(Received for publication, June 17, 1994; and in revised form, October 20, 1994)
From the
Phorbol esters cause long term activation of protein kinase C
(PKC) and frequently the down-regulation of PKC protein levels in
mammalian cells. Mammalian PKC-, -
, and -
down-regulate
in response to phorbol esters when expressed in Schizosaccharomyces
pombe. However, PKC-
does not down-regulate in S.
pombe, in contrast to the behavior of this isotype in mammalian
cells. Co-expression of PKC-
or -
with PKC-
in S.
pombe renders PKC-
susceptible to down-regulation. A protein
kinase defective form of PKC-
does not down-regulate efficiently
in S. pombe but, like PKC-
, is susceptible when
co-expressed with PKC-
or full-length PKC-
. Thus,
down-regulation is a consequence of the catalytic function of certain
PKC isotypes with other isotypes being affected in trans. PKC
down-regulation parallels a striking accumulation of vesicles in S.
pombe, suggesting a direct relationship between these events.
Protein kinase C (PKC) ()is the major cellular
receptor for phorbol esters(1) , and most PKC isotypes can be
activated by phorbol esters both in vivo and in
vitro(2, 3) . Phorbol esters promote the binding
of PKC to membranes analogous to the effect of the natural PKC
activator, diacylglycerol
(DAG)(3, 4, 5, 6) . However, phorbol
esters differ from DAG primarily by duration of action. DAG production
in the membrane (for example, through activation of phospholipase C,
which hydrolyzes inositol polyphosphates(7) ) is short-lived as
the DAG is rapidly metabolized(3) . Under these circumstances,
PKC transiently translocates from the cytosol to the membrane, whereas
phorbol esters are not metabolized and cause long term membrane
association of PKC (8) .
One consequence of phorbol ester
treatment, correlated with sustained association of PKC with membranes,
is the down-regulation of the PKC proteins
themselves(9, 10) . Certain natural agonists can also
cause the down-regulation of some PKC
isotypes(11, 12) ; down-regulation can follow
physiological stimuli, possibly through the sustained production of DAG
by phospholipases, including phospholipase D(12, 13) .
However, the exact mechanism for the down-regulation of PKC has not
been described. It has been proposed that
Ca-activated neutral proteases (calpains)
specifically degrade membrane-bound PKC, based on the in vitro sensitivity of PKC to various proteases(14, 15) .
Mammalian PKC isotypes vary in their susceptibility to
down-regulation when expressed in Schizosaccharomyces
pombe(16) . PKC- protein levels were not affected by
phorbol esters. PKC-
differs structurally from other isotypes by
lacking one of the two usual cysteine repeats (17) that
correlate with phorbol ester binding(18, 19) . This
isotype does not bind, is not activated by phorbol esters in
vitro(20) , and does not translocate to the membrane in
whole cells after phorbol ester
treatment(21, 22, 23) . Thus, it is expected
that PKC-
does not respond to phorbol esters in S. pombe.
PKC-
, -
, and -
did down-regulate as they do in
mammalian cells(3) . Unexpectedly, PKC-
did not
down-regulate in S. pombe. PKC-
binds phorbol esters (2, 3) and does down-regulate after phorbol ester
treatment in mammalian cells, although the rate of down-regulation can
be slower than for other PKC isotypes(24) . Thus,
down-regulation showed a PKC isotype specificity in S. pombe that differed from that seen in mammalian cells.
Here, it is shown that down-regulation requires kinase function of certain PKC isotypes, and isotypes that cannot themselves initiate the process can be affected by susceptible isotypes. Thus, the process is of a general nature but occurs in response only to certain isotypes. Furthermore, down-regulation parallels the accumulation of vesicles, suggesting an association between these events.
PKC-, -
, and -
down-regulate in response to
TPA in both S. pombe and mammalian cells(16) .
