©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Two 17-kDa Rat Parotid Gland Phosphoproteins, Subjects for Dephosphorylation upon -Adrenergic Stimulation, as Destrin- and Cofilin-like Proteins (*)

(Received for publication, October 25, 1994; and in revised form, February 1, 1995)

Takao Kanamori (1)(§) Taro Hayakawa (1) Masami Suzuki (2) Koiti Titani (2)

From the  (1)Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464, Japan and the (2)Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-11, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We previously reported that when P(i)-loaded rat parotid slices are incubated with the beta-adrenergic agonist isoproterenol, the level of a soluble P-labeled 17-kDa protein (pp17) decreases rapidly (Kanamori, T., and Hayakawa, T.(1982) Biochem. Int. 4, 517-523). Here we show that pp17 consists of two distinct phosphoproteins (pp17a and pp17b), identify their unphosphorylated forms (p17a and p17b, respectively), and provide evidence for their beta-adrenergic stimulation-induced dephosphorylation. Since p17a and p17b were predominant forms even in nonstimulated cells, peptides were generated from them with Staphylococcus aureus V8 protease or cyanogen bromide; subsequent sequencing of these peptides and homology search allowed identification of p17a and p17b as destrin- and cofilin-like proteins, respectively. Interestingly, they were also dephosphorylated in response to cholinergic stimulation. Because destrin and cofilin are actin-depolymerizing proteins whose activities are possibly regulated by their phosphorylation/dephosphorylation, the two parotid proteins reported here might be involved in cortical F-actin disruption observed in parallel with exocytotic amylase secretion.


INTRODUCTION

In the rat parotid gland, beta-adrenergic stimulation induces exocytotic amylase secretion from the acinar cells through activation of adenylate cyclase and the resultant increase in the intracellular level of cAMP (for a review, see (1) ). In eukaryotic cells, the effect of cAMP is generally thought to be mediated through activation of cAMP-dependent protein kinase(2) . In accordance with this view, there exist several lines of evidence suggesting the involvement of cAMP-dependent protein kinase in beta-adrenergic agonist-induced amylase secretion, and endogenous protein substrates for this enzyme have extensively been studied with P(i)-loaded rat parotid slices or cell aggregates (for a review, see (1) ). During the course of these studies, some groups, including our own, unexpectedly observed that beta-adrenergic stimulation decreases the level of a P-labeled endogenous protein with an estimated molecular mass between 13.6 and 17 kDa(1, 3, 4, 5, 6) . The observed effect is probably due to dephosphorylation of the phosphoprotein, but the conclusive evidence is lacking. Here we show that our 17-kDa soluble phosphoprotein (pp17) consists of two distinct phosphoproteins, identify them as phosphorylated forms of destrin- and cofilin-like proteins, provide evidence for their beta-adrenergic stimulation-induced dephosphorylation, and discuss their possible involvement in parotid amylase secretion.


EXPERIMENTAL PROCEDURES

Materials

Urea, acrylamide, and N,N`-methylenebisacrylamide were purified with the mixed-bed resin (AG 501-X8). Nonidet P-40 was purified according to Procedure 1 in (7) .

Slice Experiments

Rat parotid slices were prepared and loaded with P(i) (DuPont NEN) as described in (8) , except that slices were incubated with P(i) for 90 min. The P(i)-loaded slices were placed in vials containing about 40 volumes each of P(i)-free KRB (118 mM NaCl, 5 mM KCl, 2.5 mM CaCl(2), 1.2 mM MgSO(4), 25 mM NaHCO(3)) supplemented with 5 mM beta-hydroxybutyrate and 10 mM glucose. Unless otherwise stated, slices were incubated at 37 °C for 15 min in the absence or presence of the beta-adrenergic agonist l-isoproterenol (10 µM) under an atmosphere of 95% (v/v) O(2), 5% (v/v) CO(2). In some experiments, slices were not loaded with P(i) and the 90-min incubation with P(i) was omitted; experiments were done with ordinary KRB (KH(2)PO(4) concentration, 1.2 mM) supplemented as described above.

Partial Purification of pp17

The procedure described below was carried out at 0-4 °C unless otherwise stated; purification of pp17 was followed by SDS-polyacrylamide gel electrophoresis (PAGE) (^1)and autoradiography of the resultant gels.

Each portion of frozen parotid tissue was placed in a glass-Teflon homogenizer containing 10 volumes of Solution A consisting of 20 mM Tris-HCl (pH 7.5), 0.18 M sucrose, 50 mM NaF, 1 mM Na(3)VO(4), 2 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 12 µg/ml chymostatin, and 2 µg/ml pepstatin A, thawed, and homogenized. The phosphorylation state of pp17 in the homogenates was stable for up to 24 h at 0 °C (data not shown). The homogenates were filtered through two layers of gauze and centrifuged at 100,000 times g for 90 min; the supernatant fluids were collected, stored at -30 °C, and referred to as ``100,000 times g supernatant fractions.''

