Alexandre Crépin

Gabriel Bidaux

Fabien Vanden-Abeele

Etienne Dewailly

Vincent Goffin #

Natalia Prevarskaya

Christian Slomianny
email: christian.slomianny@univ-lille1.fr

Prolactin stimulates prostate cell proliferation by increasing endoplasmic reticulum content due to SERCA 2b over-expression

Keywords: Prolactin, prostate, calcium, SERCA, proliferation PRL, prolactin, ER, endoplasmic reticulum, [Ca 2+ ] cyt , cytosolic calcium concentration, SERCA 2b, sarcoendoplasmic calcium ATPase 2b, TNT, Tris-NaCl-Tween buffer, HBSS, Hank siRNA, small interference RNA, TG, thapsigargin, PCNA, Proliferating Cell Nuclear Antigen, TRPM8, transient receptor potential

Prolactin (PRL) has been shown to be involved in the differentiation and proliferation of numerous tissues, including the prostate gland. Moreover, variations in calcium concentration within the endoplasmic reticulum ([Ca 2+ ] ER ) may play a role in cell growth.

However, few studies have focused on the regulation of calcium homeostasis by prolactin.

This study evaluates the regulation of calcium pools as well as the possible role of [Ca 2+ ] ER variations as a signal for growth modulation by PRL. We show that PRL stimulates the proliferation of normal SV40 immortalized epithelial prostatic (PNT1A) cells with a maximum effect at a dose of 100 ng/ml. We also show that PRL (100 ng/ml) increases the [Ca 2+ ] ER when measured either by indirect quantification with Fura-2AM after application of 1 µM thapsigargin or by direct quantification with Mag-Fura-2AM within the endoplasmic reticulum. Western blot analysis shows that the sarco-endoplasmic calcium ATPase 2b (SERCA 2b) is over-expressed in PNT1A cells treated with 100 ng/ml PRL for 24 hours. A siRNA SERCA2a/b, used to downregulate endogenous SERCA 2b expression, reduces both PNT1A cell proliferation and [Ca 2+ ] ER . We thus identify [Ca 2+ ] ER and SERCA 2b as protagonists in PRL-induced proliferation.

INTRODUCTION

Due to the constant increase in life expectancy, benign prostate hyperplasia and prostate cancer have become very common diseases. Prostate cancer is now the second cause of cancer-induced death among men. It has been clearly established that the growth, differentiation and programmed cell death of prostatic cells are regulated by androgens [START_REF] Feldman | The development of androgen-independent prostate cancer[END_REF].

For this reason, the main treatment for prostate tumours consists of cell growth inhibition by suppressing the action or production of endogenous androgens by chemical or surgical castration. This only results in a temporary regression of the disease, since almost all tumours continue to progress.

Therefore, other non-steroid factors or hormones, like prolactin (PRL), are considered to be important for the normal and pathological development of the prostate gland. Indeed, PRL serum levels increase with age, whereas androgen levels decrease, suggesting that the role of PRL in the development of prostate hyperplasia and cancer becomes more important with age. PRL is a polypeptidic hormone, belonging to the cytokine super-family, produced mainly in the pituitary gland, but is also synthesized by peripheral tissues like mammary or prostate glands. Studies by several teams including ours have shown that PRL is one of the non-steroidal factors involved both in prostate cell proliferation [START_REF] Van Coppenolle | Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation[END_REF][START_REF] Nevalainen | Prolactin and prolactin receptors are expressed and functioning in human prostate[END_REF] and in the development of benign prostate hyperplasia and prostate cancer [START_REF] Van Coppenolle | Pharmacological effects of the lipidosterolic extract of Serenoa repens (Permixon) on rat prostate hyperplasia induced by hyperprolactinemia: comparison with finasteride[END_REF][START_REF] Van Coppenolle | Effects of hyperprolactinemia on rat prostate growth: evidence of androgeno-dependence[END_REF].

Previous research in our laboratory has demonstrated the crucial role played by Ca 2+ pools in the prostate [START_REF] Humez | Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha[END_REF][START_REF] Legrand | Ca2+ pools and cell growth. Evidence for sarcoendoplasmic Ca2+-ATPases 2B involvement in human prostate cancer cell growth control[END_REF][START_REF] Humez | Epidermal growth factor-induced neuroendocrine differentiation and apoptotic resistance of androgen-independent human prostate cancer cells[END_REF]. Indeed Ca 2+ homeostasis modulation is a mechanism common to a number of signal transduction pathways regulating a wide range of cell phenomena. Cytosolic Ca 2+ is known to control numerous cell functions, including contraction, gene expression and proliferation [START_REF] Berridge | Calcium signalling and cell proliferation[END_REF]. The maintenance of Ca 2+ levels within the lumen of the endoplasmic reticulum is another critical factor for cell growth [START_REF] Waldron | Endoplasmic reticulum calcium pump expression and control of cell growth[END_REF]. The endoplasmic reticulum is a Copyright 2006 Biochemical Society dynamic environment perfectly designed for protein synthesis, post-traductional modifications and folding. Moreover, the maintenance of high intraluminal Ca 2+ is essential for these processes [START_REF] Michalak | Calreticulin, a multifunctional Ca2+ binding chaperone of the endoplasmic reticulum[END_REF]. However the role of calcium signalling in the PRL signal transduction has yet to be fully elucidated.

In the present study, we show that PRL stimulates the proliferation of PNT1A prostatic cells via a mechanism involving the increase in the endoplasmic reticulum Ca 2+ content due to the over-expression of SERCA 2b proteins. Using siRNA strategy to downregulate the endogenous SERCA 2b expression in PNT1A cells, we show that a reduction in Ca 2+ store content leads to a proportional decrease in cell growth. Thus, our study provides the first direct evidence that SERCA expression and endoplasmic reticulum Ca 2+ content are major players in the PRL signal transduction controlling prostate cell growth.

Considering the recent evidence of PRL involvement in the aberrant proliferation of prostate tumours, our results provide a new insight into the mechanisms of the age-related cancer development.

MATERIALS AND METHODS

Cell culture -The immortalized human prostate normal cell line PNT1A obtained from the European Collection And Cell Culture (ECACC) was maintained in culture in RPMI 1640 medium (Life technologies, Inc.) supplemented with 10% fetal calf serum (Seromed, Poly-Labo, Strasbourg, France) and 5 mM L-glutamine (Sigma, L'Isle d'Abeau, France) and 1% kanamycine (Sigma, L'Isle d'Abeau, France). Cells were grown at 37°C in a humidified atmosphere containing 5% CO 2 . The medium was replaced every 48 h.

Proliferation assays -The assay medium was the same as that used for culture, but supplemented with 1% fetal calf serum. The 96-well plates (Nunc) had a final volume of 200 µl or 24-well plates (Nunc) had a final volume of 1 ml and were assayed 48 h after seeding. Copyright 2006 Biochemical Society PNT1A were cultured for 4 days of treatment with or without reagents, as indicated. The medium was replaced every 48 h. Proliferation was determined with a MTS reagent kit. siRNA cell transfection -PNT1A cells were transfected overnight with 40 nM siRNASERCA 2a/b, using JetSI TM ENDO transfection reagent (Polyplus transfection SA, Illkrich, France). Ready-to-use siRNASERCA 2a/b (processing option: A4) with a sense sequence of: 5'-CAAAGUUCCUGCUGAUAUA(dTdT)-3' was synthesized by Dharmacon Inc (Lafayette, USA). Control siRNA experiments were performed either by applying the transfection reagent alone (vehicle) or by transfecting siRNATRPM8 [START_REF] Thebault | Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated Biochemical Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870 Copyright 2006 Biochemical Society channels in LNCaP human prostate cancer epithelial cells[END_REF]. Ca 2+ measurements using Fura-2 AM -Cells were grown on glass coverslips and before each imaging experiment, the culture medium was replaced by a Hank's Balanced Salt Solution (HBSS) containing (in mM): NaCl, 142; KCl, 5.6; MgCl2, 1; CaCl 2, 2; Na2PO 4 , 0.34; KH2PO 4 , 0.44; HEPES, 10; glucose, 5.6; and buffered to pH 7.4 with NaOH. Cells were loaded with Fura-2 by exposure to 2 µM fura-2 acetoxymethyl ester (Fura-2 AM) (Calbiochem, Meudon, France) in HBSS for 1 h at 37°C. After incubation, cells were washed three times in the same dye-free solution. When a Ca 2+ -free medium was required, CaCl 2 was omitted and replaced by equimolar MgCl 2 . Intracellular Ca 2+ was measured by an imaging system (Quanticell 900, Visitech, UK). The glass coverslip was mounted in a chamber on a Nikon microscope equipped for fluorescence. Fura-2 fluorescence was excited at 340 and 380 nm and emitted fluorescence was measured at 510 nm. The [Ca 2+ ] cyt was derived from the ratio of the fluorescence intensities for each of the excitation wavelengths (F 340 /F 380 ), and from the Grynkiewicz equation. Thapsigargin (TG), the most potent selective inhibitor of Sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) is often used to estimate Ca 2+ pool content. In a Ca 2+ -free medium, TG induces an increase in [Ca 2+ ] cyt , due to Ca 2+ release from intracellular stores. Endoplasmic reticulum (ER) Ca 2+ content could be estimated in Fura-2

Direct quantification of [Ca 2+

] ER -For imaging Ca 2+ within the ER, PNT1A cells were loaded with 2 µM of the AM-ester derivative of Mag-Fura 2 for 45 min at 37°C. After incubation with the dye, the cells were briefly rinsed in a high K+ solution (in mM): 125 KCl, 25 NaCl, 10 HEPES, 0.1 MgCl2 [pH 7.2], and then exposed for 2 min to an "intracellular buffer" at 37°C and 5 mg/ml digitonin. Digitonin-permeabilized cells were continuously perfused with digitonin-free "intracellular buffer" supplemented with 0.2 mM Mg-ATP and free [Ca 2+ ] clamped to 170 nM using Ca 2+ /EGTA buffer. The Mag-Fura 2 fluorescence ratio was calibrated using exposure to 10 µM ionomycin and 15 mM Ca 2+ or 10 mM EGTA, assuming a dissociation constant for Ca 2+ -Mag-Fura 2 at room temperature of 53 µM [START_REF] Hofer | Quantification of intraluminal free [Ca] in the agonist-sensitive internal calcium store using compartmentalized fluorescent indicators: some considerations[END_REF].

