Inserm U469 `Molecular and Cellular Endocrinology: Signaling and Pathology', 141 rue de la Cardonille, 34094 Montpellier CEDEX 05, France
Author for correspondence (e-mail: dominique.joubert{at}ccipe.cnrs.fr)
Accepted 13 August 2003
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Summary |
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Key words: Protein kinase C, Targeting, Pituitary, Cell-cell contact
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Introduction |
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Functional alterations of PKC frequently occur in human tumours including pituitary tumours: most of the tumours exhibit variable levels in PKC accumulation in comparison with normal tissue (Alvaro et al., 1992; Hagiwara et al., 1990
; O'Brian et al., 1989
; Prevostel et al., 1995
; Shimizu et al., 1991
) and a mutated form of PKC
(the D294G-PKC
mutant) has been discovered by our group in a subpopulation of human pituitary (Alvaro et al., 1993
) and thyroid tumours (Prevostel et al., 1995
) which in addition, exhibit an accumulation of PKC
. Consistent with this, Schiemann et al. (Schiemann et al., 1997
) have shown that pituitary tumours with no increase in PKC
accumulation do not contain the D294G mutation. More recently, Fagin and colleagues (Knauf et al., 2002
) failed to confirm the presence of the D294G mutation in human thyroid tumours by using single-strand conformation polymorphism (SSCP) and specific allele oligonucleotide hybridization (SAOH) analyses of genomic DNA. As proposed by Fagin and colleagues, this may reflect the highly heterogenous distribution of cells containing the mutated form of PKC
within the tumour. However, in addition to its potential interest in tumourigenesis, the D294G mutation turned out to be a powerful tool to investigate the mechanisms underlying the selectivity of PKC
subcellular targeting. Indeed, upon activation, PKC
is no longer selectively targeted to cell-cell contacts (Vallentin et al., 2001
), indicating that translocation to cell-cell contacts is a highly regulated process, probably mediated through a specific targeting motif(s).
The D294G mutation is located within the third PKC variable region (V3), also called the hinge region. Very little is known about the function of this region. In a previous study, we have established that the D294G mutation induces a selective loss in the recognition of substrates with properties of anchoring proteins (Prevostel et al., 1998
). Recently, Parsons et al. (Parsons et al., 2002
) have identified a binding motif for ß1 integrin within the PKC
V3 region that is critical for directed tumour cell migration. Thus, these observations are strong arguments in favour of a major role of the V3 region in PKC functions.
In the present study, we establish that the specificity of PKC targeting to cell-cell contacts does exist in the pituitary gland and that it is neither cell-type-, nor isozyme-specific since PKC epsilon (PKC
) is, similarly to PKC
, selectively targeted to cell-cell contacts upon TRH or TPA stimulation. In contrast, activated PKC delta (PKC
) exhibits no selectivity in plasma membrane compartmentalisation. The comparison of PKC
,
and
protein sequences, together with the transient transfection of GFP-tagged PKC
,
and
mutants clearly demonstrates the importance of the GD(E)E motif in the specificity of targeting to cell-cell junctions. This motif is located within the V3 region of both PKC
and
; it is absent in PKC
and mutated in the D294G PKC
mutant. The use of PKC
deletion mutants indicates, however, that the C2 domain is also required for the targeting to cell-cell contacts.
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Materials and Methods |
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Analysis of endogenous PKC ,
and
subcellular localisations
In rat and transgenic mice pituitary glands
Pituitary glands from adult rats or mice were incubated for 1 hour at 37°C with HamF10 medium supplemented with foetal calf and horse serums (see GH3B6 medium) with or without 100 nM PMA or TRH. Pituitary glands were fixed with 4% paraformaldehyde in phosphatebuffered saline (PBS), pH 7.4 for 30 minutes, washed 3 times with PBS pH 7.4 and cut sagittally with a vibratome into 40 µm thick sections. Theses sections were carefully rinsed in PBS and subsequently incubated overnight at 4°C with several antibodies diluted in PBS pH 7.4 containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA): a rabbit polyclonal anti-PKC (mouse sections), a mouse monoclonal PKC
(dilution: 1/250) (rat sections), a rabbit polyclonal anti-PKC
(dilution: 1/500), and a rabbit polyclonal anti-laminin (dilution: 1/250). After 3 washes for 10 minutes at room temperature in PBS pH 7.4 containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA), the sections were incubated for 1 hour at room temperature with the secondary goat antirabbit or anti-mouse antibodies conjugated to indocarbocyanine 3 (Cy3) or Alexa (dilution: 1/1000). Immunostained sections were then mounted in Mowiol (Calbiochem, La Jolla, CA) and examined by confocal microscopy. Controls consisted of omitting the primary antibodies.