To each 100,000 times g supernatant fraction containing 3 mg of protein was added 12 µl of 2-mercaptoethanol. The samples were adjusted to 1.2 ml by the addition of Solution A. A portion (0.5 ml) from each sample was applied to a DEAE-cellulose (DE53) column (0.9 times 1.6 cm, 1 ml of bed volume) equilibrated with Solution B consisting of 20 mM Tris-HCl (pH 7.5), 50 mM NaF, 0.1 mM Na(3)VO(4), 2 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 4 µg/ml leupeptin, 6 µg/ml chymostatin, and 0.1% (v/v) mercaptoethanol. The effluents were discarded. Each column was then loaded with an additional 0.65-ml portion of the sample and washed with 2 ml of Solution B. The 0.65- and 2-ml fractions of the effluent from each column were pooled, mixed with 40 µl of 10% (w/v) SDS, incubated for 3 min in a boiling water bath, dialyzed with a device for dialysis (9) against three changes of 30 volumes of Solution C consisting of 0.1% (w/v) SDS, 0.1% (v/v) 2-mercaptoethanol, and 10 mM NH(4)HCO(3) for a total of 30 h at room temperature, and lyophilized. The resultant samples were each dissolved in 0.4 ml of H(2)O and mixed with 3.6 ml of ethanol; the mixtures were stored overnight at -30 °C and centrifuged at 2,000 times g for 20 min at room temperature; the precipitates were washed with 90% (v/v) ethanol and dried (each pellet contained about 1.2 mg of protein).

The following procedure is based on our previous finding that, in the presence of urea and Nonidet P-40, SDS-solubilized proteins can be adsorbed by cation exchangers according to their own charge(10) . To minimize modification of proteins by cyanate derived from urea(11, 12) , we purified urea with AG 501-X8, and added methylamine to urea-containing solutions. The dried samples prepared above were each dissolved at room temperature in 0.25 ml of Solution D consisting of 25 mM Mes/methylamine (pH 5.8), 1% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 0.1 mM EDTA, mixed with 0.5 ml of Solution E consisting of 25 mM Mes/methylamine (pH 5.8), 9 M urea, 4% (w/v) Nonidet P-40, 5% (v/v) 2-mercaptoethanol, and 0.1 mM EDTA, and cooled to 4 °C. Portions (0.7 ml each) from the resultant samples were applied to phosphocellulose (P11) columns (0.46 times 1.5 cm, 0.25 ml of bed volume) equilibrated with Solution F consisting of 25 mM Mes/methylamine (pH 5.8), 0.33% (w/v) SDS, 6 M urea, 2.67% (w/v) Nonidet P-40, 0.1% (v/v) mercaptoethanol, and 0.1 mM EDTA. The columns were each washed with 0.5 ml of Solution F, 2 ml of Solution G consisting of 25 mM Mes/methylamine (pH 5.8), 6 M urea, 0.1% (v/v) mercaptoethanol, and 0.1 mM EDTA, 3 ml of Solution H consisting of 25 mM P(i)/methylamine (pH 5.9), 6 M urea, 0.1% (v/v) mercaptoethanol, 0.1 mM EDTA, and 0.03 M NaCl, and 2 ml of Solution I consisting of 25 mM P(i)/methylamine (pH 5.9), 0.1% (v/v) mercaptoethanol, and 0.1 mM EDTA; the effluents were discarded. Each column was then washed at room temperature with 1.5 ml of Solution I containing 0.2% (w/v) SDS; the effluents were saved, dialyzed against Solution C as described above, and lyophilized. The resultant samples were each dissolved in 0.3 ml of H(2)O and mixed with 2.7 ml of absolute ethanol; proteins were precipitated, collected, and washed as described above (each pellet contained about 0.3 mg of protein). As described under ``Results,'' the samples thus obtained contained pp17 and were referred to as ``partially purified pp17 fractions.'' In some experiments where larger quantities of the partially purified pp17 fractions were required, they were prepared with proportionately increased volumes of samples, columns, and elution buffers.

Gel Electrophoresis

SDS-PAGE was carried out as described in (13) . Unless otherwise stated, separating gels contained 15% (w/v) acrylamide and were 2-mm thick. Gels were calibrated with phosphorylase a (94 kDa), bovine serum albumin (68 kDa), catalase (58 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), lysozyme (14.3 kDa), and BDH molecular mass markers (2512-16949 Da). When SDS-PAGE was used for preparing the samples subjected to amino acid sequencing, separating gels were cast at least 1 day in advance and prerun in the separating gel buffer containing 0.1 mM sodium thioglycolate overnight(14) ; stacking gels were composed of 0.8% (w/v) AGAROSE 421F (Funakoshi, Tokyo, Japan) and cast after the prerun(14) .

Two-dimensional PAGE was done as described in (15) with the following modifications: (i) nonequilibrium pH gradient electrophoresis (NEPHGE) gels (3 times 115 mm) contained 8 M urea, 2% (w/v) Servalyt 5-9, 10 mM lysine, 10 mM arginine, and 12 mM aspartic acid(16) , but not Nonidet P-40; (ii) NEPHGE gels were prerun at 200 V for 30 min and at 300 V for 2 h and run at 300 V for 7 h at room temperature; (iii) samples were dissolved in 8 M urea containing 2% (w/v) Servalyt 5-9, 10 mM lysine, 10 mM arginine, 12 mM aspartic acid, and 2% (v/v) 2-mercaptoethanol; (iv) SDS-PAGE was done as described in (13) (1-mm-thick gels were used). When two-dimensional PAGE was carried out on a larger scale, NEPHGE gels of 4-mm diameter were used in the first dimension and 2-mm thick slab gels in the second.

