©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase C (PKC)-induced PKC Down-regulation
ASSOCIATION WITH UP-REGULATION OF VESICLE TRAFFIC (*)

(Received for publication, June 17, 1994; and in revised form, October 20, 1994)

Nigel T. Goode (1)(§) M. A. Nasser Hajibagheri (2) Peter J. Parker (3)(¶)

From the  (1)Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom and the (2)Electron Microscopy Unit and (3)Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, P. O. Box 123, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Protein kinase C (PKC) (^1)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.


MATERIALS AND METHODS

Cell Strain and Culture

The S. pombe strain h, ade6.704, leu1-32, ura4-D18 was cultured in synthetic Edinburgh minimal medium supplemented with 2% (w/v) glucose and 100 µg/ml adenine but free of leucine and uracil (minimal selective medium) to ensure maintenance of the expression plasmids(25) . Transformants (by electroporation using the manufacturer's instructions (Bio-Rad Pulse Controller)) were spread onto minimal selective plates containing 1 M sorbitol and 1 µM thiamine. Individual colonies were picked into minimal selective medium containing thiamine and cultured overnight until late log phase. After extensive washing, the cultures were diluted into fresh minimal selective medium without thiamine to allow expression of the PKC. After a further 24 h, cultures were re-diluted into medium with or without 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA).

Expression Plasmids

The PKCs were expressed in S. pombe using the pREP series of plasmids in which expression is controlled by the nmt1 promoter, which is tightly repressed by thiamine(26, 27) . Since the expression of some mammalian PKCs in S. pombe produces a marked growth inhibition(16) , this property of repression was required for routine strain propagation. All subcloning methods used were as described(28) . PKC-, -, and - were subcloned into the pREP3X vector, which contains the LEU2 gene(16) . PKC-Delta was constructed by excising the whole of the C3 and part of the C4 domain of the kinase from pREP3X-PKC-(16) . The PKCs for the supertransformations were subcloned into the pREP4 vector in which selection occurs via the URA4 gene(26) . The PKC- cDNA was subcloned from sp64-PKC- (29) as a BamHI-filled HindIII fragment into BamHI-SmaI-digested vector. PKC- was excised from pREP3X-PKC- as an SphI-BamHI fragment and ligated into pREP4 digested with the same enzymes. The PKC- cDNA was excised as an XbaI fragment from pBluescript-PKC- (22) and, after filling the recessed ends, was ligated into BalI-SmaI-digested pREP4.

Protein Extracts and Western Blotting

Denatured protein extracts were made from equivalent numbers of cells cultured in the presence or absence of TPA (without thiamine) for 30-36 h essentially as described(25) . SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described elsewhere(24, 30) . Polyclonal rabbit isotype-specific PKC-, -, and - antibodies (22, 24) were followed by donkey anti-rabbit horseradish peroxidase-conjugated antibodies and the ECL detection system (Amersham International). The monoclonal PKC- antibody, 36G9(19) , was followed by sheep anti-mouse horseradish peroxidase-conjugated antibody and ECL. Autoradiographs were scanned by densitometer (Pharmacia Biotech Inc.) to quantify relative protein levels.

Electron Microscopy

Cells were cultured in minimal medium without thiamine for 44 h. At 24 h, 3 h, and 30 min before collection, TPA was added to separate cultures. After the 44-h incubation, 10 ml of each culture was prepared for electron microscopy as described(16) . The sections were examined with a Jeol 1200 FX electron microscope.


RESULTS AND DISCUSSION

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-Delta) 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. Delta) can be induced to down-regulate, but isotype-specific kinase function is required to initiate the process.


Figure 2: PKC-Delta down-regulates when co-expressed with full-length PKC-. A cell line expressing PKC-Delta 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-Delta and - are shown. PKC-Delta has a molecular mass of 64 kDa. Lanes 1-4, PKC-Delta + vector; lanes 5-8, PKC-Delta + 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-Delta and - was determined. Co-expression of PKC- with PKC-Delta 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 -Delta). 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-Delta 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-alpha 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-Delta 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-alpha 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-Delta, -, and - did not accumulate vesicles even after TPA treatment(16) . To determine if down-regulation of PKC-Delta correlated with vesicle accumulation, PKC-Delta cells supertransformed with vector, PKC-, or PKC-, were examined after culture with or without TPA. PKC-Delta 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-Delta, 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-Delta 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-Delta cells induces vesicle accumulation. PKC-Delta-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-Delta transformants. The cells (as used for Fig. 4) are PKC-Delta + vector (A) and PKC-Delta + 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.


FOOTNOTES

*
This work was supported in part by a Royal Society research grant. 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 reprint requests should be addressed.

To whom correspondence should be addressed. Tel.: 44-71-269-3450; Fax: 44-71-269-3092.

(^1)
The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; TPA, 12-O-tetradecanoylphorbol-13-acetate.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.