knapton HJ, Tolmachova T, Collinson LM, Goulding D, Authi KS, Seabra MC. Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome. J Clin Invest 2002;110:247–57. 35. Fukuda M. Versatile role of Rab27 in membrane trafficking: focus on the Rab27 effector families. J Biochem (Tokyo) 2005;137:9–16. 36. Kurabuchi S, Hosoi K. Immunocytochemical localization of mK1, a true tissue kallikrein, in the mouse parotid gland: sexual dimorphism and effects of castration and hypophysectomy. Odontology 2004; 92:73–6. 37. Fink E, Bhoola KD, Snyman C, Neth P, Figueroa CD. Cellular expression of plasma prekallikrein in human tissues. Biol Chem 2007;388:957–63.
38. Tazawa K, Kurihara Y, Kamoshida S, Tsukada K, Tsutsumi Y. Localization of prostate-specific antigen-like immunoreactivity in human salivary gland and salivary gland tumors. Pathol Int 1999;49:500–5. 39. Tanimura A, Nezu A, Tojyo Y, Matsumoto Y. Isoproterenol potentiates alpha-adrenergic and muscarinic receptormediated Ca2+ response in rat parotid cells. Am J Physiol 1999;276:C1282–7. 40. Tojyo Y, Tanimura A, Nezu A, Matsumoto Y. Activation of beta-adrenoceptors does not cause any change in cytosolic Ca2+ distribution in rat parotid acinar cells. Eur J Pharmacol 1998;360(1):73–9. 41. Rubin RP, Adolf MA. Cyclic AMP regulation of calcium mobilization and amylase release from isolated permeabilized rat parotid cells. J Pharmacol Exp Ther 1994;268(2):600–6.
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Redistribution of Rab27-specific effector Slac2-c, but not Slp4-a, after isoproterenol-stimulation in rat parotid acinar cells Akane Imai a,*, Mitsunori Fukuda c, Sumio Yoshie b, Tomoko Nashida a, Hiromi Shimomura a a
Department of Biochemistry, School of Life Dentistry at Niigata, The Nippon Dental University, 1-8, Hamaura-cho, Chuo-ku, Niigata 951-8580, Japan b Department of Histology, School of Life Dentistry at Niigata, The Nippon Dental University, 1-8, Hamaura-cho, Chuo-ku, Niigata 951-8580, Japan c Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan
article info
abstract
Article history:
Small GTPase Rab27 has been implicated in the regulation of different types of membrane
Accepted 31 December 2008
trafficking, including melanosome transport and various regulated secretion events. We have previously shown that Rab27 and its effectors, Slac2-c/MyRIP and Slp4-a/granuphilin-a, are
Keywords:
involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar
Parotid acinar cells
cells. The ability of Rab to interact with the specific effectors is important. However, little is
Slac2-c
known about the fate of these effectors after b-adrenergic stimulation in parotid acinar cells.
Slp4-a
The present study investigated changes in intracellular redistribution of Slac2-c and Slp4-a in
Isoproterenol-stimulation
parotid acinar cells after IPR treatment. Subcellular fractionation studies detected Slac2-c and
Redistribution
Slp4-a in the apical plasma membrane (APM) and secretory granules under resting conditions. After 5 min of IPR treatment, Slac2-c was rapidly recruited to the luminal site, but after 30 min, the amount of Slac2-c in the APM fraction was reduced by approximately 80% compared to the increased level after 5 min of IPR treatment. Such reductions in Slac2-c are likely caused by the translocation of Slac2-c from the APM to the cytosol. In addition, we found that Slac2-c in the cytosolic fraction, but not other fractions, disappeared in the presence of Ca2+. Since Slac2-c contains multiple PEST-like sequences (i.e., potential signals for rapid protein degradation), we suggest that Slac2-c is Ca2+-dependently proteolyzed in the cytosol after exocytosis. In contrast, intracellular localization and expression levels of Slp4-a in parotid acinar cells were unaltered even after b-stimulation, indicating completely different fates for the two Rab27 effectors after b-stimulation. Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Parotid gland acinar cells secrete serous saliva containing amylase. These cells contain numerous secretory granules
holding proteins such as amylase, and exocytosis of these proteins is dramatically induced by isoproterenol (IPR), a b-adrenergic stimulant.1,2 Amylase release requires several steps: budding of secretory granules at the
* Corresponding author. Tel.: +81 25 267 1500; fax: +81 25 267 1134. E-mail address: [email protected] (A. Imai). 0003–9969/$ – see front matter . Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2008.12.007