Ratio imaging measurements of Mag-Fura 2 fluorescence were made using a commercial imaging system (Quanticell 900, Applied Imaging, UK).

Western blot -Following the treatments (PRL or siRNA treatment), PNT1A cells were lyzed with RIPA buffer (Triton X100 1%, Na deoxycholate 1%, NaCl 0.15M, PO4Na2/K pH 7.2 20mM) + 5mM EDTA + anti-proteases cocktail (Sigma P 8340, St Quentin Fallavier, France) for 30 minutes on ice. Insoluble material was discarded by centrifugation at 10,000g for 10 minutes at 4°C and the amount of proteins was assessed by the BCA method (Pierce, Chemical Co., Rockford, IL). Equal amounts of proteins were subjected to 16 or 10% SDSpolyacrylamide gel electrophoresis. Finally, the proteins were transferred onto nitrocellulose membrane using a semi-dry electroblotter (Biorad). After the transfer, the membrane was stained with Ponceau Red and the tracks were cut into thin strips (2mm wide) that were further processed for immuno-detection. The strips were blocked for 30 minutes at room temperature in TNT (15 mM Tris buffer pH 8, 140mM NaCl, 0.05% Tween 20) + 5% skimmed dry milk and then incubated with: mouse antibody anti-Actine (pan Ab-5, for anti-Actine, 1/1000 for anti-PCNA, 1/5000 for anti-SERCA 2b or 1/10000 for anti-Calreticulin) for 1 hour at room temperature. After thorough washes in TNT, the strips were treated with the corresponding horseradish peroxydase-linked secondary antibodies (antimouse AP192P or anti-rabbit AP182P, Chemicon, Temecula, CA) diluted in TNT + milk (1/20,000) for 1 hour at room temperature. After several washes in TNT, the strips were processed for chemiluminescent detection using the Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL), according to the manufacturer's instructions. The blots were finally exposed to X-Omat AR films (Eastman Kodak Company, Rochester, NY). The intensity of the signals was evaluated by densitometry and semi-quantified using the ratio between the rate of protein of interest divided by the actin or calnexin rate for each experiment. Each experiment presented was repeated at least twice.

Indirect immunofluorescence -PNT1A cells were grown on glass coverslips. After a 10 minute cold (-20°C) acetone permeabilization, the cells were blocked with PBS-gelatine (Phosphate buffer saline, gelatine 1.2%) for 30 minutes at 37°C and then incubated with a mouse antibody anti PNCA diluted in PBS-gelatine (1/100) for 1 hour at 37°C. After thorough washes in PBS-gelatine, the slides were treated with Alexa fluor 488-labeled secondary antibody (Molecular Probes, USA) diluted in PBS-gelatine (1/4000) for 1 hour at room temperature with blue-evans (1/50 000). After two washes in PBS-gelatine and in PBS,

Reagents and chemicals -Human recombinant prolactin (PRL) was kindly provided by

Vincent Goffin [START_REF] Paris | Bacterial production and purification of recombinant human prolactin[END_REF]. Fura-2 AM and Mag Fura-2 AM were obtained from Calbiochem (Meudon, France) and thapsigargin (TG) from Sigma (France).

Data analysis -The data were analyzed using Origin 5.0 (Microcal Software Inc., Northampton, MA, USA). Results were expressed as mean + standard error of the mean (sem). Each experiment was repeated several times: n indicates the number of cells used to express the mean and N represents the number of experiments. Statistical analysis was performed using the student's t-test (P<0.05 considered as significant) and ANOVA tests followed by Tukey-Kramer post-tests.

RESULTS

PNT1A cell growth is stimulated by PRL -PRL stimulates PNT1A cell growth in a dosedependent manner (Fig. 1 A). 50 -1000 ng/ml PRL increased cell growth after 4 days' treatment, as estimated by the MTS reagent kit. The maximum effect was observed at a 100 ng/ml dose. At this concentration, PRL induced a proliferation increase of 122.2% + 6.5% compared to control conditions. We also checked the effect of PRL on the expression of PCNA (Proliferating Cell Nuclear Antigen), a proliferation marker. Western blot analysis (Fig. 1 B) showed that PCNA was weakly expressed in PNT1A cells under control conditions. PCNA proteins were 2.3 fold over-expressed in PNT1A cells treated for 24 hours with 100 ng/ml PRL (Fig. 1 B) corresponding to the maximum effect observed in the proliferative assay. The PRL-induced increase in the PCNA expression was confirmed by immunocytochemical assays (Fig. 1 C). Indeed, in cells treated with 100 ng/ml PRL for 24 hours, the PCNA nuclear positive cells were more numerous (45%) than in control cells Copyright 2006 Biochemical Society (22%). These data were confirmed using another proliferation marker, the nuclear antigen Ki67 (data not shown).

The ER Ca 2+ content increases in PNT1A cells pre-treated with PRL -As the maintenance

of luminal Ca 2+ within the endoplasmic reticulum has been demonstrated to be essential for cell growth in different cell models [START_REF] Legrand | Ca2+ pools and cell growth. Evidence for sarcoendoplasmic Ca2+-ATPases 2B involvement in human prostate cancer cell growth control[END_REF][START_REF] Waldron | Endoplasmic reticulum calcium pump expression and control of cell growth[END_REF][START_REF] Short | Intracellular Ca2+ pool content is linked to control of cell growth[END_REF], we investigated whether the Ca 2+ pool content was modulated in PRL-stimulated PNT1A cells. The basal cytosolic Ca 2+ level was not affected when PNT1A cell growth was stimulated by PRL (Fig. 2 A). Under control conditions, the [Ca 2+ ] cyt content of PNT1A cells was 49.7 nM ± 1.2 nM. No significant increase in [Ca 2+ ] cyt was observed in cells treated with 100 ng/ml PRL (50.2 nM ± 1.2 nM).

However, the Ca 2+ release from the intracellular stores induced by 1 µM Thapsigargin in cells treated for 24 hours with 100 ng/ml PRL was greater than that in control cells (Fig. 2 B). It is important to point out the remarkable similarity between the extent of cell proliferation (Fig. 1 A) and the amplitude of the TG-induced Ca 2+ release from intracellular stores (Fig. 2 B). The maximum amplitude of TG-induced Ca 2+ release (669.2 nM ± 44.2 nM), corresponding to 156.1 % ± 10.3 % of the TG response observed under control conditions (428.8 nM ± 18.7 nM), was measured in cells with the highest growth rates (treated with 100 ng/ml PRL). This result suggests that there is a close connection between the Ca 2+ pool content and the proliferation of PNT1A cells. Moreover, 50 µM AG490, a potent and specific inhibitor of Jak2 kinase activity (the major PRL receptor associated kinase) rendered PRL incapable of stimulating PNT1A cell growth and the amplitude of the TG-induced Ca 2+ release from intracellular stores in cells treated with AG490 and PRL was the same as under control conditions (data not shown). The higher [Ca 2+ ] ER in PRL treated cells, estimated by TGinduced Ca 2+ release method, has been confirmed using the direct quantification of luminal [Ca 2+ ] ER (Fig. 2 C). The luminal [Ca 2+ ] ER was significantly higher in PRL-treated cells (ratio 0.95 ± 0.02) than in control cells (ratio 0.72 ± 0.02). The functional activity of the Copyright 2006 Biochemical Society sarcoendoplasmic calcium ATPase has been confirmed using the direct quantification of luminal [Ca 2+ ] ER (Fig. 2 D) using the classical protocol to measure the Ca 2+ re-uptake into ER after inositol triphosphate (IP3)-induced release [START_REF] Cheng | The exit from G(0) into the cell cycle requires and is controlled by sarco(endo)plasmic reticulum Ca2+ pump[END_REF]. As demonstrated by the d[Ca 2+ ]/dt max , the luminal [Ca 2+ ] ER is more rapidly refilled in PRL-treated cells (8.5 µM.s -1 ) than in control cells (2 µM.s -1 ) confirming that the sarcoendoplasmic calcium ATPase are more active in PRL-treated cells than in control cells. Thus, our results clearly demonstrate that PRL induces the increase in the ER Ca 2+ content, suggesting that the cell growth stimulation by PRL could be linked to the high ER Ca 2+ luminal concentration.

SERCA2b protein expression in PNT1A cells is regulated by PRL -We further investigated

the effect of PRL on the expression of SERCAs (Sarco(endo)plasmic reticulum Ca 2+ -ATPase) proteins known to play an important role in the maintenance of luminal [Ca 2+ ] ER [START_REF] Vanden Abeele | Bcl-2dependent modulation of Ca(2+) homeostasis and store-operated channels in prostate cancer cells[END_REF]. Moreover, it has been suggested that SERCA proteins regulate cell growth by controlling the expression of growth and transformation related genes [START_REF] Stokes | Calcium transport across the sarcoplasmic reticulum: structure and function of Ca2+-ATPase and the ryanodine receptor[END_REF]. We therefore investigated the effect of PRL on the expression of three SERCA isoforms: SERCA2a (the cardiac/slow skeletal muscle isoform), SERCA2b (the ubiquitously expressed isoform) and SERCA 3. In our experiments, neither SERCA2a nor SERCA 3 protein expression were detected under control conditions or in PRL-treated cells. However, western blot analysis showed that SERCA2b was constitutively expressed in control cells and over-expressed (3.6 fold) in cells treated with 100 ng/ml PRL for 24 hours (Fig. 3 A). This PRL-induced upregulation of SERCA2b expression in PNT1A cells seemed to be dependent on tyrosine kinase activity. After a brief (3 hour) PRL treatment, western blot analysis showed that Jak2 kinase (the major PRL receptor associated kinase) was more strongly phosphorylated in PRLtreated cells than in control cells (Fig. 3 C). When cells were treated with 100 ng/ml PRL in the presence of 50 µM Jak2 kinase inhibitor AG490, we did not observe an up-regulation of The increased effectiveness of these proteins may be the primary reason for growth progression. An alternative or additional possibility is that high Ca 2+ concentration within the ER may promote important growth factor-initiated, InsP 3 -mediated Ca 2+ signals that are generated through the release of Ca 2+ from the ER and that are essential for cells to progress through cell cycle and to maintain growth. We therefore investigated the potential variation in PNT1A Ca 2+ pool content linked to PRL-increased proliferation. Our results show that the PRL induced proliferation of PNT1A cells is at least partially due to [Ca 2+ ] ER increase and the SERCA 2b over-expression by the Jak2 pathway. We could not observe any Ca 2+ modifications in the cytoplasm or any modifications in the SERCA 2b activity following quick PRL treatment. Thus, we suggest that PRL promotes, via the Jak2-Stat5 transduction signal, the SERCA 2b gene transcription which increases the [Ca 2+ ] ER and then cell growth.