Densitometric analysis of electropherograms was carried out with a model IX-3010 flat-bed scanner (Canon, Tokyo, Japan) connected to a Macintosh LC microcomputer (Apple Computer); images were analyzed with the public domain software NIH Image 1.52, which was written by Wayne Rasband (National Institutes of Health, Bethesda, MD); values are presented as means ± S.D. and the statistical significance of measures was determined by Student's paired t test.

Peptide Mapping

Proteins were digested in gels for 3 h with Staphylococcus aureus V8 protease (ICN Biochemicals) as described in (17) with the following modifications: (i) the gel-soaking buffer contained 5% (v/v) 2-mercaptoethanol, but not EDTA; (ii) the protease was added to the slots at a rate of 50 ng/mm^2 slot surface; (iii) generated peptides were resolved by SDS-PAGE as described above (separating gels contained 17.5% (w/v) acrylamide). Proteins were also cleaved in gels with cyanogen bromide (CNBr) as described in (18) , and generated peptides were separated by Tricine/SDS-PAGE (19) (spacer and separating gels contained 10 and 16% (w/v), respectively, acrylamide; separating gels were calibrated as described above). In each case, gels were stained with silver or subjected to electrophoretic transfer. When Tricine/SDS-PAGE was used for preparing samples subjected to amino acid sequencing, spacer and separating gels were cast at least 1 day in advance and prerun as in the preparative SDS-PAGE; stacking gels were composed of 0.8% (w/v) AGAROSE 421F and cast after the prerun(14) .

Electrophoretic Transfer

After PAGE, proteins in the gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P) as described in (20) with the following modifications: (i) all the transfer buffers contained 15% (v/v) methanol; (ii) the cathodic buffer was supplemented with 0.012% (w/v) SDS and 10 mM 2-mercaptoethanol(21) ; (iii) electrotransfer from 1-mm thick gels was carried out for 2.5 h at 0.8 mA/cm^2 and then for 2 h at 40 V; electrotransfer from 2-mm thick gels, for 3.5 h at 0.8 mA/cm^2 and then for 2 h at 40 V. After transfer, PVDF membranes were stained with Ponceau S (22) or Coomassie Blue(23) ; the stained spots or bands of interest were excised with a razor blade and stored at -30 °C.

Analysis of Phosphoamino Acids

Coomassie Blue-stained proteins on PVDF membranes were hydrolyzed, in vapor phase contact with 5.7 M HCl, at 110 °C for 2.5 h as described in (24) . Phosphoamino acids were separated on cellulose thin-layer sheets by two-dimensional electrophoresis at pH 1.9 and then at pH 3.5(25) .

Dephosphorylation of Proteins Immobilized on PVDF Membranes

The Ponceau S-stained proteins on PVDF membrane pieces were destained in water, and several spots of each protein were used. The PVDF membrane pieces, including blank sections containing no protein spots, were each incubated in 0.75 ml of 0.2% (w/v) polyvinylpyrrolidone (M(r) 40,000) at room temperature for 30 min; thereafter each membrane piece was washed 6 times with 1 ml of H(2)O, incubated at 37 °C for 10 h in a reaction mixture (200 µl) consisting of 0.1 M Mes/NaOH (pH 5.5), 1 mM MgCl(2), 2 units/ml potato acid phosphatase (Sigma), 20 µg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride, and washed once for 10 min with 1 ml of 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and twice for 10 min with 2 ml of H(2)O. Each membrane piece was then incubated for 90 min at room temperature in an elution buffer (100 µl) consisting of 50 mM Tris-HCl (pH 9.0), 1% (w/v) Triton X-100, 2% (w/v) SDS, and 1% (v/v) 2-mercaptoethanol(26) , and the eluates were saved.

Sequence Analysis of Peptides

Peptides electroblotted onto PVDF membranes were sequenced with an Applied Biosystems 470A protein sequencer connected on-line to a 120A PTH analyzer. Sequence homology searches were made using the protein sequence data base of the National Biomedical Research Foundation on a VAX 3600 computer with the WORDSEARCH program(27) . The alignment procedure used the SEGMENT program(28) .

Other Procedures

Silver staining of proteins or peptides in polyacrylamide gels was done as described in (29) . Autoradiography was done with Konica x-ray film (Type A) at room temperature; fluorography, with the same film and intensifying screens (Cronex Lighting-Plus) at -80 °C(30) . Protein concentrations were determined as described in (31) , with bovine serum albumin as a standard.


RESULTS

Partial Purification of pp17

Our previous study indicated that pp17 is a minor soluble phosphoprotein in the rat parotid gland (4) . Before its characterization and identification, we partially purified pp17 from the 100,000 times g supernatant fractions prepared from isoproterenol-stimulated and nonstimulated rat parotid slices. The procedure involved DEAE-cellulose and phosphocellulose chromatographies as described under ``Experimental Procedures.'' Under the conditions used, pp17 was not retained by the DEAE-cellulose columns; it was recovered in the flow-through fractions with about 40% of the total protein applied (data not shown).

