1 Abteilung Molekulare Neurobiologie, Max-Planck-Institut für
Experimentelle Medizin, D-37075 Göttingen, Germany
2 Abteilung Membranbiophysik, Max-Planck-Institut für Biophysikalische
Chemie, D-37077 Göttingen, Germany
* Author for correspondence (e-mail: brose{at}em.mpg.de)
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Summary |
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Key words: Protein kinase C, Phorbol ester, Munc13, Secretion, Synapse
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Introduction |
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Under equilibrium conditions, biological membranes contain very little
DAGs. Their production is stimulated upon activation of a multitude of
cellular signalling cascades, and DAGs produced by these mechanisms act as key
second messengers to modulate the function of at least six different types of
target protein, the most prominent of which belong to the protein kinase C
(PKC) family (see below) [for reviews of DAG signalling see
(Wakelam, 1998;
Hodgkin et al., 1998
;
Goni and Alonso, 1999
)].
Key enzymes in most of the DAG-generating signalling processes are the
members of the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C
family (PI-PLCß, PI-PLC, PI-PLC
, PI-PLC
). Depending
on the PI-PLC subtype, different cellular signalling molecules induce
enzymatic activity via specific cell-surface receptors (see below).
This enzymatic activity results in the hydrolysis of phosphatidylinositol
4,5-bisphosphate, which contains mainly polyunsaturated fatty acids, to
inositol 1,4,5-trisphosphate and polyunsaturated DAGs. It appears that these
polyunsaturated DAGs resulting from PI-PLC activity rather than the more
saturated forms generated by alternative enzymatic pathways (see below) are
most relevant as intracellular messengers targeting PKCs (for reviews, see
Wakelam, 1998;
Hodgkin et al., 1998
). Whether
non-PKC DAG targets show the same preference for polyunsaturated DAGs is not
known but is likely, given the similarity in pharmacological characteristics
of the respective binding sites (for reviews see
Kazanietz et al., 2000
;
Kazanietz et al., 2002). Polyunsaturated DAG second messenger molecules are
inactivated by the activity of diacylglycerol kinases
(Wakelam, 1998
;
Hodgkin et al., 1998
).
Activation of PI-PLCß isozymes is initiated by ligand binding to
G-protein-coupled receptors. Relevant receptor systems include metabotropic
receptors for classic neurotransmitters, monoamine receptors and receptors for
numerous peptide signalling molecules. Apart from the Gq type
subunits coupled to these receptors, certain Gß
subunits
can also activate PI-PLCß. PI-PLC
activation involves
phosphorylation by growth-factor-activated receptor protein tyrosine kinases
or by non-receptor protein tyrosine kinases such as Lck/Fyn or c-Src. In the
latter case, tyrosine kinase activation is mediated by G-protein-coupled
receptors and involves Gs
or Gi
subunits.
PI-PLC
isozymes can also be activated in a
protein-tyrosine-kinase-independent manner, involving phospholipid-derived
second messengers. Activation of PI-PLC
isozymes is triggered by
binding of Ca2+ to the EF-hand and C2 domains of
PI-PLC
, followed by the association of the PH domain with
phosphatidylinositol 4,5-bisphosphate. PLC
contains an RA domain that
binds to Ras. Activation of Ras (e.g. following growth factor signalling)
leads to translocation of PLC-
to the plasma membrane and enzyme
activation. In addition, PLC
is activated by G
12 (for
a review, see Rhee, 2001
).
