(Received for publication, June 5, 1995; and in revised form, August 8, 1995)
From the
We have examined the mechanism for the selective down-regulation
of protein kinase C (nPKC
) in rat pituitary
GH
C
cells responding to thyrotropin-releasing
hormone (TRH) stimulation. Among various low molecular weight protease
inhibitors examined, only a cysteine protease inhibitor (calpain
inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the
down-regulation of nPKC
. Furthermore, the introduction of a
synthetic calpastatin peptide, an exclusively specific inhibitor of
calpain, into the cells also reduced the down-regulation, suggesting
the involvement of calpain among all the intracellular cysteine
proteases in this process. In accordance, we observed TRH-induced
translocation of m-calpain from the cytosol to the membrane and the
concomitant up-regulation of calpastatin isoforms; presumably, the
former represents activation of the protease initiating the kinase
degradation, while the latter constitutes a negative feedback system
protecting the cells from activated calpain. These results suggest that
in GH
C
cells, TRH mobilizes both protease
(m-calpain) and inhibitor (calpastatin) as a strictly regulating system
for the nPKC
pathway mediating TRH signals.
Protein kinase C (PKC) ()isozymes play pivotal roles
as major serine/threonine kinases in signal transduction cascades
involved in agonist-induced responses of various cells(1) . The
isozymes can be categorized into three groups: conventional PKC (cPKC),
novel PKC (nPKC), and atypical PKC (aPKC), based on their structural
and enzymatic properties(2) . Although their distinct tissue
distribution has suggested different functional roles for each
isozyme(2, 3) , their isozyme-specific functions in
physiological terms have not yet been fully resolved(4) . We
have focused our attention on the cellular function of PKC isozymes in
the responses elicited by thyrotropin-releasing hormone (TRH), a
hypothalamic hormone, in rat pituitary GH
C
cells and demonstrated that nPKC
provides a major
rate-limiting step in the secretion of prolactin (PRL) in response to
agonist stimulation (5, 6, 7, 8) .
GHC
cells possess at least six PKC isozymes, i.e.
and
II cPKC,
,
, and,
nPKC,
and aPKC
(5, 8) . The suppression of their
activities by PKC inhibitors results in a reduction in PRL secretion
evoked by TRH(6) , indicating a mediator role in
stimulus-response coupling. In accordance, TRH induces the
translocation of all isozymes except
from the cytosol to
membranes(8) . However, only the
isozyme undergoes
subsequent down-regulation(5, 7, 8) ,
indicating that this isozyme is the most extensively involved.
Consistently, the introduction of an expression vector carrying
nPKC
cDNA into the cells induces an increase in PRL secretion,
whereas increasing the amount of other isozymes,
,
II, and
, has no effect(8) . These data suggest that nPKC
among all the PKC isozymes present in GH
C
cells
plays a central role in the secretory process and that down-regulation
following translocation is a hallmark for the thorough activation of
the enzyme.
Intracellular down-regulation of PKC following activation in general seems to involve facilitated proteolysis rather than transcriptional suppression(9) . The protease(s) responsible for this process, however, have not yet been identified, although previous studies on phorbol ester-evoked cPKC down-regulation implies the involvement of calpain (10, 11) , serine proteases(12, 13) , and/or possibly other proteases (14) . Consequently, the physiological significance of this down-regulation remains uncertain. Possibly, the proteolysis of PKC may produce a signal affecting the cellular situation. For instance, an active kinase fragment may be produced from PKC by limited proteolysis (15) as an intermediate product in down-regulation, depending on the protease involved. This kinase that acts independently of cofactors, including phospholipids, would phosphorylate substrates that may not be accessible to intact PKC. Alternatively, the loss of a specific PKC activity may lead to an alteration in cellular functions. Due to our limited knowledge about the mechanism of down-regulation, these possibilities remain obscure.
In the present study, we aimed
to elucidate the mechanism of selective nPKC down-regulation in
GH
C
cells by identifying the responsible
protease as an initial step in addressing these questions in this
particular system.
