(Received for publication, October 25, 1994; and in revised form, February 1, 1995)
From the
We previously reported that when P
-loaded rat parotid slices are incubated with
the
-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
-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.
In the rat parotid gland, -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
-adrenergic
agonist-induced amylase secretion, and endogenous protein substrates
for this enzyme have extensively been studied with
P
-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
-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
-adrenergic
stimulation-induced dephosphorylation, and discuss their possible
involvement in parotid amylase secretion.
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 NaVO
, 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
g for 90 min; the supernatant fluids were collected,
stored at -30 °C, and referred to as ``100,000
g supernatant fractions.''
To each 100,000 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
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
VO
, 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
HCO
for a total of 30 h at room temperature, and lyophilized. The
resultant samples were each dissolved in 0.4 ml of H
O and
mixed with 3.6 ml of ethanol; the mixtures were stored overnight at
-30 °C and centrifuged at 2,000
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 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
/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
/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
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.
Two-dimensional PAGE was done as
described in (15) with the following modifications: (i)
nonequilibrium pH gradient electrophoresis (NEPHGE) gels (3 115
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.
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 g supernatant fraction (3 mg of protein) prepared from nonstimulated
P
-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
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
g supernatant fraction from isoproterenol-stimulated
P
-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.
Figure 2:
Analysis of pp17 by two-dimensional PAGE. P
-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
; slices
were prepared with ordinary KRB instead of P
-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
-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
-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
-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
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.
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
-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
-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.
Figure 7:
Carbachol-induced dephosphorylation of the
destrin- and cofilin-like proteins. P
-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.
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 -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
-adrenergic or cholinergic stimulation, this layer of cortical
actin disappears in parallel with secretion of
-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,
-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
,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 g supernatant fraction to the particulate
fraction in response to the
-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
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
adrenoreceptors in
isoproterenol-induced dephosphorylation of each phosphoprotein (data
not shown). It was also confirmed that 2 mMN
,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 -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.