In GH3B6 cells
the GH3B6 cells were seeded on 12 mm round coverslips in 1 ml medium and grown for 24 hours. They were washed three times with PBS pH 7.4 before being fixed for 15 minutes with 4% paraformaldehyde in PBS pH 7.4 (v/v). The cells were then washed twice with PBS, permeabilised in 0.2% Triton X-100 for 5 minutes, washed in PBS and incubated for 30 minutes in PBS supplemented with 1% BSA (PBSA). The coverslips were then incubated overnight at 4°C with the primary mouse anti-PKC (dilution: 1/250), and rabbit anti-PKC
(dilution: 1/500) antibodies. They were then washed three times for 10 minutes with PBSA, and further incubated for 1 hour at room temperature with the secondary antibodies, which were respectively, a goat anti-mouse (Fab')2 Cy3 conjugated for PKC
and a goat anti-rabbit Cy3-conjugated antibody for PKC
(dilution: 1/1000). The coverslips were then mounted in Mowiol and examined by conventional microscopy (Zeiss) by using a x100 objective.
Construction of plasmids encoding GFP-tagged PKC
The GFP-tagged proteins transiently expressed in GH3B6 cells are schematically represented in Fig. 1A,B. The corresponding PKC constructs were generated as previously described (Vallentin et al., 2000
). Briefly, PKC
deletion mutants were amplified by polymerase chain reaction (PCR) using the full-length wt, or D294G, PKC
cDNAs subcloned in the pBabe vector as a template. For the GFP-tagged full-length wt and D294G PKC
constructs the synthetic oligonucleotides used have been described by Vallentin et al. (Vallentin et al., 2000
). To generate the (V1-C1)+V3, (V1-C1)+V3D294G, C1+V3, C1+V3 D294G constructs, the PCR amplified (V1-C1) and C1 were ligated in frame to the wt and D294G mutated V3 domains by using a XbaI restriction site. The oligonucleotides used were as follows: forward: 5'-GGAATTCCGGAGCAAGAGGTGGTT-3' for (V1-C1) and (V1-C1) in both (V1-C1)+V3 and (V1-C1)+V3D294G; 5'-GGAATTCCATGCGCTTCGCCCGCAAAGGG-3' for
V1, 5'-GGAATTCCATGAACGTGCACGAGGTGAAG-3' for
V1+PS),
V1+PS)-RD,
V1+PS)-RD+V3,
V1+PS)-RD+V3D294G and C1 in both C1+V3 and C1+V3D294G; 5'-GGAATTCCATGATTCCGGAAGGGGACGAG-3' for V3; 5'-GGAATTCCATGATTCCGGAAGGGGGCGAG-3' for V3D294G; 5'-TGCTCTAGAGCAATTCCGGAAGGGGACGAG-3' for V3 in (V1-C1)+V3 and C1+V3; 5'-TGCTCTAGAGCAATTCCGGAAGGGGGCGAG-3'; for V3D294G in (V1-C1)+V3D294G and C1+V3D294G. Reverse: 5'-GGGGTACCCCTACTGCACTCTGTAAGATG-3' for
V1 and
V1+PS); 5'-GGGGTACCCCCCGCCCCCTCTTCTCAGT-3' for (V1-C1) and C1; 5'-TGCTCTAGAGCACCGCCCCCTCTTCTCAGT-3' for (V1-C1) in both (V1-C1)+V3 and (V1-C1)+V3D294G, and C1 in both C1+V3 and C1+V3D294G; 5'-GGGGTACCCCCGTGAGTTTCACTCGGTC-3' for
V1+PS)-RD+V3,
V1+PS)-RD+V3D294, V3, V3 in (V1-C1)+V3 and C1+V3, V3D294G, V3D294G in (V1-C1)+V3D294G and C1+V3D294G. All PCR amplified DNA fragments were gel purified and subcloned in frame to GFP within the EcoRI and KpnI restriction sites of the pEGFP-N1 plasmid.
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The constructs encoding the GFP-tagged PKC and
were obtained by using a similar procedure, with the exception that fulllength PKC
and
cDNAs were subcloned within the XhoI and KpnI restriction sites of the pEGFP-N1 plasmid. The synthetic oligonucleotides used in the PCR amplification were as follow: forward 5'-CCTCGAGGATGGCACCGTTCCTGCGC-3' and reverse 5'-GGGGTACCCCTTCCAGGAATTGCTCATATTT-3' for PKC
; forward 5'-CCTCGAGGATGGTAGTGTTCAATGGCCT-3' and reverse 5'-GGGGTACCCCGGGCATCAGGTCTTCACCA-3' for PKC
. All constructs were sequenced.