To examine the localization of calpain, the cells were stimulated
with TRH for various periods, harvested in Ca/EGTA
buffer (pCa of 7, 20 mM Pipes/KOH, pH 7.2, 115 mM NaCl, 2 mM MgCl
, 1 mM CaCl
, 2.5 mM EGTA, 230 µM
leupeptin, 2 mM phenylmethanesulfonyl fluoride, 50 mM 2-mercaptoethanol), and fractionated as above. Cytosol and
membrane fractions were subjected to Western blot analysis using
anti-calpain isozyme antibodies. The localization of calpastatin was
examined in a similar manner except that ``homogenization
buffer'' was employed instead of Ca
/EGTA buffer.
Figure 1:
Effect of various protease inhibitors
on the TRH-induced down-regulation of nPKC in
GH
C
cells. Cells cultured in serum-free medium
were preincubated for 1 h with vehicle (lanes1 and 2), 100 µM phenanthroline (lane3), 10 µM phosphoramidon (lane4), 100 µM pepstatin (lane5), 10 µMN
-tosyl-L-lysyl chloromethyl ketone (TLCK) (lane 6), 10 µMN-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) (lane7), 100 µM leupeptin (lane8), 10 µM ALLNal (lane9), or 100 µM E-64d (lane10), followed by stimulation with (+) or
without(-) 300 nM TRH for 6 h in the presence of the
inhibitors. The cell extracts were subjected to Western blot analysis
using anti-nPKC
antibody. The arrow indicates the
relative molecular weight of nPKC
, 90,000, as confirmed using
recombinant nPKC
expressed in GH
C
cells(8) . The lowerpanel shows the
densitometric quantification of the Western blot
results.
The effect of ALLNal was dose dependent (Fig. 2A).
The concentration of inhibitor giving 50% inhibition was about 5
µM, consistent with previous
studies(27, 28) . The possibility that ALLNal
influences the translocation of nPKC from the cytosol to membranes
that occurs prior to down-regulation was excluded because the inhibitor
had no effect on the amount of nPKC
in the membrane fraction of
TRH-treated cells (Fig. 2B).
Figure 2:
Effect of ALLNal. PanelA, dose-dependent effect of ALLNal on the down-regulation
of nPKC. Cells preincubated with the indicated concentration of
ALLNal were stimulated with (+) or without(-) TRH for 6 h
and subjected to Western blot analysis using anti-nPKC
antibody.
Densitometric quantification as in Fig. 1is shown. PanelB, effect of ALLNal on the translocation of nPKC
.
Cells preincubated with 10 µM ALLNal were stimulated with
(+) or without(-) TRH for 3 min. The cells were then
collected in homogenizing buffer and fractionated as described under
``Experimental Procedures.'' Both the cytoplasm and membrane
fractions were subjected to Western blot analysis using anti-nPKC
antibody. The arrow indicates the relative molecular weight of
nPKC
(90,000).
Figure 3:
Effect of CS and scramble peptides on the
down-regulation of nPKC. PanelA, inhibition of
purified rabbit m-calpain by the CS and scramble peptides. The effects
of calpastatin (closedcircles) and scramble (opencircles) peptides at various concentrations
were assayed using [
C]casein as described under
``Experimental Procedures.'' PanelB,
incorporation of rhodamine-labeled CS peptide into cells. Cells were
grown on glass coverslips and treated with rhodamine-labeled CS peptide
in F-12 medium for 1 h. After washing with ice-cold
Mg
-, Ca
-free phosphate-buffered
saline, the cells were fixed with 4% paraformaldehyde for 15 min. Phase
contrast (upper) and fluorescence (lower) images were
observed using a Zeiss Axiophot microscope. Bar, 12 µm. PanelC, effect of CS and scramble peptides on the
down-regulation of nPKC
in cells. Cells were preincubated with
either buffer only (lanes1 and 2), 50
µM CS peptide (lane3), or 50 µM scramble peptide (lane4) for 1 h. Stimulation
with TRH and Western blot were performed as described in the legends to Fig. 1. The arrow indicates the relative molecular
weight of nPKC
(90,000). The lowerpanel shows
the densitometric quantification of the Western blot results. Three
independent experiments were performed to confirm the
observation.