The intracellular calcium ATPase and the Ca 2+ pool content have already been closely associated with cell growth and have been shown to be modulated by hormones or growth factors like platelet-derived growth factor [START_REF] Magnier-Gaubil | BJ20060870 Copyright 2006 Biochemical Society Relationship between calcium influx and sarco-endoplasmic reticulum Ca2+ATPase regulation[END_REF]. We have shown that the Epidermal Growth factor modulates prostate cancer LNCaP cell growth and is correlated with intracellular calcium pool content [START_REF] Legrand | Ca2+ pools and cell growth. Evidence for sarcoendoplasmic Ca2+-ATPases 2B involvement in human prostate cancer cell growth control[END_REF]: The rate of cell growth is correlated with an increase in the calcium pool filling state, whereas growth-inhibited cells show a reduced calcium pool load. In addition, we have also demonstrated that the Insulin Growth Factor, which increases prostate cancer LNCaP cell growth, induces an increase in [Ca 2+ ] ER and is associated with an overexpression of SERCA 2b [START_REF] Humez | Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha[END_REF]. To our knowledge, in this study we show for the first time that PRL stimulates cell proliferation via the over-expression of SERCA 2b and the subsequent upregulation of the ER Ca 2+ content.

To study the consequence of a reduction in [Ca 2+ ] ER on the cell growth and to confirm the involvement of SERCA 2b in PRL-induced proliferation, we used the downregulation of Copyright 2006 Biochemical Society SERCA 2b protein by siRNA strategy. Our results show that a reduction in Ca 2+ store content leads to a proportional decrease in PNT1A cell growth. The correlation between the decrease in growth rate and the Ca 2+ store content decrease has already been demonstrated in other cell models. However only artificial pharmacological agents have been used in these studies. [START_REF] Short | Intracellular Ca2+ pool content is linked to control of cell growth[END_REF] have shown that depletion of the [Ca 2+ ] ER stores by the Ca 2+ pump inhibitor, thapsigargin or 2,5-di-tert-butyl-hydroquinone (tBHQ) promotes growth arrest in DDT1MF-2 smooth muscle cells. In a previous study, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum Ca 2+ concentration and induces growth arrest in DU-145 androgen-independent human prostate cancer cells [START_REF] Vanoverberghe | Mechanisms of ATP-induced calcium signalling and growth arrest in human prostate cancer cells[END_REF]. In our experiments, we also found that cytosolic Ca 2+ in siRNA SERCA 2b-treated cells was higher than in control cells. This could be due to the reduced calcium pool load or to a compensation mechanism for Ca 2+ signalling, via the up-regulation of membrane Ca 2+ channels and exchangers. [START_REF] Seth | Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes[END_REF] have shown that the reduction of SERCA expression by gene silencing is associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na + / Ca 2+ exchanger in cardiac myocytes. Store deficiency was in this study compensated for by Ca 2+ fluxes through the plasma membrane.

To summarize, our study identifies and characterizes, for the first time, the intracellular Ca 2+ homeostasis mechanisms involved in the PRL regulation of the PNT1A prostate cell growth. We have shown that PRL increases the intracellular Ca 2+ stores in these proliferative cells through the over-expression of SERCA 2b calcium pumps. Using siRNA technology, we have specifically down-regulated SERCA 2b expression and shown that the intracellular Ca 2+ stores are proportionally reduced. As a consequence, this reduces the PNT1A cell proliferation. We suggest that [Ca 2+ ] ER exerts a strict control over cell growth and that it is a crucial element in the PRL signal transduction.

Biochemical

  Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870 Copyright 2006 Biochemical Society loaded cells by measuring the difference between basal [Ca 2+ ] cyt and maximum [Ca 2+ ] cyt at the TG-induced peak.

Biochemical

  Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870 Copyright 2006 Biochemical Society Neomarkers, Fremont, CA), rabbit antibody anti-PCNA (sc-56 S, Santa Cruz), rabbit antibody anti-SERCA 2b (poly clonal rabbit IgG, gift from Prof. Frank Wuytack) or rabbit antibody anti-Calreticulin (Stressgen Biotechnologies, San Diego, CA) diluted in TNT + milk (1/500

  the slides were mounted with Mowiol® and watched through a confocal microscope (Zeiss LSM 510). The acquisition parameters were identical throughout the whole experiment to allow comparison of the intensity of the fluorescence level. The intensity of the signals was Biochemical Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870 Copyright 2006 Biochemical Society directly quantified on the confocal microscope with LSM examiner software (AIM 3.2, Zeiss, Le Pecq, France). Each experiment presented was repeated at least three times.

Biochemical

  Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870 Copyright 2006 Biochemical Society proteins within the ER lumen are involved in the folding and correct assembly of proteins.

Figure 1 :

 1 Figure 1: PRL stimulates cell growth. A: Cells were seeded in 96-well plates

Figure 2 :

 2 Figure 2: PRL increases ER Ca 2+ content. A: Effect of 1 µM Thapsigargin (TG) on the [Ca 2+ ] cyt due to a Ca 2+ release from intracellular stores (ER). The effect of TG was recorded in a Ca 2+ -free HBBS medium. (•) control. PRL (○) 50 ng/ml. (■) 100 ng/ml. (▲)

Figure 3 :

 3 Figure 3: PRL induces SERCA 2b over-expression by Jak2 pathway. A: Western

Figure 4 :

 4 Figure 4: siRNA SERCA 2a/b reduces cell growth. A: Western blot analysis of

Biochemical Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870

Biochemical Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870

Biochemical Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870

Biochemical Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870

Biochemical Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870

Biochemical Journal Immediate Publication. Published on 12 Sep 2006 as manuscript BJ20060870 Copyright 2006 Biochemical Society

ACKNOWLEDGMENTS

Financial support to Alexandre CREPIN was from INSERM (Institut National de la Santé et de la Recherche Médicale) and Conseil Régional du Nord Pas-de-Calais.

SERCA2b expression (Fig. 3 A). Treating PNT1A cells with AG490 alone had no effect on SERCA 2b expression under control conditions (data not shown). We also studied calreticulin expression, the luminal Ca 2+ binding/storage chaperon, to confirm that SERCA2b overexpression was specific to PRL and was not due to the proliferation increase in protein synthesis. We therefore compared the expression of calreticulin in both control and PRLtreated cells, using semi-quantitative western blot analysis. PRL treatment inducing SERCA 2b over-expression did not modify calreticulin expression (Fig. 3 B).

SERCA2b protein expression in PNT1A cells is closely linked to cell proliferation -siRNA

technology was used to downregulate the endogenous SERCA 2b expression in PNT1A cells with a siRNA SERCA2a/b. Western blotting with anti-SERCA 2b antibody on the first and 3nd days after transfection demonstrates that the level of SERCA 2b protein was 70% lower than control (Fig. 4 A), indicating the effectiveness of siRNA in downregulating endogenous SERCA 2b expression. Then, we tested the siRNA SERCA2a/b effect on PNT1A proliferation. We observed a 13.6% reduction in the proliferation of the SiRNA SERCA2a/btreated cells compared to control (Fig. 4 B). The result was also confirmed by the PCNA expression method. Western blot analysis shows a down-regulation of PCNA expression in siRNA-treated cells (Fig. 4 C).We next investigated whether the Ca 2+ pool content was modulated in siRNA SERCA 2a/b-treated PNT1A cells. The basal cytosolic Ca 2+ level was affected andan increase in [Ca 2+ ] cyt was observed in cells treated with siRNA SERCA2a/b (111.7 nM ± 10.1 nM) compared to controls (64.9 nM ± 2.8 nM) (Fig. 5 A). The Ca 2+ release induced by 1 µM thapsigargin in siRNA SERCA 2a/b-treated cells was about 52 % smaller than that in control cells (Fig. 5 B). Because of the higher basal cytosolic Ca 2+ level, the resulting driving force may reduce the Ca 2+ release in siRNA SERCA 2a/b-treated cells.

Therefore, these results were confirmed using the direct quantification of luminal [Ca 2+ ] ER (Fig. 5 C). The luminal [Ca 2+ ] ER was lower in siRNA SERCA 2a/b -treated cells (ratio Copyright 2006 Biochemical Society 12 0.64 ± 0.02) than in control cells (ratio 0.87 ± 0.04). These results show that in PNT1A normal human prostate cells, SERCA2b is a common target of physiological stimuli controlling cell growth by PRL .

DISCUSSION

It has been established that prolactin [START_REF] Nevalainen | Prolactin and prolactin receptors are expressed and functioning in human prostate[END_REF][START_REF] Nevalainen | Androgen-dependent expression of prolactin in rat prostate epithelium in vivo and in organ culture[END_REF] and its receptors [START_REF] Van Coppenolle | Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation[END_REF][START_REF] Nevalainen | Prolactin and prolactin receptors are expressed and functioning in human prostate[END_REF][START_REF] Leav | Prolactin receptor expression in the developing human prostate and in hyperplastic, dysplastic, and neoplastic lesions[END_REF][START_REF] Nevalainen | Expression and hormone regulation of prolactin receptors in rat dorsal and lateral prostate[END_REF][START_REF] Peirce | Quantification of prolactin receptor mRNA in multiple human tissues and cancer cell lines by real time RT-PCR[END_REF] are expressed in the prostate. These data have provided significant support for the existence of an autocrine/paracrine PRL loop in prostate cells. [START_REF] Ahonen | Prolactin is a survival factor for androgen-deprived rat dorsal and lateral prostate epithelium in organ culture[END_REF] have shown that prolactin was capable of inducing proliferation and survival of prostate epithelial cells in the long term organ culture of rat prostate tissue explant. In addition, [START_REF] Wennbo | Transgenic mice overexpressing the prolactin gene develop dramatic enlargement of the prostate gland[END_REF] have reported dramatic prostate enlargement in prolactin transgenic mice. In our previous studies we have established that a chronic hyperprolactinemia, caused by sulpiride, induced an enlargement and an inflammation of the lateral rat prostate [START_REF] Van Coppenolle | Effects of hyperprolactinemia on rat prostate growth: evidence of androgeno-dependence[END_REF]. PRL stimulates the proliferation of LNCaP prostate cancer cells [START_REF] Van Coppenolle | Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation[END_REF]. In this study, we have shown that PRL stimulates the proliferation of the normal SV40 immortalized prostatic PNT1A cells. Our experiments are consistent with those reported by [START_REF] Janssen | In vitro characterization of prolactin-induced effects on proliferation in the neoplastic LNCaP, DU145, and PC3 models of the human prostate[END_REF], showing that PRL induces proliferation in LNCaP, DU145 and PC3 cancer cells. These authors have also found that PRL stimulates the proliferation of PC3 cells in a dose-dependent manner. However, the mechanisms by which PRL stimulates prostate cell proliferation are yet to be completely understood.