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endoplasmic reticulum (ER) and cis-Golgi; maturation; transport to the apical surface; docking; priming; and fusion between the secretory granule and apical plasma membrane.2,3 Many Rab proteins are implicated in these steps. Rabs are small GTP-binding proteins like Ras, and display GTPase activity. More than 60 distinct Rab proteins have been identified in mice and humans,4–10 forming a large family of putative membrane trafficking proteins. In addition, Rabs interact with specific effectors, and the complex regulates different steps (or types) of membrane trafficking in cells.4,6,7 Several Rabs (Rab3D, Rab4, Rab8, Rab11, Rab26 and Rab27) are reportedly involved in exo- and endocytosis of secretory granules in parotid acinar cells.11–19 We have previously provided evidence that Rab27 and its effectors, synaptotagmin-like protein (Slp)4-a)/granuphilin-a, and Slp homologue lacking C2 domains (Slac2)-c/MyRIP, are involved in the control of IPR-induced amylase release.19– 21 The Slac2-c/Rab27 complex and binding of Slac2-c to actin regulate IPR-induced amylase secretion.19 Slp4-a controls amylase release through interactions between Slp4-a and syntaxin 2 or 3, target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs), depending on Munc18-2.20 We have recently reported the redistribution of Rab27 in rat parotid acinar cells after IPR stimulation.22 Rab27B on secretory granules translocates to the apical plasma membrane after 5 min of IPR treatment, then diffuses into the cytosol due to the function of GDP dissociation inhibitor (GDI) after 30 min. In preparation for the next round of exocytosis, GDP-Rab27 is converted to the GTP-bound form by a specific guanine nucleotide exchange factor (GEF), then is recruited to secretory granules. In contrast, however, nothing is known about the recycling pathway of Rab27 effectors, which are also required for amylase release. We thus considered elucidation of the behaviours of Rab27 effector proteins in parotid acinar cells after b-stimulation as important. This study investigated the intracellular distribution of Slac2-c and Slp4-a in parotid acinar cells after IPR treatment.
2.2. Preparation of the homogenate and membrane fraction from parotid acinar cells All animal protocols were approved by the Laboratory Animal Committee of The Nippon Dental University School of Life Dentistry at Niigata. Parotid acinar cells and subcellular fractions were prepared as described previously.23 Acinar cells were homogenized in buffer containing 5 mM Hepes– NaOH (pH 7.5), 50 mM mannitol, 0.25 mM MgCl2, 25 mM bmercaptoethanol, 0.1 mM ethylene glycol-bis (2-aminoethylether) tetraacetic acid (EGTA), 2 mM leupeptin, 2.5 mg/ml trypsin inhibitor, 0.1 mM 4-amidinophenylmethanesulfonyl fluoride hydrochloride (PMSF), 5 mM benzamidine and 2 mg/ml aprotinin, using a glass homogenizer and Teflon pestle. The supernatant fraction prepared by centrifugation at 300 g for 10 min at 4 8C was used as homogenate sample, and subjected to subcellular fractionation. Five subcellular fractions (basolateral plasma membrane, BLM; apical plasma membrane, APM; intracellular membrane, ICM; cytosol; and secretory granules, SG) were obtained. Secretory granule membrane (SGM) was prepared by treating SG with hypotonic medium (10 mM Tris–HCl, pH 7.2, containing protease inhibitors as described above). Protein assay was performed using a protein assay kit (Bio-Rad Laboratories, Inc., Alfred Nobel Drive Hercules, CA, USA).
2.3.
Western blotting
SDS–PAGE and immunoblot analysis were performed as described previously.24 Samples were separated by 7.5 or 10% SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis). Proteins in the gel were transferred to Immun-blot PVDF (polyvinylidine difluoride) membranes (Bio-Rad) using a semi-dry blotter. Membranes were probed with anti-Rab27B polyclonal antibody (1:5000), anti-Slp4-a polyclonal antibody (1:2000) or anti-Slac2-c polyclonal antibody (1:2000). Membranes were washed, then incubated with HPO-conjugated secondary antibody, followed again by washing. Immunoreactive bands were visualized using an ECL or ECLplus detection system. Intensity of immunoreactive bands on film was quantified using Image Gauze LAS-1000 software (Fuji Film, Tokyo, Japan).
2.
Materials and methods
2.4.
2.1.