Apart from the PI-PLC pathway, DAGs are produced from phosphatidylcholine,
which predominantly contains saturated and mono-unsaturated fatty acids, by
two subsequent reactions involving phosphatidylcholine-specific phospholipase
D (PC-PLD) and phosphatidic acid phosphohydrolase (for reviews, see
Wakelam, 1998;
Hodgkin et al., 1998
). As is
the case for PI-PLCs, the two mammalian PC-PLDs (PC-PLD1 and PC-PLD2) are
activated by a plethora of cellular signalling cascades, often involving
cell-surface receptors for signalling molecules (for reviews, see
Exton, 1998
;
Liscovitch et al., 2000
;
Cockcroft, 2001
). However, the
saturated/mono-unsaturated phosphatidic acid intermediates generated by PC-PLD
activity appear to be mainly responsible for the cellular signalling events
that are triggered by PC-PLD activation, whereas the
saturated/mono-unsaturated DAGs resulting from the subsequent phosphohydrolase
reaction may be irrelevant for signalling, at least as far as activation of
PKCs is concerned. Thus, the activity of specific phosphatidic acid
phosphohydrolases may lead to signal termination by inactivating
saturated/mono-unsaturated phosphatidic acid
(Wakelam, 1998
;
Hodgkin et al., 1998
). In
general, PC-PLD activation by cell-surface receptors is indirect and often
mediated by G-protein-coupled receptors and G-protein activation. Depending on
the cellular process, PC-PLD stimulation involves intermediate activation of
Arf- and Rho-type small GTPases as well as of PKCs
and ß. In
addition, lipid-derived signalling molecules such as phosphatidylinositol
4,5-bisphosphate or oleate, which are the products of regulated kinase or
lipase activities, stimulate PC-PLD
(Exton, 1998
;
Liscovitch et al., 2000
;
Cockcroft, 2001
).
Two additional cellular pathways of DAG production, both utilizing
phosphatidylcholine, involve phosphatidylcholine-specific PLC (PC-PLC) and
phosphatidylcholine-ceramide cholinephosphotransferase (for a review, see
Wakelam, 1998). In both
cases, mostly saturated/mono-unsaturated DAGs are produced, the signalling
role of which is questionable (see above). Moreover, direct evidence for an
involvement of DAGs generated by these pathways in mammalian cellular
signalling events is sparse, particularly where signalling cascades that are
triggered by the activation of cell surface receptors are concerned.
Considering the various possible sources of DAGs in mammalian cells, it is
evident that polyunsaturated DAGs resulting from PI-PLC activity are the most
relevant DAG second messengers. Irrespective of the PI-PLC isozyme involved,
induction of enzymatic activity causes the formation of DAG and inositol
1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate, in turn, leads to the
mobilisation of Ca2+ from intracellular stores; DAG is able to bind
to C1 domains of a large number of proteins with diverse function.
As mentioned above, the most prominent DAG targets belong to the PKC family of
serine/threonine kinases. Binding of DAG, often in synergy with
Ca2+, leads to membrane translocation and activation of PKC
isozymes (Newton, 1995;
Newton, 1997
;
Newton, 2001
). After
activation, PKCs are thought to regulate a multitude of intracellular
processes, ranging from cell proliferation to neurotransmitter and hormone
secretion. Modulation of cellular processes by DAG and by the functionally
analogous phorbol esters (natural diterpene secondary metabolites of
Euphorbiaceae and Thymelaceae, see below) has often been
attributed exclusively to activation of PKCs. This is surprising because most
eukaryotic cells contain five alternative types of DAG targets [chimaerins,
protein kinase D1 (PKD1), RasGRPs, Munc13s and DAG kinase
(Fig. 1) (for reviews see
Kazanietz, 2000
;
Kazanietz, 2002
;
Kazanietz et al., 2000
)], and
the pharmacological tools that are frequently used to study PKC function are
not sufficiently specific to exclude the involvement of other DAG targets in
cellular processes that are thought to be mediated by modulatory effects of
DAG or phorbol esters on PKCs (Betz et
al., 1998
; Kazanietz,
2000
; Kazanietz et al.,
2000
; Way et al.,
2000
; Rhee et al.,
2002
). Indeed, a number of observations indicate that the effects
of DAG and phorbol esters are not mediated by PKCs but rather involve three
alternative DAG targets in at least three key cellular processes: (1) DAG- and
phorbol-ester-mediated subcellular translocation of PKD1 is essential for
protein transport from the trans-Golgi network to the cell surface
(Matthews et al., 1999a
;
Maeda et al., 2001
;
Rey et al., 2001
;
Baron and Malhotra, 2002
;
Van Lint et al., 2002
); (2)
activation of the Ras/Raf/MEK/ERK pathway in T lymphocytes is triggered by
G-protein-coupled receptors and tyrosine-kinase-coupled receptors and is
dependent on DAG-(or phorbol-ester-) induced activation of RasGRP rather than
PKCs (Dower et al., 2000
;
Jones et al., 2002
); (3)
stimulatory effects of DAG and phorbol esters on neurotransmitter secretion
from nerve cells are mediated by DAG/phorbol-ester receptors of the
Unc-13/Munc13 family and not, as previously believed, by PKC isozymes
(Betz et al., 1998
;
Lackner et al., 1999
;
Miller et al., 1999
;
Nurrish et al., 1999
;
Rhee et al., 2002
).