Ca 2+ homeostasis modulation is involved in a number of signal transduction pathways regulating a wide range of cell phenomena. Cytosolic Ca 2+ is known to control numerous cell functions, including contraction, gene expression and proliferation [START_REF] Berridge | Calcium signalling and cell proliferation[END_REF]. However, it has been documented that Ca 2+ pools control numerous cell functions, including protein synthesis and cell proliferation [START_REF] Waldron | Endoplasmic reticulum calcium pump expression and control of cell growth[END_REF][START_REF] Gill | Calcium pools, calcium entry, and cell growth[END_REF] and the maintenance of Ca 2+ levels within the lumen of the endoplasmic reticulum is critical for cell growth [START_REF] Waldron | Endoplasmic reticulum calcium pump expression and control of cell growth[END_REF]. Indeed, a range of Ca 2+ -binding


Nidhal Pdfauthor=isaac Jardin

Ben Amor

Aghleb Bartegi

José A Pariente

Ginés M Salido

Juan A Rosado

Nidhal Isaac Jardin

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Annika Rogstam

Sara Linse

Anders Lindqvist

Peter James

Ludwig Wagner

Tord Berggård
email: tord.berggard@bpc.lu.se

Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin

Keywords: phosphate-buffered saline; EGTA, ethylene glycol-bis- Secretagogin, SNAP-25, EF-hand, Calcium binding, Conformational change, Sensor, Buffer

Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumor marker. In this study we show that secretagogin binds four Ca 2+ ions (Log 10 K 1 = 7.1±0.4, Log 10 K 2 = 4.7±0.6, Log 10 K 3 = 3.6±0.7, Log 10 K 4 = 4.6±0.6 in physiological salt-buffers) with a [Ca 2+ ] 0.5 of around 25 µM. The tertiary structure of secretagogin changes significantly upon Ca 2+ binding, but not upon Mg 2+ -binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca 2+ -binding, but not upon Mg 2+ -binding. These properties suggest that secretagogin belong to the "sensor" family of Ca 2+ -binding proteins. However, in contrast to the prototypical Ca 2+ sensor calmodulin which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25, a protein involved in Ca 2+induced exocytosis in neurons and in neuroendocrine cells. K D was determined to 1.2 x 10 -7 M in the presence of Ca 2+ and 1.5 x 10 -6 M in the absence of Ca 2+ . The comparatively low Ca 2+ affinity for secretagogin, the fact that it undergoes Ca 2+ -induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca 2+ signaling to exocytotic processes.

INTRODUCTION

Secretagogin is a recently described protein which is expressed in pancreatic islets [1] and the central nervous system, particularly neuroendocrine cells [2,3]. The secretagogin mRNA was identified as one of the most abundant transcripts in pancreatic islets, highlighting the potential importance of secretagogin in this tissue [1]. Secretagogin is downregulated in pancreatic islets exposed to high concentrations of glucose [4], in human non-functional (NF) pituitary adenomas [5] and in adenocarcinomas [6]. These results suggest that secretagogin may be involved in suppressing cell growth and it has been proposed as a novel biomarker for diagnosis of different forms of cancer [7].

Secretagogin belongs to the hexa EF-hand (HEF) protein family; a group of Ca 2+binding proteins within the calmodulin superfamily. Well-known members of the HEF group are calbindin D 28k and calretinin. A high-resolution structure was recently presented for calbindin D 28k [8] showing a single globular domain comprising all six EF-hands in agreement with earlier fragment complementation studies [9,10]. Secretagogin exists in three different forms, two of which are characterized by a single amino acid exchange (Q/R) at codon 22 (secretagogin Q-22 and secretagogin R-22) [11]. The third form, setagin, does not bind Ca 2+ and consists of only 49 amino acids [11]. Many EF-hand proteins belong to one of two classes: signalling-or buffering-proteins. EF-hand proteins that function as Ca 2+ -buffers or transporters have high Ca 2+ affinities (K D < 0.1 µM) and undergo only minor conformational changes upon binding of Ca 2+ (for a review see [12]). An important biological function of Ca 2+ -buffer proteins is to maintain non-toxic levels of free intracellular Ca 2+ and to restore the low resting level after signalling events. The signalling proteins (also called Ca 2+ sensors) have lower Ca 2+ affinities (K D > 1 µM) and undergo significant Ca 2+ -induced conformational changes, which result in exposure or modulation of binding surfaces for target proteins. The target proteins are often activated or attenuated upon complex formation. For example, a Ca 2+ sensor protein may activate protein kinases that initiate cytoplasmic and nuclear actions. These processes are tightly controlled by the Ca 2+ concentration in the cell and the Ca 2+ -binding affinity of the EF-hand protein. In this report we have studied the Ca 2+binding properties of secretagogin, its conformational response upon Ca 2+ -binding and the binding of secretagogin to other proteins.

EXPERIMENTAL PROCEDURES

Proteins. Human recombinant calbindin D 28k was expressed in Escherichia coli and purified to homogeneity as described [13] Human recombinant secretagogin and SNAP-25 were expressed as GST-fusion proteins in Escherichia coli. The cells were pelleted and resuspended in ice-cold PBS, and then lysed by passage through a French Pressure cell.

Insoluble material was pelleted, and the supernatants were loaded onto glutathione-sepharose columns, which were then washed thoroughly with PBS at 4˚C. Thrombin was added to the columns, which were then incubated on a rocking table at room temperature for 12 h for secretagogin and 2h for SNAP-25. The thrombin activity was stopped with phenylmethylsulfonyl fluoride, and the cleaved proteins were eluted with PBS. The proteins were further purified on a Pharmacia HiLoad ® 26/60 Superdex 75 prep grade gel filtration column. The concentration of purified SNAP-25 and secretagogin were determined by amino acid analysis after acid hydrolysis. Ca 2+ -free (apo) protein was prepared in a similar manner as described previously for calbindin D 28k [13]. 1 H NMR spectroscopy was used to verify that the apo samples were free from EGTA.

Far UV CD spectroscopy 9 µM (0.3 mg/mL) secretagogin in 1 mM DTT, 0.5 mM EGTA, 2 mM Tris/HCl, pH 7.5 was used to record the far UV-CD spectra in the far UV-range (185-250 nm). 5 mM MgCl 2 , pH 7.5 was then added, and the same measurements were performed again. Finally, 1 mM CaCl 2, pH 7.5 was added, and the same measurements were repeated. CD spectra were obtained using a Jasco J-720 spectropolarimeter at 25°C (thermostated).

Near UV CD spectroscopy and Near UV absorbance spectroscopy.

A protein solution (37 µM secretagogin, in 1 mM DTT, 0.5 mM EGTA, 2 mM TRIS, pH 7.5) was used to record the CD and UV spectra in the near UV range (250-300 nm). 5 mM MgCl 2 , pH 7.5 was then added, and the same measurements were performed again. Finally, 1 mM CaCl 2 , pH 7.5 was added, and the same measurements were repeated. UV absorbance measurements were done in a GBC UV/Vis 920 spectrophotometer. CD spectra were obtained using a Jasco J-720 spectropolarimeter at 25°C (thermostated). Fluorescence cuvettes made of quartz with path lengths of 10 mm were used.

ANS and Trp Fluorescence spectroscopy. Fluorescence spectra were obtained using a Perkin Elmer Luminescence Spectrometer LS 50 B connected to a Julabo F25 water bath. All data were collected at 25.0 °C using a quartz cuvette (10 mm path-length). For tryptophan fluorescence spectra, the excitation wavelength was 295 nm and the emission was scanned between 300 and 450 nm. The excitation slit was 3 nm and the emission slit 3 nm. The 8anilino-1-naphtalenesulfonic acid (ANS) fluorescence spectra were recorded between 400 and 600 nm with excitation at 385 nm. The excitation slit was 3 nm and the emission slit 8 nm. A protein solution (5 µM secretagogin, in 1 mM DTT, 0.5 mM EGTA, 2 mM Tris/HCl, 0.15 mM KCl, pH 7.5) was used to record the intrinsic (tryptophan) fluorescence emission spectrum or the 8-anilino-1-naphtalenesulfonic acid (ANS) fluorescence spectra, in which case the protein was saturated with 120 µM ANS. Then, 5 mM MgCl2, pH 7.5 was added, and the measurements were performed again. Finally, 1 mM CaCl 2 , pH 7.5 was added, and the measurements were repeated. 2+ -binding constants by the chelator method. The protein (30 µM) was titrated with Ca 2+ in the presence of a chromophoric chelator (25 µM) in 2 mM Tris/HCl, pH 7.5, 0.15 M KCl. Experiments were performed using the chelators 5,5'-Br 2 -BAPTA and 5N-BAPTA. The chelator concentration was determined by absorbance, the Ca 2+ concentrations by atomic absorbtion spectroscopy, and the protein concentration by amino acid analysis after acid hydrolysis. The chelator-protein solutions (1.25 ml) were titrated with 4 µl aliquots of a 2.94 mM CaCl 2 solution, and a few 4 µl aliquots of a 10.7 mM CaCl 2 solution were added at the end of the titration. The titration was monitored as a change in absorbance at 263 nm (5,5'-Br 2 -BAPTA) or 430 nm (5N-BAPTA). Two to three sets of data were obtained for each chelator-protein combination. Parallel experiments were performed in a buffer without salt (2 mM Tris/HCl, pH 7.5). The data were analyzed using the Caligator software [14] using a model with a chelator of known Ca 2+ affinity and up to four macroscopic Ca 2+ binding constants for the protein. Limited proteolysis of secretagogin. Secretagogin was dissolved in 50 mM Tris/HCl, 150 mM KCl, containing either 1 mM CaCl 2 or 1 mM EDTA at a protein concentration of 1 mg/ml.