Site directed mutagenesis
The point mutated PKC and
were created by using the QuickChangeTM site-directed mutagenesis kit according to the manufacturer's standard protocol. The pairs of synthetic oligonucleotides used to obtain the mutated PKCs were as follows: forward 5'-GACAACCGAGGAGGGGAGCACCGAGCC-3' and reverse 5'-GGCTCGGTGCTCCCCTCCTCGGTTGTC-3' for E374G-PKC
; forward 5'-GTCGGAATATACCAGGGAGAGGAGAAGAAGACA-3' and reverse 5'-TGTCTTCTTCTCCTCTCCCTGGTATATTCCGAC-3' for F314E-PKC
. Mutated PKC were fully sequenced by Genome express (Meylan, France).
Analysis of GFP tagged PKC subcellular location in living cells
Cells were cultured as previously described (Vallentin et al., 2000). Transient transfection experiments were carried out with ExGen 500 according to the manufacturer's standard protocol. Briefly, the cells were seeded at 300,000 cells per well in a 24-well dishes (Falcon) 18 hours before transfection. Immediately before transfection, fresh culture medium (1 ml) was added to the cells. ExGen 500 stock solution (1.25 µl) was diluted in 12.5 µl NaCl 150 mM. The plasmid DNA (250 ng/well) was diluted in 12.5 µl of NaCl 150 mM. These solutions were then mixed together. After 30 minutes, the transfection mixture was added to the cells. The 24-well dishes were then centrifuged for 5 minutes at 280 g and maintained for 48 hours at 37°C. Subcellular localisation of GFP-tagged PKC was monitored under basal or TPA stimulated conditions (100 nM for 1 hour). At the time of the observation, the culture medium was replaced by a phosphate-buffered saline solution (pH 7.4). The localisation of the fusion proteins was examined by conventional microscopy. All experiments were performed at least three times.
Western blot analysis
Transfected GH3B6 cells were washed in PBS before being lysed and boiled in 150 µl of Laemmli buffer. Equal amounts of proteins were loaded for 12% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. Non-specific binding sites were blocked with Tris-buffered saline (50 mM Tris, 150 mM NaCl) containing 10% milk powder for 1 hour at room temperature. The membrane was then incubated with a monoclonal anti-GFP antibody (1/1000) at 4°C overnight. After washing in Tris-buffered saline containing 0.1% Tween, membranes were incubated with a goat anti-mouse horseradish peroxydase-conjugated antibody. The immunoreactive bands were revealed using a chemiluminescence detection kit. Fig. 1C,D attest that the GFP-tagged PKC were expressed at the expected size.
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Results |
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The targeting selectivity to cell-cell contacts is not restricted to the classical PKC isoform
The distinct subcellular location of each PKC isoforms is supposed to be closely linked to their different biological functions (Csukai and Mochly-Rosen, 1999), raising the question of whether specific targeting to cell-cell contacts is restricted to the
isoform, and thus to its function in the pituitary. As shown in Fig. 3B, PKC
is, similarly to the
isoform, located in the cytoplasm in basal conditions and selectively translocated to cell-cell contacts upon TRH or TPA stimulation of pituitary glands of transgenic mice expressing GFP specifically in GH cells. Upon TPA stimulation, the presence of PKC
at cell-cell contacts was observed in GFP-positive as well as in GFP-negative cells. Upon TRH stimulation, translocation of PKC
to cell-cell contacts also occurred although to a lesser extent.
In TPA stimulated GH3B6 cells, PKC and
were both located at cell-cell contacts (Fig. 4), indicating that this cell line is a valuable model for investigating the molecular basis underlying PKC subcellular targeting.
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The observation that the selectivity of targeting to cell-cell contacts is not restricted to PKC suggests that a common mechanism is involved in the targeting of both isoforms, despite distinct activation modes: calcium-dependent for
and calcium-independent for
.