However, TPA does not cause the down-regulation of PKC-
in S.
pombe(16) in contrast to the effect in mammalian
cells(24) . All four of these enzymes are phorbol
ester-dependent protein kinases when purified from S.
pombe(16) . Thus, the ability to down-regulate does not
simply reflect PKC activity or phorbol binding but is specified by the
isotype. Most mammalian cells express several PKC
isotypes(2, 3) , and down-regulation of PKC-
in
these cells may result from the function of other isotypes. This
hypothesis was tested by expressing additional PKCs with PKC-
in S. pombe. S. pombe was used because of its low level of
endogenous TPA-dependent PKC activity(16) , which reduces the
possible effects of one isotype upon another.
A stable strain of S. pombe expressing PKC- (16) was
supertransformed with vectors to express PKC-
or -
. In vector
control cultures, PKC-
does not down-regulate in response to TPA (Fig. 1, lowerpanels, lanes1-4 and 9-12). Co-expression of
PKC-
did not lead to PKC-
down-regulation, and PKC-
itself did not down-regulate (Fig. 1, lanes13-16). In contrast, TPA treatment of cells
co-expressing PKC-
with PKC-
led to a marked decrease in
PKC-
protein levels in parallel with the down-regulation of
PKC-
(Fig. 1, lanes5-8; Table 1). Thus, PKC down-regulation is a consequence of the
action of only some PKC isotypes (
), and other isotypes (
)
can be affected in trans.
Figure 1:
PKC- down-regulates when
co-expressed with PKC-
. A stable cell line expressing PKC-
was transformed with vector, PKC-
, or PKC-
. Two
representative isolates are shown for each experimental condition.
Extracts were made after 30 h of culture in the presence or absence of
TPA (±). The positions of the PKCs are shown and also indicate
the antibody used on the duplicate membranes. Lanes1-4, PKC-
+ vector; lanes
5-8, PKC-
+ PKC-
; lanes 9-12,
PKC-
+ vector; lanes 13-16, PKC-
+
PKC-
. PKC-
and -
migrate with a molecular mass of 80
kDa, whereas PKC-
migrates as several species around 90
kDa.
It is unlikely that
down-regulation simply follows relocation of the enzyme to the membrane
since both PKC- and -
translocate to membranes after TPA
treatment in mammalian cells (2, 3) and both show
TPA-dependent kinase activity on extraction from S. pombe(16) . A function of PKC-
and not of PKC-
must
drive down-regulation. To establish whether this function involved the
active kinase domain of PKC-
, a kinase-defective form of PKC-
(PKC-
) was transformed into S. pombe. In contrast to
full-length PKC-
, the inactive form did not efficiently
down-regulate in response to TPA (Fig. 2, lanes1-4). However, the kinase-defective mutant did
down-regulate when co-expressed with full-length PKC-
(Fig. 2, lanes5-8; Table 1).
Thus, the response requires kinase activity, as distinct from the
physical presence, of certain isotypes (e.g.
). Other
isotypes (e.g.
) or indeed catalytically inactive mutants (e.g.
) can be induced to down-regulate, but
isotype-specific kinase function is required to initiate the process.
Figure 2:
PKC- down-regulates when
co-expressed with full-length PKC-
. A cell line expressing
PKC-
was transformed with vector or with full-length
PKC-
. Two isolates are shown for each condition. Western blot
analysis, using the PKC-
antibody, was performed on extracts from
cells cultured for 30 h in the presence or absence of TPA (±).
The positions of PKC-
and -
are shown. PKC-
has a molecular mass of 64 kDa. Lanes 1-4,
PKC-
+ vector; lanes 5-8, PKC-
+ PKC-
.
