Signalling Pathways Regulating the Dephosphorylation of Ser729 in the Hydrophobic Domain of Protein Kinase Cepsilon upon Cell Passage*

Karen EnglandDagger, John Watson§, Gary Beale, Maria Warner, James Cross, and Martin Rumsby

From the Department of Biology, University of York, York YO10 5DD, United Kingdom

Received for publication, October 16, 2000, and in revised form, November 22, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have recently demonstrated that in quiescent fibroblasts protein kinase C (PKC) epsilon 95 is phosphorylated at Ser729, Ser703, and Thr566 and that upon passage of quiescent cells phosphorylation at Ser729 is lost, giving rise to PKCepsilon 87. Ser729 may be rephosphorylated later, suggesting cycling between PKCepsilon 87 and PKCepsilon 95. Here we show that the dephosphorylation at Ser729 is insensitive to okadaic acid, calyculin, ascomycin C, and cyclosporin A, suggesting that dephosphorylation at this site is not mediated through protein phosphatases 1, 2A or 2B. We demonstrate that this dephosphorylation at Ser729 requires serum and cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine, and Ro-31-8220. These results suggest that the phosphorylation status of Ser729 in the hydrophobic domain at Ser729 is regulated independently of the phosphorylation status of other sites in PKCepsilon , by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser729.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The PKC1 family of related phospholipid-dependent serine/threonine kinases are involved in the control of many cellular processes, including cell growth and differentiation (1, 2). To date 11 PKC isoforms have been identified: the conventional PKCs (alpha , beta I, beta II, and gamma ), which are regulated by calcium and diacylglycerol, the novel PKCs (delta , epsilon , and theta ), which are calcium independent but dependent upon diacylglycerol, and the atypical PKCs (lambda , iota , and zeta ), which are both diacylglycerol- and calcium-independent. Another isoform, PKCµ, is known as protein kinase D (3).

Recent work from many groups has highlighted the importance of phosphorylation in the regulation of PKC activity (4-7). Initially phospholipid-dependent kinase 1 phosphorylates a conserved site on the lip of the catalytic region that corresponds to Thr566 in PKCepsilon (8, 9). Phosphorylation at this site is important for the enzymatic activity of PKC (4). Two further phosphorylation sites have been identified in the C-terminal region of the enzyme at the turn and hydrophobic motifs (4-6). Phosphorylation at the turn motif (Ser703 in PKCepsilon ) is believed to be mediated through autophosphorylation (5). There is some debate as to whether the hydrophobic site (Ser729 in PKCepsilon ) becomes phosphorylated as a result of autophosphorylation or by a separate kinase. Phosphorylation at this hydrophobic site may be modulated by PKCzeta and appears to be sensitive to rapamycin (10, 11). Although phosphorylation at these two C-terminal sites is not essential for the catalytic activity of PKC, they seem to regulate the stability of the enzyme (12-16).

PKCepsilon is the only isoform that has oncogenic potential (17, 18) that may be mediated through its interaction with Raf 1 kinase (19, 20). PKCepsilon is also unique in having actin and Golgi-binding domains (21-25). PKCepsilon from fibroblasts migrates on SDS-PAGE as two distinct forms, with molecular sizes of 95 and 87 kDa (PKCepsilon 95 and PKCepsilon 87) that differ in their intracellular localization. In quiescent cells the PKCepsilon 95 form predominates, whereas after passage PKCepsilon 87 becomes the major species. We have recently reported that these forms differ in their phosphorylation status at Ser729 (26). Thr566 and Ser703 are phosphorylated in both these forms of PKCepsilon , and the protein has complete N and C termini (26). The formation of PKCepsilon 87 upon cell passage is not due to new protein synthesis. We have therefore suggested that a phosphatase responsible for dephosphorylation of Ser729 is activated upon cell passage. Removal of the Ser729 phosphate from the hydrophobic domain may reduce the stability of the enzyme, rendering it more accessible to phosphatase attack and potentially to degradation (27). Alternatively, the change in localization of PKCepsilon on passage may make it accessible to a Ser729 phosphatase. Therefore regulation of phosphorylation at Ser729 may prove to be yet another level of control for PKC.

The regulation of how PKC becomes dephosphorylated has not been well studied. Most work has been carried out using TPA to induce activation that leads to dephosphorylation and degradation. Our system involving cell passage allows us to examine the control of the putative Ser729-specific phosphatase rather than to look at the complete dephosphorylation of the protein. Our results suggest that dephosphorylation of PKCepsilon is a two-stage process. A specific phosphatase removes the phosphate at Ser729 followed by either the removal of other phosphate groups or rephosphorylation at Ser729 and recycling of the enzyme to the 95-kDa form.