A critical role for the GD(E)E motif located within the V3 variable region of PKC and
in the selectivity of targeting to cell-cell contacts
The point mutation leading to the substitution of an aspartic acid by a glycine in the hinge region of PKC (position 294) is sufficient to induce a loss in the specificity of targeting to cell-cell contacts (Vallentin et al., 2001
). The sequence alignment of PKC
and
reveals the presence of similar motifs (GDE, GEE) in their V3 regions (see Fig. 1A) If this motif is really involved in the selectivity of targeting, the GEE to GGE substitution in PKC
might have the same effect as the GDE to GGE substitution in the D294G mutant of PKC
, that is a loss of cell-cell contact targeting selectivity. We therefore introduced the E374G mutation in PKC
and tested its impact on the selectivity of targeting. Both GFP-tagged mutated and wt PKC
were transiently transfected into GH3B6 cells. As shown in Fig. 5, the E374G mutation of PKC
was, similarly to the D294G mutation of PKC
, sufficient to abolish the selectivity of targeting to cell-cell contacts upon TPA stimulation. This demonstrates that the GD(E)E motif plays a crucial role in the selectivity of PKC targeting to cell-cell contacts.
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The F314E point mutation in PKC V3 region does not induce cell-cell contact targeting
Further comparison of the subcellular localisation and proteic sequences of PKC ,
and
indicated that the V3 region is not the only region responsible for cell-cell targeting. The subcellular location of GFP-tagged PKC
in GH3B6 cells is shown in Fig. 5, in comparison to those of PKC
and
. In contrast to the GFP-tagged PKC
and
, the GFP-tagged PKC
is targeted to the entire plasma membrane upon activation. An analysis of the proteic sequence of the V3 region of PKC
indicated the presence of a GFE motif instead of GD(E)E (Fig. 1A). This, together with the observation that PKC
is not selectively targeted to cell-cell contacts, argues for the importance of the GD(E)E motif in the selectivity of targeting. However, the F314E mutation introduced in the V3 region of PKC
in order to restore the GD(E)E motif, was not able to target PKC
to cell-cell contacts (Fig. 5). This therefore indicates that the GD(E)E motif is essential but is not on its own sufficient to confer the selectivity of targeting. This motif could therefore be part of a binding domain for an anchoring/cargo protein, the expression or post-transductional properties of which are determined by the cell-cell contact formation (see Discussion).
The PKC V3 region does not contain the whole information for the selectivity of targeting
According to our previous data, the PKC V3 region alone is not spontaneously located at cell-cell contacts (Vallentin et al., 2000
). Thus, either V3 does not contain the whole targeting sequence or it does contain the targeting sequence but it is not able to accumulate at cell-cell contacts. Since the C1 domain alone can act as a plasma membrane-targeting module in response to TPA (Oancea et al., 1998
), we postulated that its association with V3 might suffice for cell-cell contact specificity of targeting upon TPA stimulation. However, as shown in Fig. 6A, the C1 domain did not translocate when associated to the wt (or the D294G mutated) V3 region, although, consistent with the results of Oancea et al. (Oancea et al., 1998
), C1 alone was efficiently translocated to the plasma membrane upon TPA stimulation. Therefore, the association of V3 to the C1 domain may induce conformational changes that may abolish the ability of C1 to interact with TPA.
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Another possible explanation is that the V3 region inhibits the translocation of the C1 domain. Consistent with this, our previous data have established that the V3 region is involved in the cytoplasmic sequestration of PKC. In the presence of V1 and the pseudosubstrate (PS), translocation of C1-V3 (or C1-V3D294G) is restored but without any selectivity for cell-cell contacts, which was of course expected for C1-V3D294G but not for C1-V3 (Fig. 6B). This not only mean that V1-PS might play an important role in the translocation of C1 as it does for the entire protein (Vallentin et al., 2000
), but that the V3 region does not contain the whole information to target PKC
to cell-cell contacts. Interestingly (Fig. 5B, bottom), when C1 was associated with V1, it did not translocate either. This suggests that the mechanisms by which V1 and V3 block translocation of C1 may be related to each other.
The PKC cell-cell contact targeting motif is located within the C2-V3 region
The above results together with our previous data suggest that the V1 region may be important for translocation/localisation of PKC to/at the plasma membrane but is not required for the cell-cell contact targeting selectivity. This is consistent with the fact that PKC
and
are both selectively translocated to cell-cell contacts, despite the fact that they exhibit strong differences within their V1 regions. In agreement with this, Fig. 7A shows that although the removal of the V1 domain alone abolishes the TPA-induced translocation of PKC
to the plasma membrane, a further deletion of the pseudosubstrate is able to restore the selective translocation of PKC
to cell-cell contacts. Thus the N-terminal part of the protein, including both V1 and the pseudosubstrate, is not required for the selectivity of targeting to cell-cell contacts, even though it is involved in the control of translocation. Fig. 7B also establishes that the catalytic domain is also not required for the selectivity of PKC
to cell-cell contacts. Indeed, the PKC
region, including C1 to V3, translocates selectively to cell-cell contacts. As expected, the same region bearing the D294G mutation has lost targeting selectivity to cell-cell contact and accumulates at the entire plasma membrane. The amino acid sequence that determines the selectivity of PKC
targeting to cell-cell contacts is thus probably located within the C2-V3 region.