Fig. 1shows the elution profile of pp17 on the phosphocellulose chromatography. pp17 was retained by the columns (lanes 1-7); about 75% of the total protein applied to each column was eluted during the sample application and the column-washing procedure with Solutions F-I (data not shown); pp17 was eluted with 0.2% (w/v) SDS in Solution I (lanes 8-11) together with about 25% of the total protein applied to each column (data not shown).


Figure 1: Partial purification of pp17 by phosphocellulose chromatography. The 100,000 times g supernatant fraction (3 mg of protein) prepared from nonstimulated P(i)-loaded rat parotid slices was processed for DEAE-cellulose chromatography. The flow-through fraction from the DEAE-cellulose column was treated as described under ``Experimental Procedures,'' and the resultant sample (0.7 ml) was applied to a phosphocellulose column (0.46 times 1.5 cm) equilibrated with Solution F. The column was washed with 0.5 ml of Solution F. The fractions collected upon sample application and washing with Solution F were pooled (lane 1). The column was then washed once with 2 ml of Solution G (lane 2), three times with 1 ml of Solution H (lanes 3-5), twice with 1 ml of Solution I (lanes 6 and 7), and twice with 1.5 ml of Solution I containing 0.2% (w/v) SDS (lanes 8 and 9). The flow-through fraction and the faction collected upon washing with Solution G were each mixed with 9 volumes of absolute ethanol, and precipitated proteins were collected. Each 1-ml fraction collected upon washing with Solutions H and I was mixed with 10 µl of 10% (w/v) SDS; these fractions and the 1.5-ml fractions collected upon washing with Solution I containing 0.2% (w/v) SDS were dialyzed against Solution C and lyophilized. Likewise, the 100,000 times g supernatant fraction from isoproterenol-stimulated P(i)-loaded rat parotid slices was processed; the effluent from the phosphocellulose column was collected upon washing with the first 1.5-ml portion of Solution I containing 0.2% (w/v) SDS and treated as described above (lane 10). One-third of the protein eluted into each fraction was processed for SDS-PAGE, and an autoradiogram of the resultant gel is shown (lanes 1-10). Lane 11 shows the Coomassie Blue-stained gel area corresponding to lane 8. The positions of standard proteins with the molecular masses indicated in kDa are shown on the right; arrows indicate the position of pp17.



Analysis and Characterization of pp17

The partially purified pp17 fractions from isoproterenol-stimulated and nonstimulated slices were subjected to two-dimensional PAGE, and pp17 in gels was detected by autoradiography. As shown in Fig. 2c, pp17 from nonstimulated slices was resolved into two major spots (closed arrows 1 and 2) and their intensities on the autoradiogram were lowered by beta-adrenergic stimulation (Fig. 2, compare c and d). On the original autoradiogram, two minor spots were also observed (in Fig. 2c, their positions are indicated by the open arrows). The intensities of these minor spots were slightly increased when the partially purified pp17 fraction from nonstimulated slices was incubated at 37 °C for 24 h in the 8 M urea-containing sample buffer before two-dimensional PAGE (data not shown); the intensities of the two major spots were slightly lowered by the treatment (data not shown). It is likely that although we took several precautions to avoid modification of pp17 by cyanate derived from urea (see ``Experimental Procedures''), pp17 was slightly modified during its partial purification and/or two-dimensional PAGE. We hence did not study the two minor spots further. Corresponding to the two major spots on the autoradiogram (closed arrows 1 and 2 in Fig. 2c), two protein spots were observed on the silver-stained gel (closed arrows 1 and 2 in Fig. 2a); beta-adrenergic stimulation lowered the intensities of these protein spots (Fig. 2, compare a and b), increasing those of two other protein spots (closed arrows 1` and 2` in Fig. 2, a and b); the proteins indicated by closed arrows 1` and 2` seemed more basic than those indicated by closed arrows 1 and 2, respectively. The isoproterenol-induced increases in the intensities of the two protein spots were more easily detected when rat parotid slices were prepared with ordinary KRB instead of P(i)-free KRB and stimulated by the agonist in the former medium and when gels were stained with Coomassie Blue after two-dimensional PAGE (Fig. 2, compare e and f); densitometric analysis of electropherograms from three independent experiments indicated that isoproterenol increased the intensities of the spots indicated by closed arrows 1` and 2` by 32 ± 9% (p < 0.02) and 30 ± 6% (p < 0.01), respectively. These results suggest the following: (i) pp17 consists of two phosphoproteins, pp17a (closed arrow 1 in Fig. 2) and pp17b (closed arrow 2 in Fig. 2); (ii) beta-adrenergic stimulation induces dephosphorylation of pp17a and pp17b rather than other reactions such as proteolysis, generating their unphosphorylated forms, p17a (closed arrow 1` in Fig. 2) and p17b (closed arrow 2` in Fig. 2), respectively. pp17a and pp17b migrated slightly more slowly in SDS-PAGE than p17a and p17b, respectively, as generally regarded as an effect of protein phosphorylation (e.g.(32) and references therein). In Fig. 2a, two minor protein spots are indicated by the open arrows. The intensities of these minor spots were slightly increased when the partially purified pp17 fraction from nonstimulated slices was incubated, before two-dimensional PAGE, in 8 M urea under the conditions described above (data not shown); the intensities of p17a and p17b spots were slightly lowered by the treatment (data not shown). Similar spots exist in Fig. 2b (open arrows). These minor spots seemed to represent artifactual products and were not studied further. Phosphoamino acid analysis of the hydrolysates of pp17a and pp17b indicated that each protein was phosphorylated at some serine residue(s) (Fig. 3).