In this study we have examined how the dephosphorylation of PKCepsilon at Ser729 in 3T3 and 3T6 fibroblasts is regulated. We present evidence that the dephosphorylation of Ser729 is not mediated by protein phosphatases 1, 2A, and 2B but is dependent upon serum and cell readhesion, and that MAPK and mTOR (mammalian target of rapamycin) pathways are involved. PKC is also important in regulating the phosphorylation status of Ser729.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

Cells were from the European Collection of Animal Cell Cultures (Porton Down, UK) and the William Dunn Cell Bank (Oxford, UK). Cell culture plastic was from Nunc (Life Technologies, Inc.). All other cell culture reagents were from Life Technologies, Inc. except serum, which was from PAA Laboratories (Linz, Austria). All other chemicals were from Sigma, and chemical inhibitors were from Calbiochem (Nottingham, UK) unless otherwise stated. Nitrocellulose membrane (Hybond C) was from Amersham Pharmacia Biotech. Dried milk powder was Marvel (Premier Beverages, Stafford, UK). BCA reagents were from Pierce.

The polyclonal PKCepsilon and PKCdelta antibodies used for Western blotting and immunoprecipitations were generated to the C-terminal peptide sequence by Professor N. Groome (Oxford Brookes University, UK) as previously described (26). Peroxidase-conjugated secondary antibodies were from Sigma.

Methods

Tissue Culture-- 3T3 and 3T6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a humidified incubator at 5% CO2 as described previously (26). Cells were passaged by rinsing with Tris saline, followed by release with Tris trypsin. Cells were resuspended in fresh medium as above, plated into fresh tissue culture flasks, and allowed to settle for 15 min before harvesting unless otherwise stated.

Inhibitor Studies-- Cells were treated with inhibitors at concentrations described in the text. Inhibitors were dissolved in Me2SO unless otherwise stated and effects compared with Me2SO control treated cells. Solvent concentrations were never greater than 0.01%. Quiescent cells were treated for 30 min before passage, and fresh inhibitor was added after passage. PKCepsilon and PKCdelta translocation inhibitor and activator peptides coupled to anttenepaedia carrier protein were generously supplied by Professor D. Mochly-Rosen (Stanford, CA).

Plasmids and Cell Transfection-- Hemagglutinin-tagged pCH3delta RD was generously donated by Professor S. Jaken (University of Vermont). 3T3 and 3T6 cells were transiently transfected with Superfect (Qiagen) and were analyzed after 48 h, were quiescent, or were passaged. Transfection was monitored by Western blotting for the hemagglutinin tag. PKCdelta antisense was created through cloning the full-length PKCdelta into the pZeo SV vector (Invitrogen). Stable transfections were selected using Zeocin (Invitrogen), and individual colonies were expanded and analyzed by Western blotting. In all cases results were compared with mock transfected cells.

Western Blotting-- Cells were scraped from flasks into lysis buffer (50 mM Tris, 0.5 mM EDTA, 2 mM EGTA, pH 7.5, with 0.5% Nonidet P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM [4-(2-aminoethyl)benzenesulfonylfluoride, 50 mM NaF, 5 mM sodium pyrophosphate, 10 µM sodium orthovanadate). Protein concentration was determined using the BCA assay (Pierce). Laemmli loading buffer was added, and equal amounts of protein (30 µg) were run on SDS-PAGE gels, transferred to nitrocellulose, and probed for PKCepsilon as described previously (26).

Immunoprecipitation-- Cells were harvested in immunoprecipitation buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% SDS, 1% Triton X-100, 1% deoxycholic acid, 0.2 mM [4-(2-aminoethyl)benzenesulfonylfluoride, 50 mM NaF, 5 mM sodium pyrophosphate, 10 µM sodium orthovanadate). Samples were precleared with protein A-Sepharose at 4 °C for 1 h on a rotary stirrer. Equal amounts of cell protein were incubated with anti-PKCepsilon antibody overnight (1:250), and this was recovered with protein A-Sepharose. After washing with immunoprecipitation buffer, the pellet was resuspended in 10% SDS and Laemmli loading buffer and resolved by SDS-PAGE.

[35S]Methionine Incorporation-- Cells were incubated in methionine-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) for 1 h. 3.7 Mbq of [35S]Met/Cys (Promix; Amersham Pharmacia Biotech) was then added for 2 h. Cells were then passaged into fresh medium and flasks, and 3.7 Mbq [35S]Met/Cys was retained in the medium. Cells were harvested at different time points, and PKCepsilon were immunoprecipitated as described above. Samples were resolved by SDS-PAGE, and gels were fixed in isopropanol/acetic acid/H20 (25/20/65, v/v) for 1 h and then incubated for 15 min in Amplify (Amersham Pharmacia Biotech) before drying and exposing to x-ray film. All data shown are typical of at least three independent experiments.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PKCepsilon 87 Is Formation upon Cell Passage Is Not the Result of New Protein Synthesis-- In quiescent cells PKCepsilon 95 predominates, whereas 15 min after passage into serum with readhesion PKCepsilon 87 becomes the major form (Fig. 1A). We have recently shown that PKCepsilon 95 and PKCepsilon 87 differ in their phosphorylation at Ser729 and that the formation of PKCepsilon 87 is most probably the result of dephosphorylation of PKCepsilon 95 at Ser729 (26). It is likely that the apparent increase in total PKCepsilon protein (Fig. 1A) is the result of increased immunoreactivity of our antibody with PKCepsilon 87 compared with PKCepsilon 95 since the polyclonal PKCepsilon antibody used in these studies is raised against the C-terminal region of PKCepsilon (-NQEEFKGFSYFGEDLMP), which includes Ser729. This observation applies to other Western blots. This is confirmed by [35S]Met/Cys incorporation studies that reveal that no new PKCepsilon synthesis occurs within 15 min of passage although synthesis is detected 48-72 h after passage (Fig. 1B). Western blots show that PKCepsilon 95 reappears and becomes the predominant PKC form within 1 h after cell passage (Fig. 1A). Because there is no synthesis of PKCepsilon over this time period (Fig. 1B), this suggests that PKCepsilon 95 detected within 1 h of passage is derived through the rephosphorylation of PKCepsilon 87.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 1.   A, Western blot for PKCepsilon from 3T3 cells. Lane 1, quiescent cells; lane 2, 15 min after passage; lane 3, 1 h after passage; lane 4, 2 h after passage; lane 5, 4 h after passage; lane 6, 8 h after passage. PKCepsilon 95 and PKCepsilon 87 are indicated. B, fluorogram of PKCepsilon immunoprecipitated from [35S]Met/Cys-labeled 3T3 cells. Lane 1, 15 min after passage; lane 2, 30 min after passage; lane 3, 1 h after passage; lane 4, 4 h after passage; lane 5, 8 h after passage; lane 6, 12 h after passage; lane 7, 24 h after passage; lane 8, 48 h after passage. PKCepsilon is indicated.