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As the relevance of non-PKC DAG receptors in the DAG second messenger pathway is appreciated only by a rather small circle of experts in DAG signalling but otherwise ignored, here we critique the widely accepted model according to which all DAG/phorbol-ester effects are caused by the activation of PKC isozymes, a view that is presented in most current textbooks and forms the (at least implicit) conceptual basis of almost all past and current pharmacological studies concerned with the determination of the role of PKC isozymes in the control of cellular function. We briefly summarise established and postulated functional roles of PKCs and discuss the characteristics of pharmacological tools that are routinely used in most studies of PKC function in vivo. Subsequently, we describe major caveats of pharmacological analyses of PKC function and discuss alternative and more powerful experimental approaches such as the use of dominant-interfering PKC variants, antisense knockdown of PKC expression or PKC gene deletion in mice, which have yielded important insights into PKC function but are not part of the methodological repertoire in most studies of PKC function. We conclude with a discussion of the functional importance of four non-PKC targets of the DAG second messenger pathway, chimaerins, PKD1, RasGRPs and Munc13s with a focus on the latter.
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PKCs as paradigmatic C1-domain-containing DAG receptors |
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In all cPKCs and nPKCs, kinase activation is closely coupled to ligand
binding by C1 and C2 domains and the resulting membrane
translocation, which in turn reverses autoinhibition caused by a
pseudosubstrate site. Essentially, the C1 and C2 domains
of cPKCs and the C1 domains of nPKCs can function as independent
membrane-targeting modules such that ligand binding by each individual domain
type leads to significant membrane association
(Newton, 2001). Indeed,
phorbol ester or DAG binding to the C1 domains of nPKCs (and to a
lesser degree also of cPKCs) is sufficient for translocation and activation.
On the other hand, Ca2+-dependent phospholipid binding by the
C2 domains of cPKCs acts synergistically with DAG binding to the
respective C1 domain. Such synergistic activation of cPKC
C1 and C2 domains leads to the translocation and tight
membrane association of the enzyme, which then causes a conformational change
that reverts autoinhibition (Newton,
2001
; Hurley and Meyer,
2001
).
For proper cellular function of PKCs, their correct spatial distribution is
essential. Such spatially specific targeting of PKCs is unlikely to be brought
about by C1- and C2-domain-mediated membrane
interactions alone. Rather, specificity of PKC membrane targeting is thought
to be achieved by isozyme-specific binding proteins that are essential for the
formation of PKC-containing subcellular signalling complexes at the
appropriate subcellular location, thus spatially restricting PKC signalling
and allowing integration of PKC-mediated signalling with other intracellular
signalling pathways. PKC-binding proteins regulate targeting of PKC to
upstream activators (e.g. InaD, Syndecan-4), to substrates (e.g. STICKs,
RACKs) or to cytoskeletal and vesicular proteins (e.g. actin) (for reviews,
see Csukai and Mochly-Rosen,
1999; Jaken and Parker,
2000
).
PKCs are thought to play essential roles in multiple cellular signal
transduction pathways of eukaryotic organisms. Genetic studies in yeast have
demonstrated that the dynamic regulation of the cell wall is the major
cellular site of PKC action in this simple organism. Deletion of PKC in
Saccharomyces cerevisiae leads to arrest of protein synthesis prior
to mitosis but after DNA synthesis. The underlying signalling pathway is
triggered by Hcs77p and involves the activation of PKC by Rho1. PKC
activation, in turn, initiates a phosphorylation cascade via a MAP kinase
module that leads to the activation of several transcription factors and
transactivation of, among others, heat shock and cell wall genes
(Mellor and Parker, 1998).
Genetic studies on PKC function in mammals are more difficult to interpret
owing to the presence of multiple genes and possible functional redundancy. In
fact, all known PKC-deletion mutants show rather mild phenotypic changes.