In addition to PKC-, the
and
isotypes also
down-regulate in S. pombe(16) . To ascertain if the trans dominant effect on down-regulation was specific to the
PKC-
/-
combination or applied more generally, the effect of
PKC-
on the down-regulation of PKC-
and -
was
determined. Co-expression of PKC-
with PKC-
markedly
increased the extent of down-regulation of the mutant PKC-
protein
after TPA treatment (Table 1). Similarly, down-regulation of
PKC-
was increased when co-expressed with PKC-
(Table 1). In both instances, PKC-
effectively
down-regulated after TPA treatment (data not shown). Thus, PKC-
,
like PKC-
, can drive the down-regulation of other isotypes
(PKC-
and -
). This trans dominant effect,
however, does not apply to all isotypes. PKC-
does not
down-regulate when expressed alone or with PKC-
in S. pombe (Fig. 3A, lanes1-4 and 5-8, respectively; Table 1). The lack of trans effect of PKC-
on PKC-
also establishes that
down-regulation is not due to a repressive effect of PKC-
on the
promoter used to express the PKCs or on PKC translation.
Figure 3:
PKC down-regulation is not due to promoter
or growth effects. A, PKC- does not cause the
down-regulation of PKC-
. The PKC-
cell line was transformed
with vector or PKC-
. Two isolates are shown for each condition.
Extracts were made from cells after culture for 30 h in the presence or
absence of TPA (±). Positions of PKC-
and -
are
indicated. The two duplicate membranes were analyzed with the PKC-
and -
antibodies, respectively. Lanes 1-4,
PKC-
+ vector; lanes 5-8, PKC-
+
PKC-
. B, growth arrest does not account for
down-regulation. A stable PKC-
S. pombe cell line (16) was cultured in nitrogen-replete (lanes
1-2) or nitrogen-deficient (lanes 3-4)
medium. After 24 h, the cultures were continued for a further 30 h in
the presence or absence of TPA (±). Extracts were then made and
examined by Western blot.
Down-regulation correlates with a TPA-dependent growth
inhibition(16) . Therefore, it was important to determine that
the loss of PKC protein does not simply follow growth arrest. PKC-
expressing S. pombe were cultured in nitrogen-deficient or
nitrogen-replete medium for 54 h. PKC-
is expressed at similar
levels in growth-arrested and growing cells (Fig. 3B, lanes1 and 3). Furthermore, in both
instances, treatment of these arrested cells with TPA led to PKC-
down-regulation (Fig. 3B, lanes2 and 4). Thus, down-regulation does not automatically follow growth
arrest but requires TPA treatment of specific isotypes.
Three
distinct PKC down-regulation responses are identified. PKC- is not
affected, as predicted by its unresponsiveness to TPA. PKC-
translocates to the membrane in mammalian cells but does not trigger
down-regulation, at least in S. pombe. Down-regulation can be
initiated by both PKC-
and -
, and, in addition to causing
homologous down-regulation, these isotypes can affect other members of
the PKC family (PKC-
and -
). Furthermore, since PKC
down-regulation is initiated by active kinase, the process appears to
constitute a direct negative feedback pathway through the destruction
of the active PKC. However, whether the ability of PKC to drive this
process is a negative or positive signal remains an open issue.
Inactive PKC- and -
mutants can down-regulate after TPA
treatment when expressed in mammalian cells(31, 32) .
This apparent contradiction with the results shown above for
PKC-
can be explained by the presence of endogenous mammalian
PKCs (which trigger down-regulation) in the cell types used.
Interestingly, others have shown that an inactive PKC-
mutant does
not down-regulate when expressed in mammalian cells(33) .
Different levels of endogenous PKCs capable of triggering and/or
sustaining down-regulation in the cell lines used in these experiments
could account for the conflicting results(31, 33) .
Efficient down-regulation would not occur in cell lines with low PKC
activity, a situation analogous to S. pombe cells that contain
low levels of endogenous PKC activity(16) .
PKC
isotype-specific functions are apparent since PKC- is clearly
distinguished from both PKC-
and -
in its inability to
down-regulate in isolation, despite these isotypes being similar in
terms of TPA regulation of activity. The implication is that the output
signals must differ. The specific molecular events have not been
elucidated, but a correlation was noted between the ability to
down-regulate and a marked vesicle accumulation in S. pombe (of which at least an element was endocytic)(16) . Cells
expressing PKC-
, -
, and -
did not accumulate
vesicles even after TPA treatment(16) . To determine if
down-regulation of PKC-
correlated with vesicle accumulation,
PKC-
cells supertransformed with vector, PKC-
, or
PKC-
, were examined after culture with or without TPA.