Macroscopic Ca

The pH was adjusted to 7.5. Sequencing grade-modified trypsin (Promega) was dissolved in 50 mM ammonium bicarbonate, yielding a stock solution with a concentration of 0.5 mg/ml. Proteolysis was initiated by mixing 700 µl of secretagogin (1 mg/ml) with 7 µl of the trypsin stock at room temperature. Aliquots of 50 µl were withdrawn at various time-points and the digestion was blocked by the addition of 2 µl soybean trypsin inhibitor (1mg/ml) (Boehringer Mannheim). Peptide fragments from the digestions were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). Spectra were acquired on a MALDI-Micro MX (Waters) in linear mode. Sinapinic acid was used as matrix and an external calibration standard was used for calibration of the spectra. The experimentally measured peptide masses were compared with the theoretical peptides calculated from the human secretagogin sequence by the FindPept tool (http://us.expasy.org/tools/findpept.html). The digested fragments were also separated by SDS PAGE (16.5 %) in Tris/Tricine buffer. Some coomassie-stained protein fragments were excised from the gel and identified by LC-MS/MS as described previously [33].

Affinity chromatography, SDS/PAGE and identification of proteins. Human recombinant secretagogin or calbindin D 28k were immobilized on CNBr activated Sepharose. A number of columns were prepared with 3 mg of immobilized protein on 1 ml of CNBr activated Sepharose. Bovine brain tissue (10 g) from 2 animals or mouse brain tissue (3 g) from 10 mice was homogenized using a mixer followed by a Potter-Elvehjem tissue grinder in 20 mL of 50 mM Tris, 150 mM NaCl, 1 mM DTT + 200 uL protease inhibitor cocktail (Sigma P8340). The homogenate was then centrifuged 3 times for 6 min at 3000 g and the supernatant was collected and ultracentrifuged at 100.000 G for 90 minutes. The pellet was collected and dissolved by homogenization by a Potter-Elvehjem tissue grinder in 20 mL of a buffer containing detergent (1 % β-dodecyl maltoside, 50 mM Tris, 150 mM NaCl, 1 mM DTT + 200 uL protease inhibitor cocktail (Sigma P8340)). The pellet fraction was then incubated for 1 hour, ultra-centrifuged at 100.000 G for 90 minutes and the supernatant was collected and divided into two equal parts. CaCl 2 (2 mM) was added to one part and EGTA (2 mM) was added to one part. The samples were ultracentrifuged again at 100.000 G for 90 minutes and the supernatants were collected and filtered through 45 µM filters. 7 mL of the supernatants was then incubated with secretagogin-or calbindin D28k-sepharose, respectively, for two hours. The affinity columns incubated in the presence of 2 mM CaCl 2 were rinsed with 20 column volumes of 50 mM Tris, 0.15 M NaCl, 1mM DTT, 2mM CaCl 2 , pH 7.5 containing 0,25 % β-dodecyl maltoside, followed by a two step elution. First, the columns were eluted with 50 mM Tris, 0.15 NaCl, 1mM DTT, 2mM EGTA, 0,25 % βdodecyl maltoside, pH 7.5 and then by 0.1 M Glycine/HCl , 0,25 % β-dodecyl maltoside, pH 2.5. The affinity columns incubated in the presence of 2 mM EGTA were rinsed with 20 column volumes of 50 mM Tris, 0.15 M NaCl, 1mM DTT, 2mM EGTA, pH 7.5 containing 0,25 % β-dodecyl maltoside, followed by a two step elution. First, the columns were eluted with 50 mM Tris, 0.15 NaCl, 1mM DTT, 2mM CaCl 2 , 0,25 % β-dodecyl maltoside, pH 7.5 and then by 0.1 M Glycine/HCl , 0,25 % β-dodecyl maltoside, pH 2.5. In all cases we checked that no material appeared in the wash (monitored by UV absorption at 280 nm) before elution.

The eluates were concentrated by ultra-filtration to 50 µL. 10 µL of the eluates from the secretagogin and calbindin D28k columns were separated on SDS/PAGE (12%). After the separation of proteins by SDS/PAGE, bands observed only on the secretagogin-gel but not on the calbindin D28k-gel, were excised from the gel and identified by LC-MS/MS as desribed previously [33].

GST pull-down assay. Logarithmically growing Rin-5F insulinoma cells were washed in ice cold PBS and then lysed in cell lysis buffer (TBS pH 7.5 containing 1% Triton X-100, 10 µg/ml aprotinin, 1 µg/ml pepstatin, 10 µg/ml leupeptin, 50 mM PMSF and 0.8 mM Pefabloc).

The cell lysate was pre-cleared by a 5 min spin at 12000g. An aliquot (30 µl) of packed fusion protein-loaded glutathione-Sepharose 4B beads (GST-secretagogin, and GST-setagin) was incubated in 500 µl Rin-5F cell lysate for 4 hours at 4°C under constant rotation. The beads were then washed 4 times in cell lysis buffer containing protease inhibitors and finally suspended in SDS sample buffer under reducing conditions. Boiled samples were applied onto a 12% SDS PAGE and after electrophoretic protein separation, gels were either stained or blotted onto a nitrocellulose membrane and incubated using a monoclonal mouse SNAP-25 antibody (Sigma; product number: S5187, clone SP12) followed by a goat anti-mouse peroxidase-conjugated antibody (DAKO ; product number P 0447).

Surface plasmon resonance studies. The interaction between calmodulin, calbindin D 28k secretagogin and SNAP-25 was studied using surface plasmon resonance technology on a BIAcore™ biosensor system. All buffers were sterile filtered using a 0.22 µm filter. The flow buffer consisted of 10 mM HEPES/KOH, pH 7.4, with 0.15 M NaCl, 0.005 % Tween 20, 0.02 % NaN 3 , containing either 2 mM CaCl 2 (buffer A) or 3.4 mM EDTA (buffer B).

Immobilization of calmodulin, calbindin D 28k secretagogin to a CM-5 sensor chip surface was performed at a constant flow rate of 5 µL/min, using buffer B as flow buffer.

Equal volumes of 0.1 M NHS and 0.4 M EDC were first mixed, and 40 µL of the mixture was allowed to flow over the sensor chip surface to activate the carboxymethylated dextran.

Secretagogin was then injected over the sensor chip (20 µL at 0.020 mg/ml in 10 mM NaAc The association and the dissociation kinetics in the absence or presence of Ca 2+ were studied using buffer A or B as flow the buffers. SNAP-25 was diluted from a concentrated stock into buffer A or B. The surface was regenerated by injecting 0.1 M HCl during 5 minutes to remove residual bound analyte.

The data was evaluated by a non-linear least-square analysis method using the software KaleidaGraph™ as previously described [9].

RESULTS

Since secretagogin has six EF-hand motifs, it is reasonable to expect that the protein binds Ca 2+ . EF-hand Ca 2+ -binding proteins are often classified as either Ca 2+ -sensor or Ca 2+buffer proteins. In order to investigate if secretagogin is likely to have a role in Ca 2+ -induced signal transmission as a Ca 2+ sensor protein or if it is likely to function as a high affinity Ca 2+ buffer protein, we used four different methods: isothermal titration calorimetry, titration with Ca 2+ in the presence of a chromophoric chelators and titration with Ca 2+ monitored by ANS and Trp fluorescence. The initial plan was to also monitor Ca 2+ binding by NMR spectroscopy (e.g. by heteronuclear 2D 15 N-1 H HSQC NMR spectroscopy). However, Ca 2+ titrations of secretagogin at high protein concentrations monitored by 1 H NMR spectroscopy indicated that the spectral resolution gets poorer as more Ca 2+ is added. At very high Ca 2+ concentrations (3 equivalents and up) precipitation is seen, hence the protein aggregates at high protein (100 µM) and Ca 2+ concentration. Therefore, any future NMR-experiments should be performed with the apo-protein, at low concentrations of Ca 2+ , or in the presence of protein-targets that stabilize the protein in Ca 2+ solutions.

Far UV CD spectroscopy

Far-UV CD spectroscopy was used to investigate the secondary structure of secretagogin in the apo, Ca 2+ -and Mg 2+ -bound states. We observed an intense negative ellipticity 208 and 222 nm in the spectra of apo, Ca 2+ -and Mg 2+ -secretagogin (Supplementary material, Fig 1). This indicates that the protein has significant α-helical character in all forms. Addition of either Ca 2+ or Mg 2+ causes only minor differences in the intensity of each of the 208, and 222 nm bands, suggesting that the binding of each ion induces only minor changes in the secondary structure of the protein. We conclude that the secondary structure is not significantly altered by the binding of Ca 2+ to secretagogin. Similar results have been obtained for the prototypical Ca 2+ sensor calmodulin which displays a modest increase in CD signal on Ca 2+ binding although the high-resolution structures of the Ca 2+ and apo states reveal the same number of residues in helical conformation [15][16][17] Near UV CD spectroscopy However, the shape of the near-UV CD spectrum of secretagogin, especially between 270 and 290 nm, is markedly altered by addition of 1 mM Ca 2+ . As near-UV CD spectroscopy is sensitive to structural changes in the vicinity of aromatic amino acid side chains, or to changes in mobility of aromatic side chains, these results suggest that the tertiary structure of secretagogin is rearranged upon Ca 2+ -binding.

UV Absorbance Spectroscopy.

The UV absorbance spectra generated in 0.5 mM EGTA and 5 mM MgCl 2 are highly similar, whereas the shape of the spectrum changes upon addition of 1 mM CaCl 2 (Fig. 1B). The UV absorbance spectrum of secretagogin in the apo and Mg 2+ form displays a maximum at 282 nm, which is shifted to 280.5 nm when Ca 2+ is added. The UV difference spectrum (not shown) induced by Ca 2+ binding shows a positive absorbance at 240-283 nm and a negative absorbance between 283 and 310 nm, indicating that a conformational change has occurred in the local environment around aromatic residues upon Ca 2+ binding. A peak seen around 243 nm may reflect increased light scattering in the Ca 2+ state or effects on Phe residues.