Figure 2: Analysis of pp17 by two-dimensional PAGE. P(i)-loaded rat parotid slices were incubated for 15 min without (panel a) or with (panel b) 10 µMl-isoproterenol. The partially purified pp17 fraction (0.15 mg of protein) prepared from each portion of slices was subjected to two-dimensional PAGE (the two NEPHGE gels were loaded onto the same SDS-PAGE gel). The resultant gel was stained with silver and the relevant areas of the gel are shown in panels a and b. Migration in NEPHGE is from left to right (toward the cathode); migration in SDS-PAGE, from top to bottom. The gel thus prepared was subjected to autoradiography; panels c and d are from the resultant autoradiogram and correspond to panels a and b, respectively. In a separate experiment, rat parotid slices were not loaded with P(i); slices were prepared with ordinary KRB instead of P(i)-free KRB and incubated for 15 min in the former medium without (panel e) or with (panel f) 10 µMl-isoproterenol. The partially purified pp17 fractions were prepared and subjected to two-dimensional PAGE as described above. The relevant areas of the Coomassie Blue-stained gel are shown in panels e and f; in panels a-f, closed arrows 1 and 2 indicate the spots of the pp17 constituents pp17a and pp17b, respectively; in panels a, b, e, and f, closed arrows 1` and 2` indicate the spots of p17a and p17b, respectively; in panels a-c, open arrows indicate the spots of artifactual, probably carbamylated, products.




Figure 3: Analysis of phosphoamino acid residues in pp17a (panel a) and pp17b (panel b). The partially purified pp17 fraction (0.6 mg of protein) prepared from nonstimulated P(i)-loaded rat parotid slices was subjected to two-dimensional PAGE. Thereafter proteins in the gel were electrotransferred onto a PVDF membrane and stained with Coomassie Blue; the individual pp17a and pp17b spots were cut from the PVDF membrane; and each protein was hydrolyzed in 5.7 M HCl at 110 °C for 2.5 h. The generated amino acids, together with added standard phosphoamino acids, were separated on thin-layer cellulose sheets by two-dimensional electrophoresis. The resultant cellulose sheets were subjected to fluorography and relevant areas of the fluorograms are shown. Migration at pH 1.9 in the first dimension was from left to right (toward the anode); migration at pH 3.5 in the second dimension, from top to bottom (toward the anode). The spots of added phosphoamino acids were detected with ninhydrin and shown by dotted circles: PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine.



To confirm that p17a and p17b were the unphosphorylated forms of pp17a and pp17b, respectively, we attempted to dephosphorylate, with acid phosphatase, pp17a and pp17b on PVDF membranes. As shown in Fig. 4, the phosphatase treatment of pp17a and pp17b indeed produced p17a (closed arrow 1` in Fig. 4d, compare c and d in Fig. 4) and p17b (closed arrow 2` in Fig. 4f, compare e and f in Fig. 4), respectively. Densitometric analysis of electropherograms similar to the one shown in Fig. 2e indicated that the molar ratio of pp17a to p17a was about 3:7 in the partially purified pp17 fraction prepared from nonstimulated P(i)-unlabeled rat parotid slices; the ratio of pp17b to p17b in the same fraction was about 2:7 (data not shown).


Figure 4: Dephosphorylation of pp17a and pp17b with acid phosphatase. The partially purified pp17 fraction (2 mg of protein) prepared from nonstimulated P(i)-unlabeled rat parotid slices was subjected to two-dimensional PAGE (two NEPHGE gels of 4-mm diameter were used; the separating gels for SDS-PAGE were prerun and the stacking gels were prepared with agarose). After two-dimensional PAGE, proteins were electrotransferred onto PVDF membranes and stained with Ponceau S; PVDF membrane regions (about 15 mm^2 each) containing no protein spots (panel b), pp17a (panel d), and pp17b (panel f) were excised, blocked with polyvinylpyrrolidone and incubated with potato acid phosphatase at 37 °C for 10 h; thereafter proteins were eluted from the PVDF membrane pieces. The partially purified pp17 fraction (15 µg of protein, dissolved in 1% (w/v) SDS) prepared from nonstimulated parotid slices was then added to each PVDF eluate (about 200 µl); the same fraction (15 µg of protein) was also added to each of the three 200-µl portions of the elution buffer (panels a, c, and e). Proteins were precipitated with 9 volumes of absolute ethanol and subjected to two-dimensional PAGE. Relevant areas of the resultant silver-stained gels are shown. For comparison, two first dimension gels were loaded onto the same second dimension gel; thus, areas shown in panels a and b, panels c and d, or panels e and f are from the same second dimension gel. In each panel, closed arrows 1` and 2` indicate the spots of p17a and p17b, respectively; an open arrowhead indicates the spot used as an aid in identifying the neighboring spots. In panels a, c, and e, closed arrows 1 and 2 indicate the spots of pp17a and pp17b, respectively; in panels b, d, and f, closed arrowheads indicate the spots of major proteins derived from the phosphatase preparation; in panel d, an open arrow indicates the spot of a possible artifactual product formed during phosphatase treatment and/or two-dimensional PAGE (see Fig. 2a and ``Results''); the dis-tinct spot marked with an asterisk was observed in this particular electropherogram.