Nevertheless, they have yielded important insights into the function of
individual PKC isozymes. (1) PKC, one of the most prominent PKC
isozymes in brain, has been shown to be important for brain functions involved
in learning and memory (Abelovic et al., 1993). Interestingly, the typical
stimulatory effects of phorbol esters on transmitter release are still
detectable in PKC
-deficient nerve cells, indicating that alternative
DAG/phorbol-ester receptors are involved in this phenomenon
(Goda et al., 1996
). (2) Lack
of PKCß leads to immunodeficiency [impaired humoral response and cellular
B cell response (Leitges et al.,
1996
)]. PKCß appears to be critically involved in
B-cell-receptor-mediated survival signalling to NF-
B
(Su et al., 2002
).
Interestingly, B-cell-receptor-mediated signalling in PKCß-deficient B
cells can still be bypassed by phorbol esters, indicating the involvement of
alternative DAG/phorbol-ester receptors in this pathway
(Leitges et al., 1996
). (3)
PKC
has been shown to be involved in the regulation of GABAA
receptor function (Hodge et al.,
1999
) and in the regulation of nociceptor function
(Khasar et al., 1999
). (4)
PKC
appears to be involved in a unique signalling pathway linking T
cell antigen receptor signalling to NF-
B activation in mature T
lymphocytes (Sun et al.,
2000
). (5) PKC
-deficient smooth muscle cells exhibit
increased apoptotic resistance (Leitges et
al., 2001a
). In addition, loss of PKC
leads to increased
antigen-induced mast cell degranulation
(Leitges et al., 2002
) and to
the prevention of B cell tolerance owing to maturation and differentiation of
self-reactive B cells (Mecklenbrauker et
al., 2002
). (6) PKC
is important for the regulation of
NF-
B transcriptional activity. As a consequence, lack of PKC
leads to impaired B cell receptor signalling, inhibition of cell proliferation
and survival and defects in the activation of ERK and the transcription of
NF-
B-dependent genes (Leitges et
al., 2001b
; Martin et al.,
2002
).
Numerous studies using alternative approaches indicate the involvement of
different PKCs in the modulation of ion channel conductance, transmitter
receptor function, smooth muscle contraction, cell migration, cell
proliferation and differentiation, apoptosis, lipogenesis, glycogenolysis, as
well as transmitter/hormone exocytosis and protein secretion [for examples
from the large number of reviews on PKC function in the literature, see
(Kanashiro and Khalil, 1998;
Dempsey et al., 2000
;
Barry and Kazanietz, 2001
;
Ventura and Maioli, 2001
)].
Apart from insights into PKC function that have been obtained in
pharmacological studies employing small molecule activators and inhibitors of
PKCs (which have particular advantages and disadvantages as discussed below),
many of the current models of PKC function originate from studies in which the
role of individual PKC isozymes was characterised using more informative
methodological approaches. These include the following: (1) overexpression of
wild-type and dominant interfering PKC mutants [e.g. PKC
as a regulator
of RelA transcriptional activity (Anrather
et al., 1999
); PKC
and PKC
as regulators of glucose
transport (Tsuru et al.,
2002
); PKC
and PKC
as regulators of
calcineurin-induced transactivation (Ishaq
et al., 2002
); (for a review, see
Dempsey et al., 2000
)]; (2)
interference with PKC expression using ribozymes [e.g. PKC
as a
regulator of glioma cell growth (Sioud
and Sorensen, 1998
)]; (3) interference with expression using
antisense oligonucleotides [for reviews of the literature with an emphasis on
therapeutically relevant approaches see
(Tamm et al., 2001
;
Goekjian and Jirousek, 2001
;
Swannie and Kaye, 2002
)]; and
(4) interference with PKC function using peptides that induce or block PKC
interactions with targeting proteins (for reviews, see
Csukai and Mochly-Rosen, 1999
;
Jaken and Parker, 2000
).