Intrinsic fluorescence spectra.

Secretagogin contains three tryptophan residues. Two of these are located in the fist helix of EF-hand 1 and one is located in the N-terminal helix of EF-hand 3. The tryptophan fluorescence spectrum of the apo form has an emission maximum at 339.5 nm. Addition of 5 mM MgCl 2 does not change the spectrum to any significant degree. Addition of 1 mM CaCl 2 , however, results in a change in the spectrum (Fig. 2A). The Ca 2+ -loaded form displays a higher fluorescence intensity than the apo-and Mg 2+ forms (by a factor of ~1.35) and a blueshift in the maximum emission from 339.5 to 334.5 nm (Fig. 2A). Addition of excess EGTA returns the emission intensity to the initial value (not shown). The blue-shift in tryptophan fluorescence shows that one or more tryptophan residues become more buried when Ca 2+ is bound. The changes in tryptophan fluorescence may reflect global structural changes. However, they could also be a consequence of more localized rearrangements around the Ca 2+ sites.

Interaction of secretagogin with ANS.

The degree of exposed hydrophobic surface in the Ca 2+ -and apo-state of human recombinant secretagogin was measured using the hydrophobic probe ANS. In the presence of secretagogin, the fluorescence intensity of ANS increases significantly accompanied by a strong blueshift both in Ca 2+ (60 nm) and EGTA (58 nm) buffers (Fig. 2B). This suggests that secretagogin has exposed hydrophobic surfaces both in the presence and absence of Ca 2+ . The fluorescence intensity is 2.3 times higher in the Ca 2+ state than in the apo state, and there is a further blueshift of 2 nm after Ca 2+ addition. The fact that a wavelength shift accompanies the intensity change indicates a true difference in the conformational response between the two forms (i.e. fluorescence quenching can not explain the observed difference). By monitoring the ANS fluorescence before and after the addition of Ca 2+ , followed by addition of excess of EGTA, we can conclude that the Ca 2+ induced ANS-fluorescence intensity change is reversible. Mg 2+ addition to secretagogin does not induce any changes in the ANS fluorescence spectrum as compared to the apo-form suggesting that the presence of this ion does not change the amount of exposed hydrophobic surface in secretagogin (Fig 2B .)

Limited proteolysis of secretagogin.

Limited tryptic digestion of human recombinant secretagogin was performed in the presence of EGTA and Ca 2+ . The reaction was quenched at different time points ranging from 2 minutes to 24 hours and analyzed by SDS polyacrylamide gel electrophoresis (PAGE) in a Tris/Tricine buffer system (Supplementary material, Fig 2). MALDI-TOF masspectrometry of the digests showed a number of masses corresponding to specific tryptic peptides of secretagogin (supplementary material, Table 1). A major fragment with a molecular mass of around 23 kDa was seen by electrophoresis both in the presence and absence of Ca 2+ (Supplementary material, Fig 2). This protein band was identified by MALDI-TOF and subdigestion followed by peptide identification by LC-MS/MS as consisting of two fragments; 48-246 and 12-204. These results suggest that major Ca 2+ independent cleavage-sites are at amino acids 12, 48, 205 and 247. Supplementary material, Fig 3 shows the mass spectra of the digests at 5 and 30 minutes, respectively, and the first and last amino acids are given for all peptides that can be assigned to a specific mass within the secretagogin sequence. Both the rate and pattern of tryptic digestion are Ca 2+ dependent, although some of the identified peptides are similar in both forms. As shown in Fig. 3A, peptides that are identified in the presence of Ca 2+ derive mainly from the N-terminal part of the protein. Major cleavage sites in this region are at amino acids 24, 56, 61 and 63. These cleavage-sites are all located in EFhand 1 or in the loop between EF-hand 1 and 2. In contrast, peptides identified in the absence of Ca 2+ derive from the whole protein with many cleavage sites in EF-hands 4, 5 and 6 (Fig. 3B).

Ca 2+ binding determined from Isothermal titration calorimetry (ITC)

ITC was used to analyze the energetics of Ca 2+ binding to secretagogin. For these experiments (and for the titration with Ca 2+ in the presence of chromophoric chelators; see below) we used two different batches of recombinant secretagogin to ensure that our results were reproducible. The purity of both batches was checked by agarose gel electrophoresis in the presence of 1 mM EDTA or 2 mM calcium lactate, SDS-PAGE, isolelectric focusing and mass spectrometry. 1 H NMR spectroscopy was used to verify that all samples were free from EGTA after preparation of apo-protein. To quantify the residual Ca 2+ -concentration in the apo-protein, we performed competition experiments with the high affinity chelator quin2. The results suggest that both our batches of recombinant secretagogin are homogenous, intact, free from EGTA, and contain 0.3 equivalents of Ca 2+ (not shown). Titration of CaCl 2 into aposecretagogin in physiological salt concentrations (0.15 M KCl) results in a biphasic curve indicating the presence of two classes of binding sites (Fig 4C andD). Data were fitted by the Origin software according to a model allowing for cooperative binding. To determine the stoichiometry of binding we varied the number of binding sites from 1 to 6. The data could not be fitted with one or two binding sites, but was well fitted with three or more binding sites. Binding of Ca 2+ to the first binding site proceeds with a negative enthalpy change, indicating an exothermic process with a ∆H of -17 kJ/mol, while Ca 2+ -binding to the second class of sites is an endothermic process with an average ∆H of +7 kJ/mol. As the data was fitted equally well with three or four Ca 2+ -binding sites in physiological salt concentrations, we also performed the experiments in low salt (Fig 4A andB). Again we observed two classes of binding sites. However, the initial exothermic process is significantly less pronounced at low salt. In low salt solutions, Ca 2+ binding to the first site is exothermic with ∆H of -3 kJ/mol, followed by an endothermic process. At low salt concentrations, the data can not be fitted with less than one high affinity site and three low affinity sites, suggesting that the protein binds 4 Ca 2+ ions. The Ca 2+ binding affinity (Ka) of the first site is too high to be accurately determined by the present experiments, but could be estimated to around 5 . 10 6 M -1 or higher. In low salt solutions, the macroscopic Ca 2+ -binding constants for the 3 low-affinity sites (K 2 -K 4 ) are obtained as Log 10 K 2 = 4.3±1.0, Log 10 K 3 = 5.5±1.0 and Log 10 K 4 = 7.0 ±1.0, respectively. The average ∆H for these three sites is +20 kJ/mol, and the data are best fitted when Log 10 K 4 > Log 10 K 3 > Log 10 K 2 , suggesting a high degree of cooperativity of Ca 2+binding to the low affinity sites.

Ca 2+ binding determined from competition with chromophoric chelators.

To further characterize the Ca 2+ binding properties of secretagogin, we used titration with Ca 2+ in the presence of chromophoric chelators to determine the macroscopic Ca 2+ -binding constants of secretagogin as described [18,19]. In a previous study we used the chelators quin2 or 5,5'Br 2 -BAPTA (K D (Ca 2+ ) of 0.12 and 2.3 µM, respectively, in 0.15 M KCl) for the determination of the Ca 2+ affinity of the related hexa EF-hand protein calbindin D 28k [13].

Since the ITC experiments indicated that secretagogin may contain a set of sites with lower affinity we used both 5,5'Br 2 -BAPTA and the chelator 5N-BAPTA, which has lower Ca 2+ affinity (Kd (Ca 2+ ) = 27 µM in 0.15 M KCl) than that of quin2 or 5'Br 2 -BAPTA. Titrations were also performed in the same buffer with no added salt (Fig. 5, Table 1). The data was analyzed by fitting each titration by a model that allows for up to four macroscopic binding constants in the protein using the Caligator software [14]. In accordance with the ITC results, the data suggest that secretagogin binds Ca 2+ to one site with comparatively high affinity (Log 10 K 1 = 7.1 ± 0.4 in 0.15 M KCl) which is manifested as a rather flat part in the beginning of the titrations in the presence of 5N-BAPTA (Fig. 5), and as a slight curvature in the presence of 5,5'Br 2 -BAPTA (not shown). The titrations in the presence of 5N-BAPTA suggest that there are also at least two sites with lower affinity in 0.15 M KCl. Incorporation of a fourth site leads to lower χ 2 , but the change is not significant given that more parameters are introduced. Thus the data from the chelator experiments are equally well fitted with 3 or 4 binding-sites. If we assume that secretagogin binds four Ca 2+ ions, as suggested by the ITCexperiments, the following affinities are obtained in physiological salt buffers: Log 10 K 1 = 7.1±0.4, Log 10 K 2 = 4.7±0.6, Log 10 K 3 = 3.6±0.7, Log 10 K 4 = 4.6±0.6. All data sets are best fitted if K 4 is at least an order of magnitude larger than K 3 implying strong positive cooperativity of Ca 2+ binding to the low affinity sites [20]. This is seen as a sigmoidal shape in the titration data after the saturation of the high affinity site. The Ca 2+ concentration required to achieve half maximal saturation of secretagogin in 0.15 M KCl is around 25 µM.

At low salt concentrations secretagogin binds Ca 2+ to four sites with the following affinities: Log 10 K 1 = 8.0±0.3, Log 10 K 2 = 5.0±0.1, Log 10 K 3 = 3.0±0.1, Log 10 K 4 = 5.5±0.3. Again, the data are better fitted if K 4 is at least an order of magnitude larger than K 3 , implying positive cooperativity among the low affinity sites. We used two batches of recombinant secretagogin, both of which gave similar results. Altogether 9 titrations (4 and 5 titrations each of the two batches of recombinant secretagogin) were performed using 5N-BAPTA.

Ca 2+ binding to secretagogin as monitored by ANS-and Trp-fluorescence.

In order to confirm that the binding constants obtained by the chelator method are in the correct range, we monitored Ca 2+ -binding by Trp-and ANS fluorescence. Although the titration monitored by Trp and ANS fluorescence does not provide the high precision obtained using the competitive chromophoric chelator method, it does confirm that the binding constants obtained by the chelator method are in the correct range (See supplementary material).

Identification of secretagogin binding proteins.