Materials
Three aliquots of parotid acinar cells or slices under 1 mm3 were preincubated at 37 8C for 10 min in 5 ml of Hanks’ balanced salt solution (HBSS) containing 0.5% bovine serum albumin (BSA), then further stimulated with 1 mM IPR for 0, 5 and 30 min, respectively. After incubation, the medium was immediately removed by centrifugation at 500 g for 1 min at 4 8C. Acinar cells were washed twice with ice-cold homogenization buffer and subjected to membrane preparation. Parotid slices were used in immunohistochemical observation.
Anti-Rab27B, anti-Slp4-a-C2B domain (C2B), and anti-Slac2-cSlp homology domain (SHD) rabbit polyclonal antibodies were prepared as described previously.19 Trypsin and collagenase (CLSPA) were obtained from Sigma (St. Louis, MO, USA) and Worthington (Lakewood, NJ, USA), respectively. Horseradishperoxidase (HPO)-conjugated secondary antibody, anti-rabbit immunoglobulin (Ig)G and anti-mouse IgG + A + M were from Sigma and Zymed (San Francisco, CA, USA), respectively. ECL and ECLplus detection systems were obtained from GE Healthcare UK Ltd. (Buckinghamshire, England). Dynabeads1 M-280 sheep anti-rabbit IgG was obtained from Dynal Biotech (Oslo, Norway). All other chemicals were commercially obtained at analytical grade.
2.5.
IPR-stimulation of parotid acinar cells
Immunofluorescence microscopy
IPR-stimulated parotid slices were fixed with 4% paraformaldehyde in 0.07 M phosphate buffer (pH 7.3) and for 8 h at room
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temperature. Fixed specimens were then rapidly frozen in isopentane precooled to 35 8C. Frozen sections were cut at 6 mm using a cryostat, mounted on APS-coated glass slides and processed for the following immunostaining procedures using the indirect fluorescence method. Individual sections were incubated overnight at 4 8C with a polyclonal antibody against, either Slac2-c (1/100 dilution) or Slp4-a (1/200 dilution). After incubation, sections were exposed to Alexa Fluor 488-labeled anti-rabbit IgG (1/100 dilution) for 2 h at room temperature. Immunostained sections were further stained with Alexa Fluor 594-labeled phalloidin (Invitrogen, Carlsbad, CA, USA), which binds specifically to F-actin. Stained sections were examined and photographed under a confocal laser scanning microscope (LSM 510; Zeiss, Germany).
2.6. Limited proteolysis of Slac2-c by trypsin and mcalpain Recombinant T7-tagged Slac2-c protein was expressed in COS7 cells and purified with the anti-T7 tag antibody-conjugated agarose (Merck Biosciences Novagen, Darmstadt, Germany), but all procedures were performed in the absence of any protease inhibitors.25 Purified proteins were incubated at 25 8C for 30 min (or 30 8C for 15 min) with various concentrations of trypsin (or m-calpain in the presence or absence of 750 mM Ca2+), as described previously.25 Digested proteins were then analyzed by 10% SDS–PAGE, followed by immunoblotting with anti-Slac2-c-SHD antibody, as described above.
2.7. Effect of calcium ions on degradation of Slac2-c and Slp4-a Homogenate of parotid acinar cells in the buffer without protease inhibitors was divided into two tubes in the presence or absence of 1 mM CaCl2 or 2 mM EGTA (Fig. 5A), or in the presence of protease inhibitors and 750 mM CaCl2 or 2 mM EGTA (Fig. 5B and C), and then incubated at 37 8C for 30 min. Subcellular fractionation was then performed as described above.
3.
Results
3.1. Subcellular distribution of Slac2-c and Slp4-a in rat parotid acinar cells before IPR stimulation At first, to investigate subcellular distributions of Slac2-c and Slp4-a before IPR stimulation in parotid acinar cells, homogenates were separated into five fractions (BLM, APM, ICM, SGM and cytosol) by centrifugation, and Western blotting was performed using specific antibodies. Slac2-c and Slp4-a were mainly detected in SGM and APM fractions before stimulation (Fig. 1). Although Slac2-c was found in all fractions, both Slac2c and Slp4-a were highly localized in the APM fraction. In contrast, we previously reported that Rab27B, a specific binding partner of Slac2-c and Slp4-a in parotid acinar cells, was detected mostly in the SGM fraction and was also found in the APM fraction to a lesser extent.21 To further confirm localization of Slac2-c and Slp4-a in rat parotid glands at an immunohistochemical level, we stained endogenous Slac2-c
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Fig. 1 – Subcellular distribution of Slac2-c and Slp4-a in rat parotid acinar cells. Lanes are: BLM, basolateral plasma membrane; APM, apical plasma membrane; ICM, intracellular membrane; SGM, secretory granule membrane; and Cyto, cytosol. Each fraction (5 mg protein/ lane) was separated by 10% SDS–PAGE and Western blotted. Positions of molecular mass markers (kDa) are shown on the left.