All the above approaches are conceptually and experimentally more stringent than the classic pharmacological studies (see below), although they have their individual caveats. Overexpression often results in levels of wild-type or dominant-negative PKC variants that exceed endogenous levels by an order of magnitude or more. As a result, overexpressed wild-type PKC isozymes may participate in signalling processes that they are usually not involved in, and mutant variants (e.g. kinase-deficient mutants) may interfere in a dominant-negative manner with signalling to other targets of signalling pathways (e.g. the DAG second messenger pathway). These problems can be accounted for by complementing data obtained in overexpression studies with data obtained using deletion mutations of the corresponding PKC isoform under investigation. Ribozymes and particularly antisense oligonucleotides often yield only partial knockdown of expression levels. Peptides, by contrast, are often used at rather high concentrations such that non-specific effects must be excluded. Irrespective of the distinct advantages of these experimental approaches, the majority of studies of PKC function in different cell biological processes are not characterised by a comparable conceptual and experimental stringency. In most of these cases, which are typically concerned with the problem of whether PKCs in general are involved in a given cellular process, commercially available pharmacological tools are used to activate or inhibit PKCs. The main caveat with these pharmacological studies is that neither the almost exclusively used phorbol-ester-derived PKC activators nor many of the commonly used PKC inhibitors are specific for PKCs (see below).
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Pharmacological tools to interfere with PKC function |
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As is the case for C1-domain-directed PKC activators,
C1-domain-directed PKC inhibitors are non-specific pharmacological
tools that bind with comparable affinity to other C1 domain
proteins. Such non-specific inhibitors include one of the PKC inhibitors used
most widely in the past, calphostin C
(Betz et al., 1998; for a
review, see Barry and Kazanietz,
2001
). For the functional separation of PKC-specific effects from
those mediated by alternative DAG/phorbol-ester receptors, some of the most
useful pharmacological tools are ATP-binding site inhibitors. Although many of
these (e.g. the indolocarbazole staurosporin, some balanol analogs,
phenylaminopyrimidines, and rottlerin) inhibit protein kinases
non-specifically, certain indolocarbazoles (e.g. Midostaurin/CGP41251,
Gö6976, Gö7612, Gö7874, UCN-01) and bisindolylmaleimides
(Gö6850, Gö6983, LY-333531, LY-379196, LY-317615) are rather PKC
specific, some even show a preference for certain isozymes
(Kanashiro and Khalil, 1998
;
Barry and Kazanietz, 2001
;
Way et al., 2000
;
Goekjian and Jirousek, 2001
;
Swannie and Kaye, 2002
).
The main problem with some of the most specific bisindolylmaleimide-derived
PKC inhibitors is their partial toxicity in certain situations. Gö6859,
for example, causes a dramatic nonspecific rundown of synaptic transmission in
primary hippocampal nerve cells without affecting phorbolester effects in this
system (Fig. 2) (Rhee et al., 2002). Given
that most PKC inhibitors are usually applied according to a preincubation
paradigm (i.e. for minutes) and at rather high concentrations, even mild
nonspecific or toxic effects of such drugs can have profound consequences.
Nevertheless, the indolecarbazol CGP41251/midostaurin and the
bisindolylmaleimide LY-333531 have advanced to late-stage clinical
developmental for the treatment of cancer and other indications, demonstrating
that these drugs act quite specifically under carefully controlled conditions
(for reviews, see Goekjian and Jirousek,
2001
; Swannie and Kaye,
2002
).
|
Apart from small molecule inhibitors of PKC, a recently emerged alternative
pharmacological approach to perturb PKC activity involves peptides that
interfere with the membrane translocation and targeting of PKCs by blocking or
inducing their interaction with anchoring proteins such as RACKs. Currently
PKC-isozyme-specific inhibitor and activator peptides for all cPKCs and nPKCs
are available. They have proven to be useful tools in dissecting signalling
processes mediated by individual PKC isozymes [e.g. in the context of PKC
effects in cardiac myocytes on contraction, ischemic cell death, MAP kinase
activation and ion channel activity (for reviews, see
Csukai and Mochly-Rosen, 1999;
Schechtman and Mochly-Rosen,
2002
)]. Unfortunately, the use of PKC-isozyme-specific interfering
peptides has been restricted to a rather small number of studies. In the
majority of pharmacological studies on PKC function, the potential of the
peptide interference method has been ignored, and experimental approaches have
been limited to the default use of classic but often problematic
pharmacological tools (i.e. phorbol esters, indolocarbazoles, and
bisindolylmaleimides) described above.