Ca 2+ -sensor proteins such as calmodulin often interact with target proteins. We investigated if secretagogin binds to specific targets by affinity chromatography using immobilized recombinant secretagogin. In order to identify bona fide interacting proteins versus sample contaminants we conducted negative controls in parallel using sepharose 4B cross-linked to human recombinant calbindin D 28k . Calbindin D 28k , was selected as control-protein because it is very similar to secretagogin based on homology searches performed using the BLAST 2 software (58 % sequence similarity). Like secretagogin, Calbindin D 28k is a hexa EF-hand protein belonging to the calmodulin protein-superfamily [3,21]. We performed the experiments using brain-homogenates from two different species (mouse and cow) and by using pull-downs from the rat insulinoma cell-line RIN-5F. All eluates were concentrated and separated by SDS/PAGE. Protein-bands which appeared to bind specifically to the secretagogin-column were excised and digested with trypsin and the resulting peptides were identified by LC-MS/MS. Experiments were performed using both cytosolic fractions (tissue homogenized without detergent) and membrane fractions (tissue homogenized in the presence of detergent in order to solubilize membrane-associated proteins). Although a small number of proteins (e.g. protein kinase C, N-ethylmaleimide sensitive fusion protein and caskin-1) were identified from secretagogin affinity chromatography experiments using cytosolic fractions, these proteins did not pass the criteria we set up for accepting a protein as a specific secretagogin binding protein. Thus, they were either not reproducibly identified or they were also found in the eluates from the calbindin D 28k control columns (not shown). When using Secretagogin is not only expressed in the brain, but also abundantly expressed in neuroendocrine cells in the pancreas. The fourth experiment was designed to investigate the binding between secretagogin and SNAP-25 in a pancreatic cell-line. For this purpose, immobilized GST-secretagogin fusion protein was used to affinity-purify ("pull-down") proteins from a membrane preparation of the beta cell line RIN 5F. GST-secretagogin protein was able to pull down endogenous SNAP-25 from RIN-5F cells. In contrast, control GST or GST-Setagin was unable to pull down SNAP-25 (Fig 6C). Setagin is 5.6 kDa splice variant of secretagogin that contains only a segment of the first EF-hand [11].

Surface plasmon resonance (SPR) was used to study the binding kinetics and affinity between secretagogin and SNAP-25 in vitro. SNAP-25 was overexpressed in E. coli and purified as described in the material and methods section. The amount of immobilized secretagogin was around 2500 RU (pg/mm 2 ). The interaction between the two proteins was studied at four different SNAP-25 concentrations ranging from 25 nM to 4.5 µM. The curves representing the three lowest SNAP-25 concentrations (25 nM, 180 nM and 900 nM) were used for fitting.

The sensorgrams indicate that the two proteins interact with high affinity in the presence of Ca 2+ . Based on the values of k on and k off (see table 2) the equilibrium dissociation constant K D in the presence of Ca 2+ was determined to 1.2 x 10 -7 M . The affinity is reduced by a factor of 8 in the absence of Ca 2+ (K D = 1.5 x 10 -6 M). The difference between the Ca 2+ and EDTA forms seems to be in k off , indicating that secretagogin dissociates from SNAP-25 more rapidly in the absence of Ca 2+ (see table 2). We also performed SPR control experiments with immobilized calbindin D 28k and calmodulin under identical conditions as above. Secretagogin binds strongly to SNAP-25 in Ca 2+ buffers and somewhat weaker in EDTA buffers ( Fig 7).

Calbindin D 28k and calmodulin, on the other hand, do not bind to SNAP-25 in the presence of Ca 2+ ( Fig 7) or in the presence of EDTA (data not shown).

DISCUSSION

An important role of Ca 2+ -buffer proteins is to equip cells with high-affinity Ca 2+binding sites in order to reduce the intracellular Ca 2+ concentration and to regulate Ca 2+ signals. For this purpose, this group of proteins does not need to undergo any Ca 2+ -induced conformational changes. Ca 2+ -sensor proteins, however, undergo conformational changes, which allow them to regulate their activities in a Ca 2+ -dependent manner. Often a hydrophobic binding surface is exposed which results in Ca 2+ -sensor/effector complexes that regulate specific cellular events. We found evidence for Ca 2+ -induced conformational changes in the tertiary structure of secretagogin by several spectroscopic methods. The results indicate that both the apo-and Ca 2+ -form of secretagogin bind significant amounts of ANS, suggesting that hydrophobic groups are exposed on the surface of the protein both in the Ca 2+ -free and Ca 2+ -loaded state. Secretagogin shares this property with the related hexa EF-hand proteins calretinin and calbindin D 28k [13,22,23]. The ANS-fluorescence studies also indicate that the Ca 2+ -loaded form of secretagogin is more hydrophobic than the Ca 2+ -free form. The exposure of hydrophobic surfaces is thermodynamically unfavourable. An obvious way of minimizing the exposed hydrophobic surfaces is by the interaction with other proteins. Thus it is possible that the hydrophobic patch at the surface of secretagogin may be instrumental for interaction with (and functional regulation of) other proteins.

Our results suggest that secretagogin binds 4 Ca 2+ ions. We find it likely that EF hands The ITC data for secretagogin stand in bright contrast to those of calbindin D 28k .

Calbindin D 28k binds four Ca 2+ ions with high affinity and positive cooperativity [13,24]. ITC experiments performed with Calbindin D 28k shows a single exothermic process that requires 4 equivalents of Ca 2+ to reach saturation and the affinity is too high to be quantified (S. Linse, unpublished observation). However, for secretagogin ITC data are clearly biphasic, and the sites act in two groups; one with high-moderate affinity and negative reaction enthalpy, and one group with lower affinity, positive enthalpy and cooperativity. The apparent Ca 2+ affinity of secretagogin (Ka) is over 10 times lower than that of the highly related hexa EF-hand proteins calbindin D 28k . At resting potential ([Ca 2+ ] = 10 -9 -10 -7 M), 0.5 -14 % of secretagogin would be saturated by Ca 2+ . This indicates that the high affinity site may provide some buffering capacity. However, Ca 2+ -buffering proteins such as parvalbumin (K D ~ 0.05 µM) usually have much higher Ca 2+ affinities suggesting that secretagogin is unlikely to function as a traditional Ca 2+ -buffer protein. If we assume that secretagogin functions as a Ca 2+ sensor protein, the affinity (K D of around 25 µM) is still rather low. For example, calmodulin, has a K D of around 5 µM (in the absence of bound targets). There are, however, several examples of Ca 2+ -sensor proteins with low affinity. The apparent Ca 2+ affinity of the Ca 2+ sensor for exocytosis in neural cells, synaptotagmin I, is 5-50 µM in the presence of phospholipid membranes [25][26][27], which is in the same range as measured here for secretagogin. This information is particularly interesting since synaptotagmin I binds to SNAP-25, the same protein that was identified in this study as a potential secretagogin binding protein. Alternatively, the requirement for higher Ca 2+ concentrations may be similar to the situation with the cytoplasmic calpains (for a review, see [28]). Calpains participate in intracellular signal processing via limited proteolysis of their targets, altering the function of these proteins. The Ca 2+ -concentration giving half-maximal activity, [Ca 2+ ]0.5, ranges from 2 to 75 µM for l-calpain (e.g., [29][30][31] and 200-1000 µM for m-calpain [30,32]. Since Ca 2+ concentrations in this range do not exist in intact living cells calpains may function at the initial, ascending limb of their Ca 2+ saturation curves, allowing only a small fraction of the calpain pool to become activated [28].

We recently completed a study in which we identified a large number of proteins in EGTA eluates from calmodulin affinity columns [33]. When comparing these results to those from the present study, it is clear that secretagogin does not bind a very large number of proteins as observed for calmodulin. Although we can not exclude the existence of other secretagogin interacting proteins, SNAP-25 was the only protein that we could identify from all tissues and species used. Unlike secretagogin, the related proteins calbindin D 28k and the promiscuous Ca 2+ sensor calmodulin were unable to bind SNAP-25. These results indicate that the interaction between secretagogin and SNAP-25 is highly specific. SNAP-25 is one of the basic components of the SNARE complex. The SNARE complex is the core-complex for membrane fusion, a process that is regulated by changes in intracellular Ca 2+ levels (for reviews, see [34][35][36]). Secretagogin is expressed in neuroendocrine cells in the brain and in the pancreatic islets [1][2][3], which are sites of exocytosis of neurotransmitters and insulin, respectively. Thus secretagogin and SNAP-25 seem to be expressed in the same type of cells.

SNAP-25 could be eluted from secretagogin columns by buffers containing Ca 2+ chelators, suggesting that the interaction is at least partially Ca 2+ -dependent. The apparent affinity of the interaction increases about 10-fold following addition of Ca 2+ . However, although the interaction is enhanced by Ca 2+ -binding, it occurs with high affinity both in the presence and absence of Ca 2+ . Does this mean that secretagogin is unlikely to function as a Ca 2+ sensor protein? EF-hand Ca 2+ -sensor proteins that are pre-bound to targets in their Ca 2+free states are not uncommon. Prebound calmodulin can for example be activated and modify associated proteins as demonstrated by its regulation of voltage gated Ca 2+ channels [37][38][39].

SNAP-25 interacts with another Ca 2+ -binding protein, the Ca 2+ sensor for exocytosis in neural cells, synaptotagmin I. Synaptotagmin I binds to SNAP-25 in the absence of Ca 2+ [40] with the apparent affinity of the interaction increasing only about 6-fold following addition of Ca 2+

[41]. Thus, the triggering of fast exocytosis by Ca 2+ binding to synaptotagmin I is not executed via Ca 2+ -induced binding of synaptotagmin I to SNAP-25 and other SNAREs [42].

Ca 2+ -independent binding of synaptotagmin I to SNAREs serve to position synaptotagmin I next to SNARE complexes after these have been assembled but before the fusion pore has opened [34]. Ca 2+ then induces a conformational change in the pre-bound synaptotagmin that results in a series of events that opens the fusion pore. Similarly, formation of a complex between secretagogin and SNAP-25 at resting Ca 2+ concentrations could, in theory, allow Ca 2+ -induced structural changes of secretagogin to be rapidly transmitted to elicit a functional change. An investigation of the possible role of secretagogin in exocytosis is an important question for the future.