Serum and Readhesion Are Required for the Formation of PKCepsilon 87 upon Cell Passage-- When quiescent cells are passaged into serum-free medium and allowed to adhere for 15 min, no formation of PKCepsilon 87 is observed (data not shown). However, when cells are passaged into increasing concentrations of serum, PKCepsilon 87 is formed in increasing amounts (Fig. 2A). This result shows that some factor(s) in serum is/are necessary for the formation of PKCepsilon 87 upon cell passage. Fibroblasts at 70% confluency contain both PKCepsilon 95 and PKCepsilon 87 (Fig. 2B). When these cells are serum-starved for 24 h, to promote entry into G0 (28), PKCepsilon 95 becomes the predominant form (Fig. 2B), as is observed in cells grown to confluency and quiescence (Fig. 2A). Readdition of serum to serum-starved cells in G0 does not promote the formation of PKCepsilon 87, although passage of these cells does (Fig. 2B). This is also the case if fresh serum is added to quiescent fibroblasts (results not shown). These findings suggest that serum is necessary but not sufficient for the formation of PKCepsilon 87 upon cell passage.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2.   A, Western blot for PKCepsilon from 3T3 cells. Lane 1, quiescent; lane m, marker lane; lane 2, passaged into medium containing 1% serum; lane 3, passaged into medium containing 10% serum; lane 4, passaged into medium containing 20% serum. PKCepsilon 95 and PKCepsilon 87 are indicated. B, Western blot for PKCepsilon from 3T3 cells. Lane 1, 70% confluent; lane 2, serum-starved for 24 h; lane 3, passaged, after serum starvation for 24 h; lane 4, cells to which fresh medium has been added for 15 min, after serum starvation for 24 h. PKCepsilon 95 and PKCepsilon 87 are indicated. C, Western blot for PKCepsilon in 3T3 cells. Lane 1, quiescent; lane 2, passaged; lane 3, passaged into flasks coated with 12 mg/ml poly-HEME to prevent adherence; lane 4, passaged into flasks coated with 24 mg/ml poly-HEME to prevent adherence. PKCepsilon 95 and PKCepsilon 87 are indicated. D, Western blot for PKCepsilon from 3T3 cells. Lane 1, quiescent; lane 2, quiescent, treated for 15 min with 2 µM nocodazole; lane 3, quiescent, treated with 200 nM cytochalasin D for 15 min. PKCepsilon 95 and PKCepsilon 87 are indicated.

Readhesion is also necessary for the formation of PKCepsilon 87 upon passage. This is shown by the finding that when quiescent fibroblasts are passaged in serum containing medium onto poly-HEME-coated plastic, or shaken to prevent adhesion, no formation of PKCepsilon 87 is observed (Fig. 2C). These results emphasize the need for both serum factors and readhesion in PKCepsilon 87 formation. We have confirmed through 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide analysis that 3T3 and 3T6 fibroblasts require both serum and readhesion to exit G0 and proliferate (data not shown). It should be noted that trypsinization of fibroblasts does not stimulate PKCepsilon 87 formation (data not shown).

Because re-entry into the cell cycle from G0 is necessary but not sufficient for PKCepsilon 87 formation (Fig. 2D), it is clear that other factors are also important in regulating the dephosphorylation of Ser729. One such factor may be the disruption of cell-substratum interactions and resulting changes in the cytoskeleton that occur upon cell passage and readhesion. Indeed, if the actin cytoskeleton is disrupted through cytochalasin D treatment of quiescent cells, PKCepsilon 87 is formed (Fig. 2D). The microtubule disrupting drug nocodazole does not produce the same effect (Fig. 2D).