and Slp4-a in acini using specific antibodies. Consistent with the results of the subcellular fractionation study described above, both proteins were predominantly localized in the subapical regions of acinar cells (Fig. 2A and J), where secretory granules were enriched. Colocalization of these Rab27 effectors with known secretory granule makers, amylase and VAMP-2, has already been shown by immunohistochemistry.19
3.2. Effect of IPR stimulation on intracellular distribution of Slac2-c and Slp4-a in acinar cells We next investigated changes in the subcellular localization of Slac2-c and Slp4-a in parotid acinar cells after IPR stimulation for 5 or 30 min. It has been reported that IPR treatment remarkably induces amylase release for 5 min and the release increases up to near maximum at 30 min.26–28 Then almost all granules have disappeared from parotid acinar cells29. Immunofluorescence micrographs demonstrated that Slac2c assembled into the luminal site after IPR stimulation for 5 min, and that Slac2-c had disappeared from the cytoplasm by 30 min (Fig. 2A–I). Slp4-a was not particularly altered by IPR treatment (Fig. 2J–R). The APM is thought to be the most important component for amylase release in parotid acinar cells, as the membrane of the secretory granule fuses with the APM in the final step of exocytosis.22 We thus further investigated the translocation of Slac2-c and Slp4-a to the APM or cytosol after 5 and 30 min of IPR stimulation using Western blotting (Fig. 3A and B). Total Slac2-c and Slp4-a in the acinar cells were shown to confirm translocation (Fig. 3C). Fig. 3A (top panel) and Fig. 3D showed that Slac2-c was increased in the APM by 5-min stimulation. After 30 min, Slac2-c was reduced by approximately 80% of the increased amount after 5 min of IPR treatment. Notably, Slac2c in the cytosol had disappeared by 30-min stimulation in Fig. 3B (top panel) and Fig. 3E. Though total Slac2-c was not changed by stimulation for 5 min, approximately 30% of Slac2c after IPR treatment for 5 min was disappeared from the acinar cells at 30 min of IPR treatment (Fig. 3C and F).
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Fig. 2 – Redistribution of Slac2-c, but not Slp4-a in parotid acinar cells after IPR stimulation. Gland slices were incubated with medium containing 1 mM IPR for 0 min (A–C and J–L), 5 min (D–F and M–O) or 30 min (G–I and P–R). Each section immunostained (green) for Slac2-c (A, D and G) and Slp4-a (J, M and P) was concomitantly stained with Alexa Fluor 594 phalloidin (red; B, E, H, K, N and Q) to confirm the luminal site of acini. Merged images are shown in C, F, I, L, O and R. Asterisks represent lumen. Bar, 7 mm.
Conversely, Slp4-a showed no marked change in the APM fraction, cytosol or total homogenate by semi-quantitative Western blotting (Fig. 3A–C). This result supported the results of immunohistochemistry shown in Fig. 2 (J, M and P).
3.3. Slac2-c in cytosol is digested through Ca2+-dependent proteolysis Why did Slac2-c fade away in the cytosol as shown in Figs. 2 and 3? We have previously found that Slac2-a, a closely related
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Fig. 3 – Effects of IPR treatment on distribution of Slac2-c and Slp4-a in the APM fraction, cytosol fraction and total homogenate. After parotid acinar cells were stimulated with IPR for 5 or 30 min, the APM fraction and cytosol were prepared as described in Section 2. (A–C) Each fraction [APM panels (A), 2 mg protein/lane; Cyto Slac2-c panel (B upper), 5 mg protein/ lane; Cyto Slp4-a panel (B lower), 10 mg protein/lane; homogenate panels (C), 5 mg protein/lane] was separated by 7.5% SDS– PAGE and Western blotted. Typical data from three independent experiments are shown. (D–F) The intensity of Slac2-cimmunoreactive bands in A–C was quantified using LAS-1000 Image Gauze software (Fuji Film) and normalized by values in the absence of IPR (0 min), respectively. Bars indicate mean W standard error for three independent experiments. *p < 0.01 (Student’s unpaired t test).