Identification of pp17a, pp17b, p17a, and p17b

To identify pp17a, pp17b, p17a, and p17b, we electrotransferred the four proteins onto PVDF membranes and subjected them to N-terminal amino acid sequence analysis; however, no sequence was obtained from each protein (data not shown). The four proteins thus seemed to be N-terminally blocked, and we attempted to obtain their internal amino acid sequences as described below.

Fig. 5a shows peptide and phosphopeptide maps obtained by digestion of the four proteins with S. aureus V8 protease. pp17a and p17a provided similar silver-stained peptide patterns (lanes 3 and 4), and slight differences were attributed to a phosphopeptide (open arrowhead in lane 3) that migrated more slowly in SDS-PAGE than its unphosphorylated counterpart (lane 4). pp17b and p17b likewise provided similar silver-stained peptide patterns (lanes 1 and 2). As expected, the pattern obtained from pp17a or p17a was different from that obtained from pp17b or p17b. When peptides were generated from p17a and p17b on a larger scale and subjected to N-terminal sequence analysis after their electrotransfer onto PVDF membranes, the 10.2-kDa peptide from p17a and the 13.5- and 9.3-kDa peptides from p17b provided sequences as shown in Fig. 6. Sequence homology searches revealed that, except for two unidentified residues, the N-terminal 34-residue sequence of the 10.2-kDa peptide from p17a was identical with the residues 52-85 of porcine brain destrin (33) (Fig. 6), suggesting that p17a was a destrin-like protein. The N-terminal sequences of the 9.3- and 13.5-kDa peptides from p17b were present in destrin (residues 52-61 and residues 52-58, respectively) from porcine brain and in cofilin (residues 52-61 and residues 52-58, respectively) from porcine brain(34) , mouse brain(35) , or human placenta (36) (see Fig. 6). Since p17b was probably distinct from p17a, p17b seemed a cofilin-like protein. It is noted that in the sequences of destrin and cofilin, the residue 52 of each protein is preceded by a glutamic acid residue in accordance with the specificity of protein cleavage by S. aureus V8 protease (see Fig. 6). Probable N-terminal blockage of porcine brain destrin has been reported(33) . We also determined the N-terminal 17-residue sequence of the 10.2-kDa peptide from pp17a; it was identical with residues 52-68 of destrin (data not shown).


Figure 5: One-dimensional peptide mapping of pp17a, p17a, pp17b, and p17b. The partially purified pp17 fraction (4.8 mg of protein) prepared from nonstimulated P(i)-loaded rat parotid slices was subjected to two-dimensional PAGE (eight NEPHGE gels of 3-mm diameter were used). The resultant gels were stained with Coomassie Blue; pp17a, p17a, pp17b, and p17b spots were excised. Panel a, peptide mapping with S. aureus V8 protease. One spot of p17a (lane 4), two spots of pp17a (lane 3), one spot of p17b (lane 2), and four spots of pp17b (lane 1) were placed in the sample wells of the stacking gel prepared for SDS-PAGE; each protein was digested in the stacking gel and generated peptides were resolved in the separating gel. The gel was stained with silver and the relevant areas are shown; lanes 5 and 6 are from a fluorogram of the gel and correspond to lanes 1 and 3, respectively. Panel b, peptide mapping with CNBr. One spot of p17a (lane 4), three spots of pp17a (lane 3), two spots of p17b (lane 2), and four spots of pp17b (lane 1) were treated with CNBr; generated peptides were separated by Tricine/SDS-PAGE; and the gel was stained with silver and subjected to fluorography. In panels a and b, closed arrowheads indicate the positions of the undigested proteins; open arrowheads, those of P-labeled peptides generated; estimated molecular masses of major peptides from p17a and p17b are shown in the middle and at the left, respectively (each method of PAGE used here was different from the one we previously used (4) and provided a value higher than 17 kDa as the molecular mass of pp17a or pp17b).




Figure 6: Alignment of partial amino acid sequences of p17a, p17b, destrin, and cofilin. The partially purified pp17 fraction (7.2 mg of protein) prepared from nonstimulated P(i)-unlabeled rat parotid slices was subjected to two-dimensional PAGE (six NEPHGE gels of 4-mm diameter were used). The resultant gels were stained with Coomassie Blue; p17a and p17b spots were excised and processed for cleavage with S. aureus V8 protease (two spots of each protein were used) or with CNBr (four spots of each protein were used). Generated peptides were separated by SDS-PAGE or Tricine/SDS-PAGE as described in the legend to Fig. 5, electrotransferred onto PVDF membranes, and subjected to N-terminal sequence analysis. The amino acid sequences thus obtained are shown in single-letter code and aligned with the sequences of porcine brain destrin (33) and cofilin (34) (only residues 1-90 of each protein are shown). SA and CN denote peptides obtained by cleavage with S. aureus V8 protease and CNBr, respectively; the numbers after SA and CN indicate their molecular masses. The letter x indicates a position where the residue could not be identified. Lowercase letters indicate amino acid residues assigned with some ambiguity. Asterisks indicate the positions where aligned residues are not identical between destrin and cofilin.