PP1, PP2A, and PP2B Are Not Important in PKCepsilon 87 Formation upon Passage-- Treatment of cells with okadaic acid (OA) and calyculin (PP1 and PP2A inhibitors (29)) or cyclosporin A and ascomycin (inhibitors of PP2B (29)) did not inhibit the formation of PKCepsilon 87 upon passage (Fig. 3) (ascomycin C data not shown). This suggests that these protein phosphatases are not involved in catalyzing the removal of the Ser729 phosphate and that an OA-insensitive phosphatase is mediating this dephosphorylation. We observed that OA increased PKCepsilon 87 formation upon cell passage. It is possible then that inhibition of PP1 and/or PP2A increases the activity of the Ser729 phosphatase. Alternatively, the inhibitors may be preventing further dephosphorylation of PKCepsilon 87 at Ser703 and Thr566, thus preserving PKCepsilon 87. In fact we occasionally detect a faster migrating PKCepsilon band (PKCepsilon 84); formation of PKCepsilon 84 is inhibited by OA, supporting the latter idea. Hansra et al. (30) have shown that TPA-induced dephosphorylation of PKCalpha could be only partially inhibited by OA and have speculated that another protein phosphatase, insensitive to OA, may be involved. These workers have also described the importance of vesicle transport in the dephosphorylation of PKCalpha , showing that incubation at 18 °C inhibited dephosphorylation (31). We find that incubation at 18 °C to inhibit vesicle transport had no effect on the production of PKCepsilon 87 (Fig. 3). In fact, similarly to OA, it increased the formation of PKCepsilon 87.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Western blot for PKCepsilon from 3T3 cells. Lane 1, passaged; lane 2, passaged, Me2SO solvent control; lane 3, passaged in the presence of NaF; lane 4, passaged in the presence of 1 nM calyculin; lane 5, passaged in the presence of 10 nM calyculin; lane 6, passaged in the presence of 1 µM okadaic acid; lane 7, passaged in the presence of 10 µM okadaic acid; lane 8, passaged in the presence of 1 µM cyclosporin A; lane 9, passaged in the presence of 10 µM cyclosporin A; lane 10, passaged and maintained at 18 °C. PKCepsilon 95 and PKCepsilon 87 are indicated.

PKCepsilon 87 Production upon Passage Is Increased by Inhibiting Calpain, PI 3-Kinase, and PKCdelta -- Treatment of fibroblasts with the calpain and proteasome inhibitor ALLN (32) increased the level of PKCepsilon 87 produced upon cell passage (Fig. 4A). This may implicate calpain in the activation of a putative Ser729 phosphatase, or, probably more likely, this reflects an inhibition of the degradation of PKCepsilon 87. Treatment of cells with the PI 3-kinase inhibitors LY294002 and wortmannin (Refs. 33 and 34; wortmannin data not shown) also caused an increase in formation of PKCepsilon 87 upon cell passage (Fig. 4B). PI 3-kinase has already been demonstrated to play a role in PKC phosphorylation through its activation of phospholipid-dependent kinase 1 (7, 8). Our findings also implicate PI 3-kinase in the control of PKCepsilon dephosphorylation and degradation.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   A, Western blot for PKCepsilon from 3T3 cells. Lane 1, passaged, Me2SO solvent control; lane 2, 3T3 cells passaged in the presence of 10 µM ALLN. PKCepsilon 95 and PKCepsilon 87 are indicated. B, Western blot for PKCepsilon from 3T3 cells. Lane 1, passaged, Me2SO solvent control; lane 2, passaged in the presence of 10 µM LY294002. PKCepsilon 95 and PKCepsilon 87 are indicated. C, Western blots for PKCepsilon and PKCdelta from 3T3 cells transfected with PKCdelta antisense. Lane 1, mock transfected, quiescent; lane 2, mock transfected, passaged; lane 3, transfected, quiescent; lane 4, transfected, passaged. PKCepsilon 95 and PKCepsilon 87 are indicated. D, Western blot for PKCepsilon from 3T3 cells transfected with pCH3RDdelta . Lane 1, mock transfected, quiescent; lane 2, quiescent, transfected; lane 3, passaged, mock transfected; lane 4, passaged, transfected. PKCepsilon 95 and PKCepsilon 87 are indicated. E, Western blots for PKCepsilon from 3T3 cells. Lane 1, quiescent; lane 2, quiescent, treated with PKCepsilon translocation inhibitor peptide; lane 3, quiescent, treated with PKCdelta translocation inhibitor peptide; lane 4, quiescent, treated with PKCepsilon translocation agonist peptide; lane 5, quiescent, treated with PKCdelta translocation activator peptide; lane 6, passaged cells untreated control; lane 7, passaged cells treated with PKCepsilon translocation inhibitor peptide; lane 8, passaged cells control, treated with scrambled peptide; lane 9, passaged cells treated with PKCdelta translocation inhibitor peptide; lane 10, passaged cells, control, treated with scrambled peptide; lane 11, passaged cells treated with PKCepsilon translocation activator peptide; lane 12, passaged cells treated with PKCdelta translocation activator peptide. PKCepsilon 95 and PKCepsilon 87 are indicated.