Conclusions

Secretagogin binds 4 Ca 2+ ions with a [Ca 2+ ] 0.5 of around 25 µM in physiological salt-buffers, which could be compared to [Ca 2+ ] 0.5 of around 1 µM for calbindin D 28k [13] and 1.5 µM for calretinin [43]. The differences in Ca 2+ affinities probably reflect important biological and evolutionary differences between these proteins. The comparatively low Ca 2+ -affinity of secretagogin makes it unlikely to function as an efficient Ca 2+ buffer protein, although the high affinity site may provide some buffering capacity. Copyright 2006 Biochemical Society (dotted line). MgCl 2 was added to a concentration of 5 mM, and a spectrum for the Mg 2+ form was recorded (dashed line). Finally CaCl 2 was added to a concentration of 1 mM, and a spectrum for the Ca 2+ form was recorded (solid line). B Near UV absorbance spectroscopy.

The UV-spectrum at room temperature was recorded for a solution of 37 µM secretagogin, in 1 mM DTT, 0.5 mM EGTA, 2 mM Tris/HCl, pH 7.5 (apo-secretagogin; dotted line). 5 mM

  Isothermal titration calorimetry. Isothermal titration calorimetry (ITC) was carried out using a VP-ITC instrument from Microcal Inc at constant temperature (25°C) in 2 mM Hepes/NaOH buffer, pH 7.5 with 0 or 150 mM NaCl. Deionized water was used in the reference cell. All solutions were thoroughly degassed by stirring under vacuum before use. 1 mM CaCl 2 was titrated into 25 µM secretagogin in the sample cell (1.4 ml). For each titration, a 2 µl injection was followed by 40 injections of 4 µl each into the sample cell. The mixture was allowed to react for 4 min between injections. Heats due to injection and dilution were obtained by titrating CaCl 2 into buffer. The data were fitted using the modified Origin software supplied by Microcal Inc. ∆H° and Log 10 K values were obtained by fitting a macroscopic binding model and fits allowing for 2, 3 or 4 binding sites were attempted.

pH 4 . 5 )

 45 . Deactivation of unreacted NHS ester was achieved by the flow of 20 µL 1 M ethanolamine hydrochloride, pH 8.5. The association and the dissociation of the analyte (SNAP-25) from the chipimmobilized components were studied at several different analyte concentrations. Control experiments were performed using a flow cell, which had been prepared using the immobilization protocol above, but without coupling protein to the chip dextran surface. The surface was regenerated by injecting 0.1 M HCl during 15 minutes to remove residual bound analyte.

Biochemical

  Journal Immediate Publication. Published on 30 Aug 2006 as manuscript BJ20060918 Secretagogin contains 19 Phe residues, 3 Trp and 5 Tyr residues. The Trp and Tyr signals from ~270-295 nm and the strong Phe signals below 270 nm indicate that secretagogin has a significant amount of tertiary structure in the absence of any bound metal ions (Fig. 1A). No significant change in the near UV-CD spectrum is seen upon addition of 5 mM MgCl 2 .

Biochemical

  Journal Immediate Publication. Published on 30 Aug 2006 as manuscript BJ20060918 Copyright 2006 Biochemical Society membrane fractions for the affinity chromatography experiments, however, synaptosomal associated protein of 25 kDa (SNAP-25) was identified as a specifically binding protein in all experiments. Below we therefore describe only the affinity chromatography experiments performed using membrane fractions.The first experiment was designed to purify proteins binding to Ca 2+ -secretagogin in mouse brain membrane fractions (Fig6A). 2 mM CaCl 2 was included when solubilising the brain extract, which was then incubated with secretagogin-sepharose or calbindin D28k-sepharose.Each column was eluted with 5 mL of a buffer containing 2mM EGTA. A protein-band which was found in the eluate from the secretagogin column, but not in the eluate from the calbindin D 28k -column, was identified by LC-MS/MS as SNAP-25 with a Mascot score of 50 (corresponding to a p value of < 0.0005, i.e. the statistical probability of a correct protein identification is 99.95 %) and 5 identified peptides (MLQLVEESK, EQMAISGGFIR, ADQLADESLESTRR, MLQLVEESKDAGIR, TLVMLDEQGEQLER).The second experiment was designed to purify proteins binding to Ca2+ -secretagogin from bovine brain membrane fractions. The secretagogin-and calbindin D 28k -columns were incubated with bovine brain homogenates in the presence of 2 mM CaCl 2 , washed and proteins where eluted with 5 mL of a buffer containing EGTA. The identity of a 30 kDa band present in the eluate from the secretagogin-column was determined to SNAP-25 by LC-MS/MS (Fig 6B). This band was not seen in the eluate from the calbindin D 28k column (Fig 6B). Three different peptides (C 8 CGLFICPCNKLK, TLVMLDEQLDR from SNAP-25a and TLVMLDEQLER from SNAP-25b) were identified with a Mascot score of 68 (corresponding to a p value of < 0.00005, i.e. the statistical probability of a correct protein identification is 99,995 %) The third experiment was designed to identify proteins binding to apo-secretagogin from mouse brain membrane fractions. Two columns with immobilized secretagogin and two columns with immobilized calbindin D 28k were prepared and incubated separately with homogenized mouse brain tissue in the presence of 2 mM EGTA. Proteins binding to these columns were first eluted by a buffer containing 2 mM CaCl 2 . No protein-bands could bee seen in the eluates from the the secretagogin columns (not shown). The columns were then eluted with glycine/HCl, pH 2.5. LC-MS/MS identified SNAP-25 in the eluates from the two secretagogin columns, but not in the eluates from the two calbindin D 28k -columns (not shown). SNAP-25 was identified from the first secretagogin column with a Mascot score of Biochemical Journal Immediate Publication. Published on 30 Aug 2006 as manuscript BJ20060918 Copyright 2006 Biochemical Society 82 (corresponding to a p value of < 10 -6 ) and with 7 identified tryptic peptides , HMALDMGNEIDTQNR) and from the second secretagogin column with a Mascot score of 193 (corresponding to a p value of < 10 - 6 ) and with 7 identified tryptic peptides (MLQLVEESK, IEEGMDQINK, EQMAISGGFIR, ADQLADESLESTR, IEEGMDQINKDMK, RADQLADESLESTR, MLQLVEESKDAGIR). These results suggest that secretagogin binds to SNAP-25 also in the absence of Ca 2+ .

3, 4 ,

 4 5 and 6 are responsible for the binding of Ca 2+ ions to secretagogin. This is because the amino acid sequences of EF-hands 1 and 2 do not conform to the EF-hand consensus sequence. EF-hand 1 has a Lys residue at a position where normally an Asp or Asn side chain provides an oxygen to coordinate Ca 2+ . The loop of EF-hand 2 has the standard Ca 2+coordinating groups, but in one position there is a positively charged Arg where normally negative or hydrophilic side-chain hydrogen bonds to a Ca 2+ -coordinating water molecule in the same loop. Therefore, these two EF-hands are most likely the ones that are non-functional in terms of Ca 2+ binding. The sequences of EF-hands 3, 5 and 6 are typical for Ca 2+ -binding EF-hands (Fig3C and D). EF-hand 4 has an Asp in position 12, which is uncommon, but found in a number of active Ca 2+ -binding sites, for example in the sarcoplasmic Ca 2+ -binding protein.

FIG 1 .

 1 FIG 1. A. Near-UV Circular dichroism. A near UV CD spectrum was first obtained for the apo protein (37 µM apo secretagogin in 1 mM DTT, 0.5 mM EGTA, 2 mM Tris/HCl, pH 7.5)

MgCl 2 FIG. 4 .FIG. 5 .FIG. 6 .FIG. 7 .

 24567 FIG 2. A. Tryptophan fluorescence. Tryptophan fluorescence spectra were recorded for a sample containing 5 µΜ secretagogin, 1 mM DTT , 0.5 mM EGTA, 0.15 M KCl, 2 mM Tris/HCl, pH 7.5 (dotted line), after which MgCl 2 , pH 7.5 was added to a concentration of 5 mM (dashed line). 1 mM CaCl 2 , pH 7.5 was then added to the same solution (solid line). B. ANS fluorescence. ANS fluorescence spectra were recorded for a sample containing 120 µM ANS, 5 µΜ secretagogin, 1 mM DTT, 0.5 mM EGTA, 0.15 M KCl, 2 mM Tris/HCl, pH 7.5 (dotted line). MgCl 2 , pH 7.5 was then added to a concentration of 5 mM (dashed line).Finally, 1 mM CaCl 2 , pH 7.5 was added to the same solution (solid line)

Table 1 .

 1 The amount of exposed hydrophobic surface in secretagogin increases upon Ca 2+ -binding and it undergoes Ca 2+ induced conformational changes, suggesting the possibility that it can transduce intracellular Ca 2+concentration changes to other proteins. Secretagogin does not interact with a large number of proteins as observed for e.g. calmodulin. Only SNAP-25, a protein involved in Ca 2+ regulated exocytosis, could be reproducibly identified as a secretagogin-binding protein. Macroscopic Ca 2+ -binding constants for secretagogin determined by competition with the chromophoric chelator 5N-BAPTA. The mean values and standard deviations are presented.

	Number of	[KCl]	Log 10 K 1	Log 10 K 2	Log 10 K 3	Log 10 K 4
	titrations
	4	-	8.0±0.3	4.9±0.1	3.0±0.1	5.5±0.3
	5	0.15M	7.1±0.4	4.7±0.6	3.6±0.7	4.6±0.6

TABLES

Table 2 .

 2 Surface plasmon resonance results.

	Immobilized			Analyte
	protein	Analyte in flow phase	Ca 2+ /EDTA	Concentrations	K d (M)	k on (s -1 M -1 )	k off (s -1 )
				Tested (µM)
	secretagogin SNAP-25 Ca 2+	0.025-0.9	1.2 10 -7 3.7 10 3 4.4 10 -5
	secretagogin SNAP-25 EDTA	0.025-0.9	1.5 10 -6 2.6 10 3 2.6 10 -4

Biochemical Journal Immediate Publication. Published on 30 Aug 2006 as manuscript BJ20060918

ACKNOWLEGEMENTS

This work was supported by grants from Stiftelsen Lars Hiertas Minne (TB), Magnus Bergvalls foundation (TB), the SWEGENE foundation (TB) and by grants from the Swedish Research Foundation (SL, PJ)

C.

E-L-K-E-A-F-R-E-F-D-K-D-G-D-G-T-I-D-F-E-E-F-L-T-M-M-A-S-L