Fig. 5b shows peptide and phosphopeptide maps obtained by cleavage of pp17a, p17a, pp17b, and p17b with CNBr; the results support the view that p17a and p17b are distinct from each other and are the unphosphorylated forms of pp17a and pp17b, respectively. When peptides were generated from p17a and p17b on a larger scale and subjected to N-terminal sequence analysis, seven peptides from p17a and three peptides from p17b provided sequences as shown in Fig. 6; the N-terminal sequence of each peptide from p17a could be aligned within the sequence of porcine brain destrin, and the residues determined without ambiguity were identical with residues 4, 6, 10, 19-22, 24-30, 75-79, and 81-89 of destrin; the N-terminal sequence of each peptide from p17b could be aligned within the sequence of porcine brain cofilin, and the residues determined without ambiguity were identical with residues 3, 4, 6, 20-30, 35-37, 75-79, and 82-89 of cofilin. It is noted that, in the sequences of destrin and cofilin, the partial sequences corresponding to the peptide fragments derived from p17a and p17b are all preceded by methionine residues in accordance with the cleavage sites of proteins by CNBr (see Fig. 6). From these results, we have concluded that p17a and p17b are destrin- and cofilin-like proteins, respectively, and that pp17a and pp17b are their respective phosphorylated forms.

Effect of Cholinergic Stimulation on the Phosphorylation States of the Rat Parotid Destrin- and Cofilin-like Proteins

The rat parotid gland is innervated by both sympathetic and parasymphathetic branches of the autonomic nervous system, and cholinergic stimulation also induces amylase secretion (for review, see (1) ). In the course of this study, it was reported that the intracellular levels of 16- and 18-kDa rat parotid gland phosphoproteins decrease in response to not only beta-adrenergic but also cholinergic stimulation (the 16-kDa phosphoprotein is easily detected one, whereas the 18-kDa phosphoprotein is the one detected with some difficulty)(1) . We hence studied the effect of carbachol, a cholinergic agonist, on the phosphorylation states of the destrin- and cofilin-like proteins in rat parotid slices and observed that 10 µM carbachol induced their dephosphorylation (Fig. 7). It has, however, been reported that certain agents induce release of norepinephrine from sympathetic nerve endings remaining in slices(37) . Although such an effect of carbachol was reported to be weak(37) , we examined the effect of propranolol, a beta-adrenergic blocker, on the above effect of carbachol and confirmed that propranolol (50 µM) had no effect (data not shown). The muscarinic cholinergic antagonist atropine (1 µM) blocked the effect of 10 µM carbachol (data not shown). These results indicate that the phosphorylation states of the parotid destrin- and cofilin-like proteins are also regulated parasympathetically through muscarinic acetylcholine receptors.


Figure 7: Carbachol-induced dephosphorylation of the destrin- and cofilin-like proteins. P(i)-unlabeled rat parotid slices were incubated for 15 min without (panel a) or with (panel b) 10 µM carbachol. The partially purified pp17 fractions were prepared and subjected to two-dimensional PAGE, as described in the legend to Fig. 2. The resultant gel was stained with silver, and the relevant areas of the gel are shown. Closed arrows 1 and 2 indicate the spots of the phosphorylated destrin- and cofilin-like proteins, respectively; closed arrows 1` and 2`, those of the unphosphorylated destrin- and cofilin-like proteins, respectively. Open arrows indicate the spots of artifactual, probably carbamylated, products.




DISCUSSION

Destrin and cofilin are actin-binding proteins that induce depolymerization of F-actin(38, 39, 40) . Our present study has shown that nonstimulated rat parotid tissue contains partially phosphorylated destrin- and cofilin-like proteins and that they are dephosphorylated in response to beta-adrenergic or cholinergic stimulation. It was recently reported that human actin depolymerizing factor (ADF) has the same amino acid sequence as porcine destrin(41) . ADF was first isolated from chick embryo brains(42) , and chick ADF is 95% identical to porcine destrin(43, 44) . Interestingly, it was found that ADF is partially phosphorylated in chick myocytes and that the phosphorylated form is completely inactive in inducing depolymerization of F-actin (45) . The presence of phosphorylated cofilin has also been reported (46, 47, 48, 49, 50) , and it is suggested that phosphorylation of cofilin inhibits its interaction with actin(45) . In a resting parotid acinar cell, actin is present as a continuous ring under the plasma membrane; upon beta-adrenergic or cholinergic stimulation, this layer of cortical actin disappears in parallel with secretion of alpha-amylase(51) . It is hence possible that the phosphorylated forms of the rat parotid destrin- and cofilin-like proteins represent their inactive forms and that, through their dephosphorylation, beta-adrenergic or cholinergic stimulation of parotid cells induces disruption of cortical actin layers. This view is consistent with the reports that, in cultured primary astrocytes, N^6,O-dibutyryl cAMP leads to disruption of cortical actin networks probably through dephosphorylation of ADF (52) and that, in thyroid cells, thyrotropin-induced dephosphorylation of cofilin and destrin is implicated in disruption of actin-containing stress fibers(49) .