Transfection of fibroblasts with full-length PKCdelta antisense reduced PKCdelta expression and increased PKCepsilon 87 formation upon cell passage (Fig. 4C). Expression of the regulatory domain of PKCdelta has been shown to be a specific inhibitor of PKCdelta (35, 36). Fibroblasts transfected with a construct containing this domain showed increased formation of PKCepsilon 87 upon cell passage (Fig. 4D). PKCdelta translocation inhibitor and activator peptides were also used to modulate PKCdelta in fibroblasts (37, 38). A PKCdelta translocation inhibitor peptide increased PKCepsilon 87 formation upon passage, whereas control and activator peptides had no effect (Fig. 4E). Peptide modulators of PKCepsilon had no effect on the formation of PKCepsilon 87 upon cell passage. Conformation of the specificity and activity of these peptides was confirmed through analysis of PKC isoform localization to membrane and cytosol fractions (data not shown).

The Production of PKCepsilon 87 upon Passage Involves MAPK, mTOR, and PKC-- The production of PKCepsilon 87 upon passage is inhibited passaging cells in the presence of PD98059, a MEK inhibitor (Ref. 39 and Fig. 5A), rapamycin, an inhibitor of mTOR (Ref. 40 and Fig. 5B), and by chelerythrine or Ro-31-8220, PKC inhibitors (Refs. 41 and 42; Fig. 5B; Ro-31-8220 data not shown). Such findings suggest a role for the MAPK pathway, mTOR, and PKC in the control of PKCepsilon 87 formation on cell passage. Inhibitors of protein kinase A, p38, and tyrosine kinases had no effect on PKCepsilon 87 formation upon passage, suggesting that these pathways are not important in this process. The inhibitor data are summarized in Table I.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5.   A, Western blot for PKCepsilon from 3T3 cells. Lane 1, quiescent; lane 2, passaged; lane 3, passaged cells, Me2SO solvent control; lane 4, 3T3 cells passaged in the presence of 100 µM PD98059. PKCepsilon 95 and PKCepsilon 87 are indicated. B, Western blot for PKCepsilon from 3T3 cells. Lane 1, quiescent; lane 2, passaged Me2SO solvent control; lane 3, passaged in the presence of 500 nM rapamycin; lane 4, passaged in the presence of 1 µM chelerythrine. PKCepsilon 95 and PKCepsilon 87 are indicated. C, Western blot for PKCepsilon from quiescent 3T3 cells. Lane 1, treated for 15 min with 250 nMalpha  phorbol; lane 2, treated for 15 min with 250 nM TPA and 1 µM chelerythrine; lane 3, treated for 15 min with 250 nM TPA. PKCepsilon 95 and PKCepsilon 87 are indicated.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effect of inhibitors on PKCepsilon 87 formation on cell passage

TPA Stimulates PKCepsilon 87 Production in Quiescent Cells-- TPA treatment of cells activates conventional and novel PKC isoforms, causing their dephosphorylation and subsequent degradation (30, 31). When quiescent fibroblasts are treated with 250 nM TPA for 15 min, formation of PKCepsilon 87 is detected, although this is at a reduced level compared with cell passage (Fig. 5C). This TPA effect can be blocked with the PKC inhibitor chelerythrine and does not occur with the inactive 4alpha -phorbol (Fig. 5C). As with PKCepsilon 87 formation upon cell passage, this effect can be inhibited by preincubation with rapamycin and PD98059 (data not shown), suggesting that the MAPK and mTOR pathways are also important here.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously shown that when fibroblasts are passaged there is a change in the phosphorylation status of PKCepsilon and that this is associated with a change in the intracellular localization of the protein. PKCepsilon 95, predominant in quiescent cells, has a perinuclear localization and is phosphorylated at Thr566, Ser703, and Ser729. PKCepsilon 87 has a cytosolic distribution and is phosphorylated at Thr566 and Ser703 (26). We have suggested that on cell passage a PKCepsilon Ser729 phosphatase may be activated. The hydrophobic (Ser729 in PKCepsilon ) site is conserved in most PKCs and in the ACG group of kinases (10). It is therefore essential to understand how phosphorylation and dephosphorylation in the hydrophobic domain is controlled. Here we have examined some factors that are important in the control of Ser729 phosphorylation status.

We have shown that the removal of the phosphate group at Ser729 from PKCepsilon 95 requires both serum and adhesion. Fibroblasts do not proliferate in the absence of serum or when prevented from adhering (28), and it is therefore likely that PKCepsilon 87 formation is dependent upon re-entry of quiescent or serum-starved cells into the cell cycle. However, we have shown that re-entry into the cell cycle alone is not sufficient for this process because if cells are serum-starved and then restimulated with serum there is no formation of PKCepsilon 87. It is possible then that PKCepsilon 87 formation is mediated through both disruption of cell-substratum interactions and readhesion of the cells to the substratum in the presence of serum. Our finding that cytochalasin D, the microfilament-disrupting drug, stimulates the formation of PKCepsilon 87 in quiescent cells supports this view. Readherence of cells after passage involves reorganization of the cytoskeleton. PKCepsilon has an actin-binding domain (21, 22), and we therefore speculate that PKCepsilon bound to actin may be activated by microfilament disruption, thereby either stimulating phosphatase activity or allowing access to a putative Ser729 phosphatase. Alternatively, disruption of the actin cytoskeleton through passage or cytochalasin D treatment could alter the localization of PKCepsilon , making it susceptible to attack by a Ser729 phosphatase.