We first examined the ability of the CS peptide
to inhibit purified calpain activity and to permeate into cells. In
test tubes, the CS peptide inhibited the proteolytic activity of
purified calpain with high affinity; 50% inhibition was achieved at a
peptide concentration of 20 nM (panelA). In
contrast, approximately 10-fold more scramble peptide was
necessary to suppress calpain activity. The permeability of the
synthetic peptide into cells was confirmed using a rhodamine-labeled
peptide (panelB); fluorescence was observed only in
cells incubated with the peptide. Although this observation does not
quantitate the actual concentration of CS peptide inside
GH
C
cells, it indicates that at least a
fraction of the extracellularly administered CS peptide entered the
cells and remained in cytoplasm.
Confirming the potent inhibitory
effect of the CS peptide on calpain and its cell permeability, we
examined its effect on intact cells (panelC). The CS
peptide significantly reduced the down-regulation of nPKC, whereas
the control scramble peptide had no effect. Because no proteases other
than calpain have been shown to be inhibited by calpastatin or
calpastatin peptide to our knowledge (32) , these data suggest
that it is indeed calpain, among various intracellular cysteine
proteases, that is involved in the proteolytic process.
Figure 4:
Calpain and calpastatin activities in
GHC
cells. PanelA, calpain
activity in cells. Cell extracts were fractionated by ion exchange
HPLC, and the enzyme activity was assayed as described under
``Experimental Procedures.'' The dottedline indicates the NaCl concentration gradient used for elution. PanelB, calpastatin activity in the cells. The assay
was performed as described under ``Experimental Procedures.''
Note the scale of activity is greater than in panelA. PanelC, Western blot analysis of
HPLC fractions using anti-µ-calpain, anti-m-calpain, and
anti-calpastatin antibodies as indicated. The bars to the right indicate the positions of marker proteins (Bio-Rad) and
their molecular weights (phosphorylase b, 106,000; bovine
serum albumin, 80,000).
The activity to inhibit calpain (panelB) and the anti-calpastatin antibody immunoreactivity showing 100-110-kDa bands on Western blotting (panelC) eluted identically, i.e. at 0.25 M NaCl, confirming the specificity of the antibody. Notably, the calpastatin activity present in the cells exceeded the sum of the calpain activities, suggesting that the protease activity is regulated rather stringently in these cells as previously reported in KB cells(22) .
Figure 5:
Effect of TRH on the cellular localization
of m- and µ-calpains in GHC
cells. PanelA, translocation of m-calpain from the cytosol
to membrane upon stimulation with TRH. Cells stimulated with TRH for
the indicated periods were fractionated in Ca
/EGTA
buffer (pCa of 7) and subjected to Western blot analysis using
anti-m-calpain antibody as described under ``Experimental
Procedures.'' PanelB, localization of
µ-calpain analyzed using anti-µ-calpain antibody as in panelA. The arrowheads show the 80-kDa
subunit of each isozyme. PanelC, the proportion of
membrane-associated calpains. The Western blot results shown in panelA (m-calpain, closedcircles)
and B (µ-calpain, opencircles) were
densitometrically quantified.
TRH also induced alterations in the calpastatin levels of
GHC
cells. The levels of both the cytoplasmic
and membrane-associated calpastatin molecules increased gradually
within 1 h (Fig. 6), and the increased levels were sustained for
more than 12 h (data not shown). The amount of calpastatin increased by
about 1.7-fold 1 h after stimulation as quantified by densitometry. In
particular, the appearance of the high molecular form, indicated in Fig. 6by the opentriangle, was notable (see
``Discussion'').
Figure 6: TRH-induced alteration in calpastatin levels. Cells stimulated with TRH for the indicated periods were fractionated and subjected to Western blot analysis using anti-calpastatin antibody as in Fig. 5. See the text for a discussion of the opentriangles. The bars indicate three forms of calpastatin. The positions of the molecular marker proteins are indicated to the right (phosphorylase b, 106,000; bovine serum albumin, 80,000; ovalbumin, 49,500; carbonic anhydrase, 32,500).