Cofilin contains a KKRKK sequence (residues 30-34) similar to the nuclear location signal sequence of SV40 large T antigen(34) . This sequence of cofilin indeed functions as a nuclear location signal(53) , and cofilin accumulates in the nucleus in response to certain stimuli such as heat shock or dimethyl sulfoxide treatment(34, 48, 54) . It is suggested that phosphorylation of cofilin masks the signal sequence and that its dephosphorylation in the cytoplasm exposes the signal sequence and causes translocation of cofilin to the nucleus(46) . In thyroid cells, however, it has recently been shown that thyrotropin-induced dephosphorylation of cofilin does not correlate with its nuclear translocation(49) . The KKRKK sequence is also present in destrin (residues 30-34) (33) and ADF (residues 30-34)(41, 43, 44) , but nuclear translocation of destrin or ADF has not yet been reported. Since the parotid destrin- and cofilin-like proteins probably contained this sequence, we explored their translocation under our experimental conditions and observed that neither of them was transferred from the 100,000 times g supernatant fraction to the particulate fraction in response to the beta-adrenergic or cholinergic stimulation (data not shown).

As shown in Fig. 3, the rat parotid destrin- and cofilin-like proteins were phosphorylated at serine residues. Results shown in Fig. 5b and Fig. 6allow us to estimate the phosphorylation sites in these proteins on the assumption that the amino acid sequences of the destrin- and cofilin-like proteins are essentially the same as those of porcine destrin and cofilin, respectively. CNBr cleavage of the phosphorylated destrin-like protein generated, as a major phosphopeptide, the 5.1-kDa P-labeled peptide (Fig. 5b, lanes 3 and 6) that probably had residue 2 at the N terminus (Fig. 6, CN5.1). From its estimated molecular mass and the amino acid sequence of porcine destrin (this protein consists of 165 amino acid residues and contains 5 methionine residues at positions 1, 18, 74, 100, and 115)(33) , the 5.1-kDa peptide seems to contain residues 2-74. The CNBr cleavage also provided the 16.7-kDa P-unlabeled CNBr peptide (Fig. 5b, lanes 3 and 6) probably containing residues 19-165 (Fig. 6, CN16.7). From these results, the major phosphorylation site(s) in the rat parotid destrin-like protein is likely to exist in residues 2-18. It is noted that only 1 serine residue, Ser-3, exists in this region of porcine destrin (Fig. 6) and that the 13.0-kDa P-labeled CNBr peptide from the phosphorylated destrin-like protein (Fig. 5b, lanes 3 and 6) is likely to contain residues 2-18 as well (Fig. 6, CN13.0). CNBr cleavage of the phosphorylated cofilin-like protein provided the P-unlabeled 18.3-kDa peptide (Fig. 5b, lanes 1 and 5) that probably had residue 19 at the N terminus (Fig. 6, CN18.3). Since porcine cofilin consists of 166 amino acid residues and contains 4 methionine residues at positions 1, 18, 74, and 115(34) , the 18.3-kDa peptide probably contains residues 19-166. It is hence likely that the phosphorylation site(s) of the rat parotid cofilin-like protein also exists in residues 1-18; it is noted that, in this region of porcine cofilin, there are 2 serine residues, Ser-3 and Ser-8 (Fig. 6). Identification of the exact phosphorylation sites in the rat parotid destrin- and cofilin-like proteins is in progress.

We previously reported that beta(1) adrenoreceptors are involved in regulation of the intracellular level of pp17, assuming that pp17 consisted of a single protein(5) . Since pp17 consisted of two phosphoproteins, we have confirmed the involvement of beta(1) adrenoreceptors in isoproterenol-induced dephosphorylation of each phosphoprotein (data not shown). It was also confirmed that 2 mMN^6,O-dibutyryl cAMP reproduced the effect of 10 µMl-isoproterenol on dephosphorylation of each phosphoprotein (data not shown).

In an attempt to obtain larger quantities of the phosphorylated destrin- and cofilin-like proteins, we incubated parotid slices with calyculin A, an inhibitor of protein phosphatases 1 and 2A(55) . Results were somewhat surprising. Calyculin A (100 nM) induced dephosphorylation of the destrin- and cofilin-like proteins, and their phosphorylated forms almost disappeared during 45 min of incubation (data not shown). These results suggest the following: (i) neither protein phosphatase 1 nor protein phosphatase 2A is directly involved in dephosphorylation of the destrin- and cofilin-like proteins; (ii) at least one of the two phosphatases is, however, involved in dephosphorylation of some unidentified phosphoprotein(s), which accumulates during incubation with calyculin A and induces dephosphorylation of the destrin- and cofilin-like proteins. It is likely that the unidentified protein exists in its dephosphorylated form in nonstimulated parotid cells and that beta-adrenergic stimulation induces its phosphorylation through activation of cAMP-dependent protein kinase. Further work is required to elucidate the exact mechanism leading to dephosphorylation of the destrin- and cofilin-like proteins and to clarify their physiological roles in parotid cells.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Aichi-Gakuin University and the Fujita Health University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-52-751-2561 (ext. 342); Fax: 81-52-752-5988.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; KRB, Krebs-Ringer bicarbonate medium; Mes, 4-morpholineethanesulfonic acid; NEPHGE, nonequilibrium pH gradient electrophoresis; CNBr, cyanogen bromide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PVDF, polyvinylidene difluoride; ADF, actin depolymerizing factor.


ACKNOWLEDGEMENTS

We thank Dr. Toshiharu Nagatsu (Fujita Health University) for his valuable advice and encouragement during this work.


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