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Discussion |
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Possible role(s) for PKC and PKC
at cell-cell contacts in the pituitary gland
To our knowledge, the only immunocytochemical analysis of the localisation of PKC subtypes in normal rat pituitary cells was performed on primary cultures, in a situation where cell-cell contacts as well as cell-extracellular matrix contacts were abolished (Naor, 1990). In the present study, we report for the first time that upon a physiological (TRH) or a pharmacological stimulation (TPA) of the pituitary gland, PKC
is selectively targeted to the contacts between GH-expressing or GH-non expressing cells. This indicates that the molecular mechanisms that determine the selectivity of PKC
targeting to cell-cell contacts is not cell-type-specific. Interestingly, the selectivity of targeting to cell-cell contacts is isozyme-specific although it is not restricted to PKC
. Indeed, whereas PKC
and the novel PKC
are selectively translocated to cell-cell contacts upon activation, PKC
is translocated to the entire plasma membrane. Together, these data raise the question of the physiological role(s) of both PKC
and
at cell-cell contacts.
The role of a kinase at a particular subcellular site is dependent on the dynamics of its own accumulation at that location, as well as that of its substrates. In a previous study, we showed that a TRH stimulation induces two phases of PKC translocation in GH3B6 cells (Vallentin et al., 2000
): a short and transient phase occurring a few seconds after the beginning of the stimulation and a long lasting phase. The dynamics of PKC
and
translocation have now to be further analysed in the intact pituitary gland in order to determine whether these two phases of translocation exist in the tissue and to clarify the respective roles of these two PKC isoforms at cell-cell contacts. A role in the regulation of hormonal secretion or cell proliferation is expected for PKC
and partly demonstrated for PKC
(Akita et al., 1994
). Furthermore, according to Akita et al. (Akita et al., 2000
), MARCKS, a regulatory component of the cytoskeletal architecture, is a major substrate of PKC
in vivo, and its phosphorylation may regulate TRH-stimulated hormonal secretion. It will, therefore, be important to know whether this phosphorylation occurs at cell-cell contacts or not. The fact that the release of hypothalamic hormones is pulsatile has also to be considered. For example, the interplay between somatostatin and growth hormone releasing hormone determines the release pattern of growth hormone (Tannenbaum, 1984). This means that the receptors for these hormones are not under a constant activation, and may explain the need for the alternation between the cytoplasmic and cell-cell contact localisation of PKC
and
. The stimulation by TRH or PMA may amplify the accumulation of PKC
and
at their physiological subcellular locations. Several reports also argue in favour of a specific role of PKC
and PKC
in regulating intercellular communication via cell-cell contacts. Indeed, according to Perez-Moreno et al. (Perez-Moreno et al., 1998
) the PKC-mediated phosphorylation of vinculin is required for the redistribution of both vinculin and
-actinin from the cytoplasm to the cell periphery and may be a crucial step for the assembly of adherent junctions. To the same extent, the TRH-induced translocation of PKC
at contacts between GH3B6 cells is associated with a redistribution of ß-catenin to the same site (Vallentin et al., 2001
). Finally, Sheu et al. (Sheu et al., 1989
) have reported that the desmosomal proteins desmoplakins are redistributed from the cytoplasm to the plasma membrane upon PKC stimulation and that this redistribution is accompanied by desmosomal formation. Desmoplakins are phosphorylated on both serine and threonine residues although it is not clear whether this phosphorylation is mediated by PKC (Pasdar et al., 1995
).
What determines the selectivity of subcellular PKC and
targeting?
The presence of a PKC isoform at the plasma membrane has long been thought to depend on its interaction with phospholipids, such as phosphatidylserine. This was the dogma until the receptor for activated kinase C (RACK-1) was discovered (Mochly-Rosen et al., 1991a; Mochly-Rosen et al., 1991b
). Since then, concepts have evolved towards the idea that accumulation of PKC at a particular cell location can be mediated by anchoring proteins (Mochly-Rosen and Gordon, 1998
). In GH3B6 cells and in the pituitary gland, activated PKC
and
are compartmentalised at cell-cell contacts. This compartmentalisation is determined by the cell-cell contact since PKC
and
do not translocate in isolated cells, despite the activation of the TRH receptors (Vallentin et al., 2000
). Two questions arise: is there a restricted amino acid sequence in PKC responsible for this selectivity and what is the cell-cell contact-induced signal responsible for the selectivity?
