?-Adrenergic stimulation of the rat parotid gland induces exocytotic amylase secretion from the acinar cells through a cAMP-mediated pathway involving cAMP-dependent protein kinase. During a search of endogenous protein substrates for the kinase, we observed that ?-adrenergic stimulation induces not only phosphorylation of several endogenous proteins but also dephosphorylation of two 17-kDa soluble phosphoproteins. We identified those two proteins as destrin- and cofilin-like proteins from their partial amino acid sequences and further observed that cholinergic stimulation also induces their dephosphorylation. Both destrin and cofilin are actin-binding proteins that depolymerize F-actin; they are structurally related (sequence identity, about 70%), and destrin is also called actin-depolymerizing factor. Each protein exists in both phosphorylated and unphosphorylated forms, and the unphosphorylated form of each is active in depolymerizing F-actin. As ?-adrenergic or cholinergic stimulation of parotid cells also induces disruption of the cortical actin layers in parallel with amylase secretion, dephosphorylation of the destrin- and/or cofilin-like protein(s) might be involved in the disruption of the cortical actin layers. At present, little is known about the exact regulatory mechanisms for their phosphorylation. As a step toward understanding better the mechanism as well as their physiological roles in parotid cells, we here determined their complete amino acid sequences and phosphorylation sites.

Male Wistar rats (about 200 g, fed ad libitum) were anaesthetized with diethyl ether and killed by decapitation; parotid glands were collected and stored at ?80°C. The two proteins were partially purified from the glands as described previously for preparation of the partially purified pp17 fraction from non-stimulated rat parotid slices, and subjected to the two-dimensional PAGE procedure of O’Farrell et al. (1977)with modifications. The first-dimension gels were 4 mm dia. and loaded with samples each containing about 0.8 mg of protein. In some cases, proteins were electrotransferred after two-dimensional PAGE on to PVDF membranes (Immobilon-P^SQ; Millipore, Bedford, MA, U.S.A.) by the method of Kyhse-Andersen (1984)with modifications. The spots of the phosphorylated and unphosphorylated forms of the two proteins were excised from Coomassie blue-stained gels or PVDF membranes. Unless otherwise stated, experiments were done with their unphosphorylated forms that predominate even in non-stimulated parotid cells. (From the data obtained upon amino acid sequencing of the peptides derived from the two proteins, it is now known that the one spot of the unphosphorylated destrin-like protein contained at least 40 pmol of the protein, and that one spot of the unphosphorylated cofilin-like protein contained at least 20 pmol of the protein. Data are not shown.) The 6 to 12 pieces of gel containing the same protein (at least 200 pmol) were then placed on an agarose (Agarose 421F; Funakoshi, Tokyo, Japan) tube gel (10×35 mm). Each protein in the gels was electrophoretically transferred to the agarose gel with the stacking buffer system in the SDS–PAGE procedure of Fling and Gregerson (1986). (Electrophoresis was at 3 mA/gel until the Coomassie-blue band reached 25 mm below the gel top. At this point, the SDS front and the band of the tracking dye bromophenol blue were slightly below and above, respectively, the Coomassie-blue band. Proteins were stacked between the SDS front and the bromophenol-blue band.) The gels were cut 1 mm below the SDS front and 1 mm above the bromophenol-blue band. The middle segments were placed in tubes each containing nine volumes of 20 mM Tris–HCl (pH 7.5) supplemented with 0.1% SDS, 0.1% 2-mercaptoethanol and 0.1 mM EDTA, and melted at about 100°C. The resultant solutions were frozen, kept for at least 2 h at ?80°C, thawed, and centrifuged at 4°C at 15,000 g for 20 min. The supernatant solutions were dialysed against two changes of 60 volumes of 10 mM NH₄HCO₃ containing 0.1% SDS and 0.1% 2-mercaptoethanol for a total of 24 h at room temperature and lyophilized. Each dried sample was dissolved in one gel volume of H₂O and mixed with nine volumes of ethanol. The mixtures were stored overnight at ?30°C and centrifuged at 2000 g for 20 min at room temperature. The precipitates were washed with 90% ethanol and dried.