Our inhibitor studies clearly show that the formation of PKCepsilon 87 upon passage is not dependent upon the more well defined protein phosphatases, PP1, PP2A, and PP2C. However, we find that the formation of PKCepsilon 87 upon cell passage is inhibited by rapamycin, indicating involvement of mTOR. mTOR has recently been implicated in the control of phosphorylation of the hydrophobic site in PKCepsilon and PKCdelta (10, 11). These authors speculate that this is mediated through the activation via mTOR of a phosphatase specific for the hydrophobic site rather than through regulation of a kinase. Our data presented here support this view and also suggest that this mTOR-controlled phosphatase is not PP1, PP2A, or PP2B.

Interestingly, OA treatment and also treatment with the calpain and proteasome inhibitor ALLN, increased the level of PKCepsilon 87 detected upon cell passage. The most likely explanation for these findings is that these inhibitors are blocking further dephosphorylation and degradation of PKCepsilon 87, thereby increasing PKCepsilon 87 levels. It seems that PKCepsilon 95 dephosphorylation occurs in two stages; firstly the removal of the phosphate at Ser729 by a specific phosphatase, followed by dephosphorylation at Thr566 and Ser703 by an OA-sensitive phosphatase. It has been shown that dephosphorylated forms of PKC are more susceptible to subsequent proteolytic degradation (13-15). PI 3-kinase inhibitors also increased PKCepsilon 87 formation upon passage. This may be through inhibition of a kinase that rephosphorylates PKCepsilon Ser729, possibly a complex including PKCzeta (9-11, 43).

Our time course results show that PKCepsilon 95 becomes the predominant form of PKCepsilon within 1 h of passage. This reappearance of PKCepsilon 95 may be mediated through rephosphorylation at Ser729 of PKCepsilon 87, the predominant form at earlier time points after passage. Our [35S]methionine-labeling experiments have shown that there is no new synthesis of PKCepsilon over this early time period, suggesting that PKCepsilon 95 must be derived from the recycling of PKCepsilon 87. Rephosphorylation of Ser729 could potentially be mediated through autophosphorylation (4) or through the activity of another kinase (10, 11).

Inhibitors of PKCdelta activity and translocation also increased PKCepsilon 87 detected upon passage suggesting that PKCdelta either inhibits PKCepsilon dephosphorylation or promotes PKCepsilon 87 dephosphorylation and degradation. Inhibition of PKCepsilon 95 dephosphorylation may be mediated through inhibiting a Ser729 phosphatase or through inhibiting the activation of PKCepsilon , hence its accessibility to phosphatase attack. There is evidence in the literature of a yin-yang relationship between PKCepsilon and PKCdelta . For example, PKCepsilon is growth promotory, whereas PKCdelta inhibits cell growth (17). PKCepsilon acts to prevent cells from cardiac ischemia, whereas PKCdelta mediates cell death (44, 45). The role of PKCdelta in the regulation of PKCepsilon dephosphorylation in fibroblasts needs to be further investigated.

PKCepsilon 87 formation upon passage is inhibited by PD98059, a MEK inhibitor. This implicates the MAPK pathway in the control of PKCepsilon Ser729 dephosphorylation. Because serum is required for this process, it is likely that the MAPK pathway is downstream of any extracellular signals in the medium that lead to cell proliferation. As already stated, it seems that signals that stimulate cell proliferation are essential for PKCepsilon 87 formation upon cell passage.

PKCepsilon phosphorylation at Ser729 is PKC-sensitive because we have demonstrated that PKCepsilon 87 production upon passage can be inhibited by the PKC inhibitors chelerythrine and Ro 31-8220. Also, PKCepsilon 87 production can be stimulated in quiescent cells through the addition of 250 nM TPA for 15 min. This suggests a role for another isoform of PKC in the regulation of PKCepsilon dephosphorylation. Alternatively, PKCepsilon may regulate the activity of a Ser729 phosphatase. PKC inhibitors would therefore inhibit this phosphatase activity through inhibition of PKCepsilon .

Our working model for PKCepsilon regulation on passage is summarized in Fig. 6. We have observed similar results in 3T6 fibroblasts and C6 glioma cells suggesting that our findings are not cell type-specific. Our data suggest that at least a two-stage process is involved in regulating dephosphorylation of PKCepsilon 95. Passage of cells or TPA treatment of quiescent cells causes dephosphorylation at Ser729. It is probable that PKCepsilon is activated upon passage and readhesion. PKCepsilon has been shown to be important in the spreading of HeLa and other cells (46, 47) where PKCepsilon is activated upon cell matrix contact and is required for actin polymerization. The loss of a phosphate at Ser729 is sensitive to re-entry into the cell cycle from G0 and readhesion of cells; it is also dependent upon the MAPK pathway. It has been reported that, once in an activated conformation, PKC becomes susceptible to phosphatase attack (27) and that loss of the C-terminal phosphorylations may regulate translocation of the kinase to the cytoplasm after activation (48). The loss of a phosphate at Ser729 is not mediated through PP1, PP2A, or PP2B. PKCepsilon 87 could then be rephosphorylated at Ser729 and recycled back to PKCepsilon 95. This may involve a membrane-associated kinase complex including PKCzeta . (43) Alternatively PKCepsilon 87 may be further dephosphorylated at sites Ser703 and Thr566, perhaps mediated by an OA-sensitive phosphatase. Complete dephosphorylation increases the instability of PKC and targets the protein for degradation (13, 14, 15). It is therefore likely that once fully dephosphorylated PKCepsilon is sensitive to degradation via calpain and the proteasome.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Summary of the regulation of PKCepsilon phosphorylation status in 3T3 cells at quiescence and on cell passage.