isoform of Slac2-c in melanocytes,30 contains multiple PESTlike sequences31 in the middle region and that Slac2-a is highly sensitive to proteases such as calpains both in vitro and in vivo.25 Inspection of the amino acid sequence of Slac2-c clearly indicated that Slac2-c also contains putative PEST sequences in the middle region (Fig. 4A). Since recombinant Slac2-c molecule was actually degraded by mild concentrations of trypsin and Ca2+-dependent m-calpain in the middle region (Fig. 4B), we expected that endogenous Slac2-c in the cytosol is digested through Ca2+-dependent proteolysis. To test this possibility, parotid homogenate was incubated with or without 1 mM of CaCl2, fractionated by centrifugation and investigated by Western blotting (Fig. 5A). The intracellular free calcium concentration in rat parotid acinar cell is maximally up to near 1 mM by muscarinic and adrenergic agonists.32 As expected, Slac2-c in the cytosol disappeared in the presence of Ca2+ (Fig. 5A, top lanes 5 and 6), whereas Slac2-c bands in the PM and SGM fractions were unaltered, irrespective of the presence of Ca2+ (Fig. 5A, top lanes 1–4). Total Slac2-c in the cells was markedly decreased in the presence of Ca2+ (Fig. 5A, top lane 8). Since degradation of Salc2-c was recovered in the presence of protease inhibitor cocktails (Fig. 5B) or a calpain inhibitor, calpastatin peptide (Fig. 5C), we concluded that Ca2+-dependent proteases, including calpains, in parotid acinar cells digest Slac2-c that is free from the membranes. By contrast, no change was observed for Slp4-a bands irrespective of the presence of Ca2+ (Fig. 5A, bottom panels), consistent with the fact that Slp4-a lacks PEST-like sequences and is highly resistant to limited proteolysis.33
4.
Discussion
Mammalian Rab27 consists of two isoforms, Rab27A and Rab27B. These isoforms are closely related to each other in terms of function and structure.34 Both Rab27A and Rab27B are localized on the SGM in parotid acinar cells, and regulate IPRinduced amylase secretion by binding to the effectors Slac2-c, Slp4-a and Noc2.19–22 In addition, we have reported the movement of Rab27B and Noc2 after IPR stimulation in parotid acinar cells.21,22 However, the fates of Slac2-c and Slp4-a after stimulation (retrieval or recycling) in parotid acinar cells or even in the well-characterized secretory cells (e.g., pancreatic b-cells and PC12 cells) have never been elucidated. The present study demonstrated that both Slac2-c and Slp4-a are present in the fractions of APM and amylasecontaining secretory granules close to the apical region (Fig. 1 , Fig. 2A and J). Nevertheless, both proteins behaved in a completely different manner after stimulation. Although intracellular distribution of Slp4-a was virtually unaltered by IPR stimulation, Slac2-c was assembled to the apical region at 5 min after IPR treatment (Fig. 2 and Fig. 3A). Thereafter, Slac2-c in the APM fraction was reduced to pre-stimulation levels by stimulation for 30 min. The most unexpected, surprising finding in this study was that Slac2-c almost fades away from the cytoplasm after IPRstimulation for 30 min (Fig. 2G) and that Slac2-c disappeared from the cytosol (Fig. 3B, top panel, E). Disappearance of Slac2c in the cytosol is most likely mediated by proteolysis, due to the following observations. First, Slac2-c is a member of the Slac2 family35 and Slac2 members contain PEST-like sequences in the middle of the molecule that are known to
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Fig. 4 – Limited proteolysis of recombinant Slac2-c by trypsin and m-calpain. (A) Schematic representation of three functional domains of mouse Slac2-c and content of 4 amino acids (Pro, Glu, Ser and Thr) in each domain. The SHD comprises 2 potential a-helical regions (SHD1 and SHD2; black boxes) separated by two zinc finger motifs (indicated by Zn2+) and functions as a specific Rab27A/Bbinding domain. The myosin VIIa-binding domain (MBD) and actin-binding domain (ABD) are indicated by shaded and hatched boxes, respectively30. Note that content of the 4 amino acids (PEST) is very high in the MBD, which is highly sensitive to proteolysis (see B). Putative proteolysis-sensitive sites are indicated by open arrowheads. (B) After incubation of T7-Slac2-c with trypsin (lanes 1–6) at 25 8C for 30 min or calpain (lanes 7–12) at 30 8C for 15 min, reactions were stopped by addition of SDS sample buffer and boiling for 3 min. Samples were then analyzed by 10% SDS–PAGE and immunoblotting with anti-Slac2-c antibody. Note that 3 major proteolytic fragments were detected with the anti-Slac2-c antibody (open arrowheads, sites 1-3). Positions of molecular mass markers (kDa) are shown on the left.