In the initial experiments, the purified proteins were reduced on membranes with tributylphosphine (Kanto Chemicals, Tokyo, Japan) and alkylated with 4-vinylpyridine (Wako, Osaka, Japan) or reduced in solution with Tris(2-carboxyethyl)phosphine (Boehringer Mannheim, Mannheim, Germany) (see below) and alkylated with 4-vinylpyridine. However, the amino acid sequencing and mass spectrometry of the peptides from the resultant proteins revealed that most Cys residues in them reacted with residual unpolymerized acrylamide during the preparative two-dimensional PAGE and were converted to Cys-S-?-propionamide residues (data not shown); similar observations have been reported. In later experiments, we therefore treated the reduced proteins with acrylamide (see below) to ensure their complete homogeneous alkylation. Acrylamide was purified with the mixed-bed resin AG 501-X8 (Bio-Rad, Hercules, CA, U.S.A.).

For the proteins recovered on the PVDF membranes, Coomassie blue was first removed with chloroform:methanol (9:1, v/v), as described by Wong et al. (1993). Each protein (3 to 6 spots) was then reduced in a tube containing 200 ?l of 0.2 M Tris–HCl (pH 7.5) supplemented with 7 M guanidine HCl, 4 mM EDTA, and 2 mM Tris(2-carboxyethyl)phosphine for 2 h at 37°C in the dark under N₂. Each tube thereafter received 2 ?l of 2-mercaptoethanol and 8 ?l of 3.5 M acrylamide and was incubated for 2 h at 37°C in the dark under N₂. The membrane pieces in each tube were then washed with H₂O (5×1 ml), dried, washed with 1-chlorobutane (2×150 ?l), and dried.

For the proteins extracted from gels, each protein derived from 6 to 12 spots was dissolved in 100 ?l of 0.35 M Tris–HCl (pH 7.5) containing 1% SDS, 4 mM EDTA, and 2 mM Tris(2-carboxyethyl)phosphine and incubated as above. Each sample received 1 ?l of 2-mercaptoethanol and 4 ?l of 3.5 M acrylamide, and was incubated as above. At the end of incubation, each sample received 100 ?l of 2 M dithiothreitol in 0.5 M Tris–HCl (pH 7.5) and was incubated for 30 min at 37°C in the dark under N₂. The resultant samples were dialysed against two changes of 1000 volumes of 10 mM NH₄HCO₃ containing 0.1% SDS and 0.1% 2-mercaptoethanol for a total of 24 h at room temperature and lyophilized. Each dried sample was dissolved in 100 ?l of H₂O, and the proteins were precipitated with ethanol as described above.

The membrane pieces carrying alkylated protein spots were wetted with methanol, rinsed with H₂O, incubated for 20 min at room temperature in a solution (70 ?l/piece) consisting of 0.1% acetic acid, 0.25% polyvinylpyrrolidone (M_r 40,000; Sigma, St Louis, MO, U.S.A.) and 0.1% methionine, and washed with H₂O (5×300 ?l/piece) and then with 10% acetonitrile (2×50 ?l/piece). The proteins on the membranes were digested in a solution (20 ?l/piece) consisting of 0.1 M NH₄HCO₃, 10% acetonitrile, and 4 ?g/ml of trypsin (modified trypsin; Promega, Madison, WI, U.S.A.) or endoproteinase Asp-N (Boehringer Mannheim) at 37°C for 20 h, and the supernatants were collected. The membrane pieces were washed with 10% acetonitrile (20 ?l/piece) and then with dimethylsulphoxide (10 ?l/piece) (dimethylsulphoxide extraction was not done for tryptic peptides). The supernatant and the washings from each tube were mixed, and the mixtures were dried.

For Asp-N digestion in solution, each alkylated protein derived from 6 to 12 spots was incubated for 18 h at 37°C in 100 ?l of 0.1 M NH₄HCO₃ containing 10% acetonitrile and 4 ?g/ml Asp-N.

For CNBr cleavage in solution, each alkylated protein derived from 6 to 12 spots was preincubated in 30 ?l of 0.5 M NH₄HCO₃ containing 5% 2-mercaptoethanol for 24 h at room temperature under N₂ (methionine sulphoxide residues were reduced during this preincubation), lyophilized after addition of 720 ?l of H₂O, and incubated in 50 ?l of 70% HCOOH containing 1% CNBr (Wako) for 20 h at 20°C in the dark under N₂. The samples thus prepared were dried.