    ACKNOWLEDGEMENTS

We thank Professor D. Mochly-Rosen (Stanford University) for the PKC translocation inhibitor and activator peptides, Professor S. Jaken (University of Vermont) for the pCH3delta RD domain vector, and Professor P. Parker (Imperial Cancer Research Fund, London) for the PKCdelta clone.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by the Biotechnology and Biological Sciences Research Council Integration of Cellular Responses Initiative. To whom correspondence should be addressed: Tumour Biology Laboratory, Department of Biochemistry, University College Cork, Lee Maltings Complex, Prospect Row, Cork, Republic of Ireland. E-mail: kengland@ucc.ie.

§ Present address: Smith and Nephew Group Research Centre, Science Park, Heslington, York YO10 5DF, UK.

Present address: Dept. of Life and Health Services, Aston Pharmacy School, Aston University, Aston Triangle, Birmingham B4 7ET, UK.

Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009421200

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; MAPK, mitogen-activated protein kinase; OA, okadaic acid; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PAGE, polyacrylamide gel electrophoresis; TPA, 12-O-teteradecanoylphorbol-13-acetate; PI, phosphatidylinositol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Goodnight, J., Mischak, H., and Mushinski, J. F. (1994) Adv. Cancer Res. 64, 159-209[Medline] [Order article via Infotrieve]
2. Dekker, L. V., and Parker, P. J. (1994) Trends Biochem. Sci. 19, 73-77[CrossRef][Medline] [Order article via Infotrieve]
3. Nishizuka, Y. (1988) Nature 334, 661-665[CrossRef][Medline] [Order article via Infotrieve]
4. Newton, A. C. (1997) Curr. Opin. Cell Biol. 9, 161-167[CrossRef][Medline] [Order article via Infotrieve]
5. Dutil, E. M., Keranen, L. M., DePaoli-Roach, A. A., and Newton, A. C. (1994) J. Biol. Chem. 269, 29359-29362[Abstract/Free Full Text]
6. Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403[Medline] [Order article via Infotrieve]
7. Garcia-Paramio, P., Cabrerizo, Y., Bornancin, F., and Parker, P. J. (1998) Biochem. J. 333, 631-636[Medline] [Order article via Infotrieve]
8. Le Good, J. A., Ziegeler, W. H., Parekh, D. B., Alessi, D. R., Cohen, P., and Parker, P. J. (1998) Science 281, 2042-2045[Abstract/Free Full Text]
9. Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. L., Chen, C.-S., Newton, A. C., Schaffhausen, B. S., and Toker, A. (1998) Curr. Biol. 8, 1069-1077[Medline] [Order article via Infotrieve]
10. Parekh, D. B., Ziegler, W., and Parker, P. J. (2000) EMBO J. 19, 496-503[Free Full Text]
11. Parekh, D., Ziegler, W., Yonezawa, K., Hara, K., and Parker, P. J. (1999) J. Biol. Chem. 274, 34758-34764[Abstract/Free Full Text]
12. Zhang, J., Wang, L., Schwartz, J., Bond, R. W., and Bishop, W. R. (1994) J. Biol. Chem. 269, 19578-19584[Abstract/Free Full Text]
13. Gysin, S., and Imber, R. (1997) Eur. J. Biochem. 249, 156-160[Abstract]
14. Bornancin, F., and Parker, P. J. (1996) Curr. Biol. 6, 1114-1123[Medline] [Order article via Infotrieve]
15. Cazaubon, S., Bornancin, F., and Parker, P. J. (1994) Biochem. J. 301, 443-448[Medline] [Order article via Infotrieve]
16. Orr, J. W., and Newton, A. C. (1994) J. Biol. Chem. 269, 27715-27718[Abstract/Free Full Text]
17. Mischak, H., Goodnight, J., Kolch, W., Martiny-Baron, G., Schaechtle, C., Kazanietz, M. G., Blumberg, P. M., Pierce, J. H., and Mushinski, J. F. (1993) J. Biol. Chem. 268, 6090-6096[Abstract/Free Full Text]
18. Cacace, A. M., Guadagno, S. N., Krauss, R. S., Fabbro, D., and Weinstein, I. B. (1993) Oncogene 8, 2095-2104[Medline] [Order article via Infotrieve]
19. Cacace, A. M., U.effing, M., Phillip, A., Han, E. K.-H., Kolch, W., and Weinstein, I. B. (1996) Oncogene 13, 2517-2526[Medline] [Order article via Infotrieve]
20. Ueffing, M., Lovric, J., Philipp, A., Mischak, H., and Kolch, W. (1997) Oncogene 15, 2921-2927[CrossRef][Medline] [Order article via Infotrieve]