A restricted amino acid sequence involved in targeting selectivity
The simplest way to address this is to distinguish the different steps of what is called `translocation'. Activation is a prerequisite for translocation and, at least for PKC, calcium has been shown to be necessary: without an increase in the [Ca]i, PKC
does not translocate. This process involves the C2 domain (Bolsover et al., 2003
; Raghunath et al., 2003
). Although PKC
and
targeting to cell-cell contacts only occurs upon stimulation, the mechanism determining the selectivity of targeting observed in pituitary cells probably coexists or pre-exists this activation process. This selectivity of targeting is however not calcium dependent since the novel PKC
is calcium independent. When PKC
and
are mutated in their V3 region (D294G and E374G mutations respectively), the selectivity is abolished although translocation still occurs. The GDE/GEE motif, affected by these point mutations, may thus be involved in the recognition of a cell-cell contactinduced signal. These are not the first data demonstrating an involvement of the V3 region in the control of PKC targeting or in the interaction with anchoring proteins. Indeed, Parsons et al. (Parsons et al., 2002
) have recently established that a 12 amino-acid motif within the PKC
V3 region (aa 313-325) is required for the direct association of PKC
with ß-integrin. However, the cell-cell contacts targeting selectivity requires an additional sequence that is, for PKC
, located in the C2 region. Indeed, the C1 module requires the presence of C2 in addition to V3 in order to be targeted to cell-cell contacts. Our data suggest that a similar sequence exists in PKC
even though it has not yet been identified. In contrast this sequence is probably not present in PKC
, another novel PKC isoform since the introduction of the GEE motif in PKC
(which possesses a GFE motif instead of the GDE/GEE motif), is not sufficient to target PKC
to cell-cell contacts.
The present work also suggests that the pseudosubstrate sequence is involved in the targeting through a mechanism distinct from the commonly accepted one implicating the binding to the catalytic core. Indeed, the deletion of the V1 region inhibits PKC translocation but the translocation is recovered by further deleting the PS (Fig. 6). To the same extent, the C1 module accumulates uniformly at the plasma membrane upon TPA stimulation but no longer translocates a when fused to the PS (data not shown). This means that an additional level in the control of translocation may well exist, such as an off/on signal involving the PS/V1 sequence. The PS sequence is already known to be able to bind proteins, such as cytoskeletal proteins (Garcia-Rocha et al., 1997
; Liao et al., 1994
; Schmitz-Peiffer et al., 1998
), lending support to our hypothesis.
A cell-cell contact-induced signal responsible for the selectivity
The D294G mutation induces a selective loss in the recognition of substrates exhibiting anchoring protein properties without affecting PKC catalytic activity (Prevostel et al., 1998
). This suggests that the selectivity of PKC
targeting to cell-cell contacts might be mediated through protein-protein interactions. The D294G mutant may not be able to interact with those proteins anymore, its translocation being probably governed only by the changes in [Ca2+]i and DAG. As soon as two cells make contact, it generates a signal able to determine translocation and selectivity of translocation of PKC
and
. This signal could be either the synthesis or the relocalisation of a PKC interacting protein. This cell-cell adhesion-dependent factor could play the role of a shuttle to target PKC at cell-cell contacts. This is not RACK-1 since we have previously shown that it is excluded from cell-cell contacts in the GH3B6 cell line and is not co-immunoprecipitated with PKC
upon PMA stimulation (Vallentin et al., 2001
). The characterisation of this factor is under investigation.
According to our hypothetical model (Fig. 8), inactive PKC is sequestered in the cytoplasm through the binding of its C2 and V3 regions with a cytoplasmic RICK (receptor for inactivated C kinase) protein. The pseudosubstrate sequence is bound to the catalytic core and to a protein that may or may not be distinct from the C2-V3 binding protein. The signal induced by intercellular adhesion, probably a RACK, is present in the cytoplasm. Upon activation (increase in [Ca2+]i), the affinity of C2-V3 for the cytoplasmic anchoring protein decreases whereas its affinity for the intercellular adhesioninduced RACK increases. The interaction of PKC with this protein induces its selective targeting to cell-cell contacts. The RACK protein may only be a shuttle that transports PKC to cell-cell contacts where it can interact either directly with DAG or with existing macromolecular complexes such as adherens junctions. Alternatively, the RACK protein may be a cargo that also mediates PKC anchoring at the cell-cell contact. This model proposes a new mechanism for the regulation of PKC activity based on a co-ordinated control of cytoplasmic sequestration and targeting to cell-cell contacts. It is based on the existence of a cell-cell contact determined signal(s).