An additional promising approach for interfering with PKC function involves
the use of antisense oligonucleotides to knockdown PKC expression. In
particular, downregulation of PKC expression using antisense
oligonucleotides appears to have significant therapeutic potential
(Dean and McKay, 1994
) (for
reviews, see Tamm et al.,
2001
; Goekjian and Jirousek,
2001
; Swannie and Kaye,
2002
). Despite its high potential and usefulness for systematic
analyses of PKC function, the antisense approach is very rarely used in basic
research and largely ignored in the majority of pharmacological studies of PKC
function.
In summary, the most frequently used pharmacological tools for PKC
activation and inhibition (i.e. phorbol esters, indolocarbazoles and
bisindolylmaleimides) are not sufficiently specific to define PKC-mediated
physiological effects unequivocally in any experimental paradigm
particularly the separation of PKC-mediated effects from those caused by other
C1 domain proteins or by other kinases remains difficult. In view
of the fact that most pharmacological studies of PKC function involve
phorbol-ester-mediated perturbations of certain cellular parameters, followed
by the addition of rather nonspecific PKC inhibitors, the involvement of
alternative DAG/phorbol-ester receptors in the observed effects must be
considered wherever phorbol esters are used as the main investigative tools.
PKC-isozyme-specific inhibitor and activator peptides and certain antisense
oligonucleoides are the most promising pharmacological tools to circumvent the
problems involved in the exclusive use of phorbol esters, indolocarbazoles and
bisindolylmaleimides to activate or inhibit PKCs. In addition, systematic
genetic studies represent an essential experimental alternative. The fact that
deletion mutations of PKC (Abelovic et al., 1993), PKCß
(Leitges et al., 1996
),
PKC
(Hodge et al., 1999
;
Khasar et al., 1999
),
PKC
(Sun et al.,
2000
), PKC
(Leitges et
al., 2001a
) or PKC
(Leitges et al., 2001b
) in
mice have rather mild phenotypic consequences indicates that there is
functional redundancy among the various PKC isozymes. To account for this
problem and ultimately to determine the role of individual PKCs, multiple
deletion mutations (e.g. of related PKC isozymes) may also be needed. Such
genetic approaches in intact animals could then be ideally complemented with
protein overexpression approaches.
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Non-PKC C1 domain proteins |
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Interestingly, all of the proven non-PKC DAG/phorbol-ester receptors
identified here function in intracellular signalling pathways that at
least according to pharmacological evidence are thought to be also
regulated by PKCs. Such functional overlap between PKC and non-PKC
DAG/phorbol-ester receptors is particularly evident in the case of the
chimaerins, which are thought to play a critical role in the regulation of the
actin cytoskeleton, cell cycle progression and malignant transformation (for
reviews, see Ron and Kazanietz,
1999; Kazanietz,
2000
; Kazanietz,
2002
; Kazanietz et al., 2002). It is similarly evident in the case
of RasGRPs, which function in the control of cell proliferation,
differentiation and transformation by regulating the Ras/Raf/MEK/ERK pathway
(Dower et al., 2000
;
Jones et al., 2002
), and in
the case of Munc13s, which are essential regulators of secretory vesicle
priming and transmitter/hormone release
(Betz et al., 1998
;
Rhee et al., 2002
; for
reviews, see Brose et al.,
2000
; Lloyd and Bellen,
2001
). Given the limited specificity of the commonly used
PKC-directed pharmacological tools (particularly of the universally used
phorbol esters), the significant number of putative non-PKC DAG/phorbol-ester
receptors and the involvement of proven non-PKC DAG/phorbol-ester receptors in
cellular processes that have often been associated with PKC function in the
past, it is very likely that several of the identified phorbol-ester and DAG
effects in mammalian cells are in fact mediated by non-PKC DAG/phorbol-ester
receptors. This view is supported by a number of studies in which
pharmacological effects of phorbol esters and other PKC-directed drugs could
not be correlated with PKC function (e.g.