PKC-
cells transfected with the control vector did not
accumulate vesicles at any point. However, cells co-expressing
PKC-
accumulated vesicles in a phorbol ester-dependent fashion
with 42% of cells affected 3 h after addition of TPA (Fig. 4).
In cells where PKC-
was co-expressed with PKC-
, vesicles
were evident in 8.5% of cells, and this percentage was increased to 31%
after treatment with TPA for 3 h (Fig. 4). Electron micrographs
of representative cultures are shown in Fig. 5. Thus, TPA
treatment of cells co-expressing PKC-
or -
with
PKC-
led to vesicle accumulation in parallel with PKC-
mutant down-regulation. It is hypothesized that the up-regulation of
membrane transport processes, including endocytosis, is central to the
down-regulation of PKC. Activated PKCs associate with membranes;
increased endocytic activity would lead to an increased rate of traffic
to and from the plasma membrane, and the PKCs are presumably targeted
for degradation in vacuoles (or lysosomes) or perhaps exposure to
proteasomes(34, 35) .
Figure 4:
Expression of PKC- or -
in
PKC-
cells induces vesicle accumulation.
PKC-
-expressing S. pombe cells were transformed with
vector, PKC-
, or PKC-
. After culture for 44 h in minimal
selective medium (containing TPA for the final period as indicated),
the cells were fixed and examined by electron microscopy. Results are
expressed as the percentage of cells with visible vesicle accumulation.
Two samples of 100 random cells were examined for each condition, and
results are the mean and range. At no stage were vesicles seen in the
vector control cells.
Figure 5:
Representative cell sections of the
PKC- transformants. The cells (as used for Fig. 4) are
PKC-
+ vector (A) and PKC-
+
PKC-
(B). Both cells were treated with TPA for 3 h.
Co-expression of PKC-
induces vesicle accumulation. CW,
cell wall; G, Golgi apparatus; M, mitochondrion; N, nucleus; PM, plasma membrane; V, vacuole; VE, vesicles.
This hypothesis requires
membrane localization of PKC as a prerequisite for down-regulation.
PKC- does not down-regulate following TPA treatment, even if
co-expressed with PKC-
, possibly reflecting the fact that
PKC-
does not translocate to the membrane in response to
TPA(21, 22, 23) . However, a chimeric
molecule containing the regulatory domain of PKC-
fused to the
catalytic domain of PKC-
down-regulated when expressed in S.
pombe(36) . The PKC-
regulatory domain of this
chimera would specify membrane localization after TPA treatment. These
data support the suggestion that PKC down-regulation is a
membrane-driven process.
It is noted that vesicles have been seen
where down-regulation was not obvious. PKC- cells accumulate
vesicles (Fig. 4), but down-regulation occurs only after TPA
treatment. Since down-regulation by necessity reflects a balance
between the rates of synthesis and destruction, the increased rate of
breakdown due to increased vesicle traffic after TPA treatment (Fig. 4) would lead to a net loss of PKC-
protein in these
cells. This effect is probably accentuated because phorbol esters cause
a tight association of PKC with membranes (3, 6) ; the
PKCs would remain attached to membranes up to and including the time of
sorting to degradative compartments. The idea that PKC down-regulation
is part of a nonspecific degradative process is supported by recent
work showing that specific proteases cannot account for
down-regulation(37) .
In summary, PKC down-regulation is a function of the kinase activity of certain isotypes. Isotypes that cannot initiate the down-regulation process can be affected in trans, predicting that some PKC isotypes contribute to the control of action of other PKCs. The strict correlation between the up-regulation of vesicle traffic and the ability to down-regulate suggests these two phenomena are related and that PKC down-regulation occurs by the same general process as PKC-mediated receptor internalization and down-regulation.