In the present study, we have shown that calpain inhibitors
with broad and narrow specificities, ALLNal and CS peptide, inhibit the
TRH-induced down-regulation of nPKC in GH
C
cells and that TRH causes both the translocation of m-calpain and
an increase in the calpastatin level. These observations indicate that
the calpain-calpastatin system is a component in TRH-induced signal
transduction and that calpain is the major protease involved in
down-regulation. This is the first demonstration of calpain involvement
in PKC down-regulation associated with physiological stimulus-response
coupling in contrast to previous studies employing phorbol ester or
calcium ionophore to induce down-regulation(10, 11) .
The present results, however, do not exclude the possible
involvement of other intracellular proteases in
down-regulation(12, 13, 14) . Because the
effect of the CS peptide was only partial (Fig. 3), a secondary
protease other than calpain may participate in the proteolytic process;
a candidate is proteasome, which was recently shown to be inhibited by
ALLNal(31, 35) . The presence of a secondary protease
is also implied by the fact that we do not observe an active nPKC
fragment corresponding to the catalytic fragment (Fig. 1)
produced in vitro by calpain-catalyzed limited
proteolysis(30) . Apparently, degradation of the active
fragment proceeds faster than its production, as shown previously in
the case of cPKC
(36) . Our observations also indicate that
nPKC
, which is enzymatically independent of calcium (30) ,
could be regulated by the calcium signaling cascade through the
proteolytic action of calpain. Whether or not proteolysis of nPKC
bears any specific physiological signals in these cells remains to be
elucidated.
The data showing the translocation of m-calpain, but not
µ-calpain, from the cytosol to membranes in response to TRH
treatment (Fig. 5) and suggesting the involvement of m-calpain
in nPKC down-regulation were rather unexpected because
µ-calpain, which requires lower calcium concentrations for
activation in vitro, has been considered as the more probable
candidate for intracellular proteolytic phenomena (37) .
However, the present observation agrees with our previous reports,
demonstrating that phorbol ester induces the synthesis and
translocation of m-calpain in COS and K562
cells(38, 39) . Possibly, the mobilization of
m-calpain is controlled by the PKC pathway. It is therefore likely that
in some cases, m-calpain is more involved in cellular stimulus-response
coupling than µ-calpain. There may exist an unknown cellular factor
associated with membranes that contributes to the specific activation
of m-calpain.
A possible reason for the selective down-regulation of
nPKC among other PKC isozymes (see the Introduction) may lie in
the difference in the time course of translocation to membrane. In
contrast to cPKC
and
II, which are translocated transiently
and are rapidly dissociated from the membrane within 1 min (data not
shown), the majority of nPKC
remains associated with the membrane
in a more sustained manner favoring colocalization with m-calpain.
Furthermore, this membrane association indicates that PKC exists under
activating conditions, in which the kinase is more susceptible to
proteolytic attack(15, 40) . Presumably, other PKC
isozymes escape from calpain action due to their relatively short
periods of association with membranes.
The up-regulation of
calpastatin isoforms, particularly of the high molecular weight form in
the membrane fraction, that is induced by TRH treatment (Fig. 6)
seems to be part of the cellular negative feedback system counteracting
activated calpain and protecting cellular components. Because the
excessive and random degradation of proteins associated with membranes
and the cytoskeleton would be highly toxic to cells and because the too
rapid proteolysis of calpain substrates including nPKC might
interfere with cellular functions, the strict regulation of calpain
activity by calpastatin should be vitally important. The three isoforms
with different relative molecular weights probably represent different
phosphorylation levels (41) or differential alternative
splicing(42) ; each isoform may possess distinct properties
affecting cellular localization and interaction with calpain. Each
might provide specific protection to a group of proteins that would
otherwise be proteolyzed by calpain. Both qualitative and quantitative
changes in calpastatin expression may play important roles, as shown
for differentiation and secretion in other
cells(43, 44, 45) . The precise role of
calpastatin in PRL secretion from GH
C
cells
remains to be elucidated. In conclusion, we propose that
physiologically stimulated nPKC
is strictly regulated at the cell
membrane by the calpain-calpastatin system.