21. Prekeris, R., Mayhew, M. W., Cooper, J. B., and Terrain, D. M. (1996) J. Cell Biol. 132, 77-90[Abstract]
22. Prekeris, R., Hernandez, R. M., Mayhew, M. W., White, M. K., and Terrain, D. M. (1998) J. Biol. Chem. 273, 26790-26798[Abstract/Free Full Text]
23. Csukai, M., Chen, C.-H., De Matteis, M. A., and Mochly-Rosen, D. (1997) J. Biol. Chem. 272, 29200-29206[Abstract/Free Full Text]
24. Lehel, C., Olah, Z., Petrovics, G., Jakob, G., and Anderson, W. B (1996) Biochem. Biophys. Res. Commun. 223, 98-103[CrossRef][Medline] [Order article via Infotrieve]
25. Lehel, C., Olah, Z., Jakon, G., and Anderson, W. B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1406-1410[Abstract]
26. England, K., and Rumsby, M. (2000) Biochem. J. 35, 219-226
27. Sweatt, J. D., Atkins, C. M., Johnson, J., English, J. D., Roberson, E. D., Chem, S.-J., Newton, A., and Klann, E. (1998) J. Neurochem. 71, 1075-1085[Medline] [Order article via Infotrieve]
28. Rozengurt, E. (1986) Science 234, 161-166[Medline] [Order article via Infotrieve]
29. Hardie, D. G. (1995) in Protein Phosphorylation: A Practical Approach (Hardie, G., ed) , pp. 109-119, IRL Press, Oxford, UK
30. Hansra, G., Bornancin, F., Whelan, R., Hemmings, B. A., and Parker, P. J. (1996) J. Biol. Chem. 271, 32785-32788[Abstract/Free Full Text]
31. Hansra, G., Garcia-Paramio, P., Prevostel, C., Whelan, R. D. H., Bornancin, F., and Parker, P. J. (1999) Biochem. J. 342, 337-344[CrossRef][Medline] [Order article via Infotrieve]
32. Vinitsky, A., Michaud, C., Powers, J. C., and Orlowski, M. (1992) Biochemistry 31, 9421-9428[Medline] [Order article via Infotrieve]
33. Vlahos, C. J., Matter, W. F., Hiu, K. Y., and Brown, R. F. (1994) J. Biol. Chem. 269, 5241-5248[Abstract/Free Full Text]
34. Powis, G., Benjouklian, R., Bergren, M. M., Gallegos, A., Abraham, R., Ashendel, C., Zalkow, L., Matter, W. F., Dodge, J., Grindey, G., and Vlahos, C. J. (1994) Cancer Res. 54, 2419-2423[Abstract]
35. Kiley, S. C., Clark, K. J., Goodnough, M., Welch, D. R., and Jaken, S. (1999) Cancer Res. 59, 3230-3238[Abstract/Free Full Text]
36. Kiley, S. C., Clark, K. J., Duddy, S. K., Welch, D. R., and Jaken, S. (1999) Oncogene 18, 6748-6757[CrossRef][Medline] [Order article via Infotrieve].)
37. Csukai, M., and Mochly-Rosen, D. (1999) Pharmacol. Res. 37, 253-259
38. Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Bio/Technol. 16, 919-924[Medline] [Order article via Infotrieve]
39. Pang, L., Sawada, T., Decker, S. J., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 13585-13588[Abstract/Free Full Text]
40. DuMont, F. J., Staruch, M. J., Melino, M. R., and Sigal, N. H. (1990) J. Immunol. 144, 251-258[Abstract/Free Full Text]
41. Herbert, J. M., Augereau, J. M., Gleye, J., and Maffrand, J. P. (1990) Biochem. Biophys. Res. Commun. 172, 993-999[Medline] [Order article via Infotrieve]
42. Davis, P. D., Elliot, L. H. I., Harris, W., Hill, C. H., Hurst, S. A., Keech, E., Kumar, M. K. H., Lawton, G., Nixon, J. S., and Wilkinson, S. E. (1992) J. Med. Chem. 35, 994-1001[Medline] [Order article via Infotrieve]
43. Ziegler, W. H., Parekh, D. B., Le Good, J. A., Whelan, R. D. H., Kelly, J. J., Frech, M., Hemmings, B. A., and Parker, P. J. (1999) Curr. Biol. 9, 522-529[CrossRef][Medline] [Order article via Infotrieve]
44. Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) J. Mol. Cell Cardiol. 31, 1937-1948[CrossRef][Medline] [Order article via Infotrieve]
45. Dorn, G. W., Souroujon, M. C., Liron, T., Chen, C.-H., Gray, M. O., Zhou, H. Z., Csukai, M., Wu, G., Lorenz, J. N., and Mochly-Rosen, D. (1999) Proc. Natl. Acad. Sci. U. S. A. U. S. A. 96, 2798-12803
46. Kim, J.-Y., Lee, Y.-S., Park, J., and Chun, J.-S. (1997) Mol. Cell. 7, 594-598
47. Chun, J., Auer, K. A., and Jacobson, B. S. (1997) J. Cell. Physiol. 173, 361-370[CrossRef][Medline] [Order article via Infotrieve]
48. Feng, X., Becker, K. P., Stribling, S. D., Peters, K. G., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 17024-17034[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.