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Acknowledgments |
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Footnotes |
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References |
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Akita, Y., Ohno, S., Yajima, Y., Konno, Y., Saido, T. C., Mizuno, K., Chida, K., Osada, S., Kuroki, T., Kawashima, S. et al. (1994). Overproduction of a Ca(2+)-independent protein kinase C isozyme, nPKC epsilon, increases the secretion of prolactin from thyrotropin- releasing hormone-stimulated rat pituitary GH4C1 cells. J. Biol. Chem. 269, 4653-4660.
Akita, Y., Kawasaki, H., Ohno, S., Suzuki, K. and Kawashima, S. (2000). Involvement of protein kinase C epsilon in thyrotropin-releasing hormone-stimulated phosphorylation of the myristoylated alanine-rich C kinase substrate in rat pituitary clonal cells. Electrophoresis 21, 452-459.[CrossRef][Medline]
Alvaro, V., Touraine, P., Raisman Vozari, R., Bai-Grenier, F., Birman, P. and Joubert, D. (1992). Protein kinase C activity and expression in normal and adenomatous human pituitaries. Int. J. Cancer 50, 724-730.[Medline]
Alvaro, V., Levy, L., Dubray, C., Roche, A., Peillon, F., Querat, B. and Joubert, D. (1993). Invasive human pituitary tumours express a pointmutated alpha-protein kinase-C. J. Clin. Endocrinol. Metab. 77, 1125-1129.[Abstract]
Bolsover, S. R., Gomez-Fernandez, J. C. and Corbalan-Garcia, S. (2003). Role of the ca2+/phosphatidylserine binding region of the c2 domain in the translocation of protein kinase calpha to the plasma membrane. J. Biol. Chem. 278, 10282-10290.
Cowell, H. E. and Garrod, D. R. (1999). Activation of protein kinase C modulates cell-cell and cell-substratum adhesion of a human colorectal carcinoma cell line and restores `normal' epithelial morphology. Int. J. Cancer 80, 455-464.[CrossRef][Medline]
Csukai, M. and Mochly-Rosen, D. (1999). Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation. Pharmacol. Res. 39, 253-259.[CrossRef][Medline]
Garcia-Rocha, M., Avila, J. and Lozano, J. (1997). The zeta isozyme of protein kinase C binds to tubulin through the pseudosubstrate domain. Exp. Cell Res. 230, 1-8.[CrossRef][Medline]
Hagiwara, M., Hachiya, T., Watanabe, M., Usuda, N., Iida, F., Tamai, K. and Hidaka, H. (1990). Assessment of protein kinase C isozymes by enzyme immunoassay and overexpression of type II in thyroid adenocarcinoma. Cancer Res. 50, 5515-5519.[Abstract]
Knauf, J. A., Ward, L. S., Nikiforov, Y. E., Nikiforova, M., Puxeddu, E., Medvedovic, M., Liron, T., Mochly-Rosen, D. and Fagin, J. A. (2002). Isozyme-specific abnormalities of PKC in thyroid cancer: evidence for posttranscriptional changes in PKC epsilon. J. Clin. Endocrinol. Metab. 87, 2150-2159.
Lewis, J. E., Jensen, P. J., Johnson, K. R. and Wheelock, M. J. (1994). E-cadherin mediates adherens junction organization through protein kinase C. J. Cell Sci. 107, 3615-3621.
Liao, L., Hyatt, S. L., Chapline, C. and Jaken, S. (1994). Protein kinase C domains involved in interactions with other proteins. Biochemistry 33, 1229-1233.[Medline]
Magoulas, C., McGuinness, L., Balthasar, N., Carmignac, D. F., Sesay, A. K., Mathers, K. E., Christian, H., Candeil, L., Bonnefont, X., Mollard, P. and Robinson, I. C. (2000). A secreted fluorescent reporter targeted to pituitary growth hormone cells in transgenic mice. Endocrinology 141, 4681-4689.
Mochly-Rosen, D. and Gordon, A. S. (1998). Anchoring proteins for protein kinase C: a means for isozyme selectivity. Faseb J. 12, 35-42.
Mochly-Rosen, D., Khaner, H. and Lopez, J. (1991a). Identification of intracellular receptor proteins for activated protein kinase C. Proc. Natl. Acad. Sci. USA 88, 3997-4000.[Abstract]
Mochly-Rosen, D., Khaner, H., Lopez, J. and Smith, B. L. (1991b). Intracellular receptors for activated protein kinase C. Identification of a binding site for the enzyme. J. Biol. Chem. 266, 14866-14868.