be sensitive to proteases such as calpains25,31 (Fig. 4A). Second, Slac2-c protein was easily digested by m-calpain or trypsin in vitro (Fig. 4B). Third, disappearance of Slac2-c was inhibited in the presence of protease inhibitors (Fig. 5B) or calpastatin peptide (Fig. 5C). Fourth, expression of some proteases has been reported in parotid acinar cells.36–38 Currently, however, whether proteolysis of Slac2-c is involved in the control of amylase release remains unknown. One possible role of Slac2c degradation is to efficiently terminate amylase release, as has been proposed for the role of Slac2-a in terminating actinbased melanosome transport in melanocytes.25 Although some researchers reported that IPR (1 mM) stimulation dose not cause any change in cytosolic Ca2+ concentration for a short period (<10 min) in rat parotid acinar cells,39,40 nothing is
Fig. 5 – (A) Proteolysis of Slac2-c, but not Slp4-a, by endogenous Ca2+-dependent proteases. After parotid homogenate was incubated with or without 1 mM CaCl2 and 2 mM EGTA, plasma membrane (PM), secretory granule membrane (SGM), cytosol (Cyto) fractions and total homogenate were prepared and determined by Western blot analysis as described in Section 2. Each lane received application of 2 mg of protein. (B) Inhibition of Slac2-c degradation in parotid homogenate by protease inhibitor (PI) cocktails, including 0.1 mM phenylmethylsulfonyl fluoride, 10 mM leupeptin and 10 mM pepstatin A. (C) Inhibition of Slac2-c degradation in parotid homogenate by 10 mM calpastatin peptide. Parotid homogenate in (B) and (C) was treated as described in Section 2. Positions of molecular mass markers (kDa) are shown on the left.
known about the intracellular Ca2+ level in parotid acinar cells after IPR-stimulation for more than 30 min. However, since it is well known that intracellular cAMP level is increased by IPRstimulation for 30 min, and cAMP has been shown to promote a concentration-dependent increase in Ca2+ mobilization from saponin-permeabilized rat parotid acinar cells,41 alteration of Ca2+ level may occur in the cells after 30 min. Further study is necessary to elucidate the exact mechanism of how Ca2+dependent proteolysis of Slac2-c is activated in parotid acinar cells after 30 min IPR stimulation. In contrast to Slac2-c, no marked changes in Slp4-a were observed in either the APM fraction or the cytosol (Fig. 2J–R and Fig. 3A). Since Slp4-a contains C-terminal tandem C2 domains (Ca2+ and/or phospholipid-binding motifs), unlike Slac2 members,35 Slp4-a may continue binding to phospholipids in the APM after IPR-induced granule secretion. Since intracellular localization of Slp4-a does not change during amylase release, action of Slp4-a may be regulated by conformational change and/or post-translational modifications. Future study is necessary to elucidate this issue. In summary, we have demonstrated the rapid redistribution of Slac2-c, but not Slp4-a, in parotid acinar cells after IPR-
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stimulation. The present results, together with our recent findings of IPR-dependent redistribution of Rab27B,22 indicate that three proteins, Rab27, Slac2-c, and Slp4-a, that are essential for amylase release behave differently in exocytotic and recycling pathways. Rapid translocation of Rab27 and Slac2-c to the luminal site mediates IPR-stimulated amylase release. Rab27 is then extracted to the cytosol by binding to GDI22 and Slac2-c is likely to be degraded by Ca2+-dependent proteases in the cytosol. Slp4-a remains in the APM after secretory granule exocytosis. Further study is necessary to clarify the particular mechanisms of Slac2-c degradation in parotid acinar cells after IPR-induced amylase release.
Acknowledgments We thank Eiko Kanno for technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, by a Research Promotion Grant (NDUF-07-10) from The Nippon Dental University, by the Naito Foundation, by the Takeda Science Foundation, by the Gushinkai Foundation and by the Uehara Memorial Foundation.
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