Naor, Z. (1990). Further characterization of protein kinase-C subspecies in the hypothalamo-pituitary axis: differential activation by phorbol esters. Endocrinology 126, 1521-1526.[Abstract]
O'Brian, C., Vogel, V. G., Singletary, S. E. and Ward, N. E. (1989). Elevated protein kinase C expression in human breast tumour biopsies relative to normal breast tissue. Cancer Res. 49, 3215-3217.[Abstract]
Oancea, E., Teruel, M. N., Quest, A. F. and Meyer, T. (1998). Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells. J. Cell Biol. 140, 485-498.
Parsons, M., Keppler, M. D., Kline, A., Messent, A., Humphries, M. J., Gilchrist, R., Hart, I. R., Quittau-Prevostel, C., Hughes, W. E., Parker, P. J. et al. (2002). Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis. Mol. Cell Biol. 22, 5897-5911.
Pasdar, M., Li, Z. and Chan, H. (1995). Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells. Cell Motil. Cytoskeleton 30, 108-121.[Medline]
Pauken, C. M. and Capco, D. G. (1999). Regulation of cell adhesion during embryonic compaction of mammalian embryos: roles for PKC and betacatenin. Mol. Reprod. Dev. 54, 135-144.[CrossRef][Medline]
Perez-Moreno, M., Avila, A., Islas, S., Sanchez, S. and Gonzalez-Mariscal, L. (1998). Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium. J. Cell Sci. 111, 3563-3571.
Prevostel, C., Alvaro, V., de Boisvilliers, F., Martin, A., Jaffiol, C. and Joubert, D. (1995). The natural protein kinase C alpha mutant is present in human thyroid neoplasms. Oncogene 11, 669-674.[Medline]
Prevostel, C., Alvaro, V., Vallentin, A., Martin, A., Jaken, S. and Joubert, D. (1998). Selective loss of substrate recognition induced by the tumourassociated D294G point mutation in protein kinase Calpha. Biochem. J. 334, 393-397.[Medline]
Raghunath, A., Ling, M. and Larsson, C. (2003). The catalytic domain limits the translocation of protein kinase Calpha in response to increases in Ca2+ and diacylglycerol. Biochem. J. 370, 901-912.[CrossRef][Medline]
Schiemann, U., Assert, R., Moskopp, D., Gellner, R., Hengst, K., Gullotta, F., Domschke, W. and Pfeiffer, A. (1997). Analysis of a protein kinase C alpha mutation in human pituitary tumours. J. Endocrinol. 153, 131-137.[Abstract]
Schmitz-Peiffer, C., Browne, C. L., Walker, J. H. and Biden, T. J. (1998). Activated protein kinase C alpha associates with annexin VI from skeletal muscle. Biochem. J. 330, 675-681.[Medline]
Sheu, H. M., Kitajima, Y. and Yaoita, H. (1989). Involvement of protein kinase C in translocation of desmoplakins from cytosol to plasma membrane during desmosome formation in human squamous cell carcinoma cells grown in low to normal calcium concentration. Exp. Cell Res. 185, 176-190.[Medline]
Shimizu, T., Usuda, N., Sugenoya, A., Masuda, H., Hagiwara, M., Hidaka, H., Nagata, T. and Iida, F. (1991). Immunohistochemical evidence for the overexpression of protein kinase C in proliferative diseases of human thyroid. Cell Mol. Biol. 37, 813-821.[Medline]
Tannenbaum, G. S. and Ling, N. (1984). The interrelationships of growth hormone (GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion. Endocrinology 115, 1952-1957.[Abstract]
Vallentin, A., Prevostel, C., Fauquier, T., Bonnefont, X. and Joubert, D. (2000). Membrane targeting and cytoplasmic sequestration in the spatiotemporal localization of human protein kinase C alpha. J. Biol. Chem. 275, 6014-6021.
Vallentin, A., Lo, T. C. and Joubert, D. (2001). A single point mutation in the V3 region affects protein kinase Calpha targeting and accumulation at cell-cell contacts. Mol. Cell Biol. 21, 3351-3363.
van Hengel, J., Gohon, L., Bruyneel, E., Vermeulen, S., Cornelissen, M., Mareel, M. and von Roy, F. (1997). Protein kinase C activation upregulates intercellular adhesion of alpha-catenin-negative human colon cancer cell variants via induction of desmosomes. J. Cell Biol. 137, 1103-1116.
Williams, C. L., Hayes, V. Y., Hummel, A. M., Tarara, J. E. and Halsey, T. J. (1993). Regulation of E-cadherin-mediated adhesion by muscarinic acetylcholine receptors in small cell lung carcinoma. J. Cell Biol. 121, 643-654.[Abstract]