Scholfield and Smith, 1989
;
Fabbri et al., 1994
;
Simon et al., 1996
;
Redman et al., 1997
;
Stevens and Sullivan, 1998
;
Hori et al., 1999
;
Honda et al., 2000
;
Iwasaki et al., 2000
;
Waters and Smith, 2000
). More
recently, several studies provided direct evidence for the functional
importance of the regulation of four non-PKC DAG/phorbol-ester receptors by
DAG and phorbol esters in distinct cellular processes.
![]() |
Functional relevance of non-PKC DAG/phorbol-ester receptors as targets of the DAG second messenger pathway |
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In common with cPKCs and nPKCs, chimaerins translocate to phospholipid
membranes in response to phorbol-ester binding
(Caloca et al., 1997).
Chimaerins are implicated in diverse cellular processes, such as cell adhesion
(Herrera and Shivers, 1994
),
cytoskeletal dynamics (Herrera and
Shivers, 1994
), lamellipodium/filopodium formation
(Kozma et al., 1996
),
phagocytosis (Cox et al.,
1997
), neuritogenesis and nerve cell development
(Leung et al., 1994
;
Hall et al., 2001
). The
Rac-GTPase-activating function of chimaerins
(Diekmann et al., 1991
) is
likely to be involved in these processes but direct evidence for a function of
chimaerins in signalling to Rac in vivo is still lacking. Current information
on the function of chimaerins is mostly derived from overexpression studies,
and additional work using complementary methods is needed to verify an in vivo
role of chimaerins in the processes mentioned above.
The C1 domains of chimaerins are high-affinity DAG/phorbol-ester
binding sites (Ahmed et al.,
1990; Ahmed et al.,
1993
; Areces et al.,
1994
; Caloca et al.,
1997
; Caloca et al.,
2001
), and chimaerins act as functional phorbol-ester receptors
when overexpressed in cells. ß2-chimaerin, for example, translocates from
a cytosolic compartment to the plasma and Golgi membranes after phorbol-ester
treatment (Caloca et al.,
2001
; Wang and Kazanietz,
2002
). This translocation is dependent on an intact C1
domain and thought to be supported in vivo by an additional interaction with a
cis-Golgi transmembrane protein, Tmp21-I
(Wang and Kazanietz, 2002
).
However, phorbol esters do not (or do only very weakly) affect the
GTPase-activating function of chimaerins
(Ahmed et al., 1993
;
Kazanietz, 2002
), which
indicates that the function of DAG binding is primarily to translocate
chimaerins to membranes, thus spatially restricting their
Rac-GTPase-activating effects. How this membrane translocation of chimaerins
relates to Rac signalling, particularly in the case of Golgi membranes, is
unknown (Kazanietz et al., 2002).
PKD1
The PKD family consists of PKD1 (also called PKCµ), PKD2 and PKD3 (also
called PKC) (for a review, see Van
Lint et al., 2002
). These enzymes form a subfamily of the AGC
superfamily of serine/threonine kinases that is structurally related to but
distinct from other AGC superfamily members such as PKCs
(Valverde et al., 1994
;
Nishikawa et al., 1997
;
Hayashi et al., 1999
;
Sturany et al., 2001
). PKD1,
the most prominent family member, contains an N-terminal apolar domain, two
C1 domains, a negatively charged central domain, a
pleckstrin-homology domain and a serine/threonine kinase domain
(Fig. 1). PKD1 is activated by
multiple signalling mechanisms. A major PKD1 activation mechanism involves
protein phosphorylation by PKC
and/or PKC
, which is likely to be
triggered by G-protein-coupled receptors followed by activation of
PI-PLCß and concomitant DAG production
(Iglesias et al., 1998
;
Matthews et al., 1999b
;
Vertommen et al., 2000
;
Waldron et al., 2001
). This
functional interaction between PKD1 and PKCs, which is a striking example of a
mechanistic coupling between two types of DAG/phorbol-ester receptors, can be
triggered in vivo by neuropeptides (via a pathway involving G-protein-coupled
receptors and PI-PLCß), growth factors (via activation of PI-PLC
)
or even oxidative stress (via Src and PI-PLC
) (for a review, see
Van Lint et al., 2002
). In
addition, PKD1 is regulated by 14-3-3 proteins
(Hausser et al., 1999
),
Gß
subunits (Jamora et al.,
1999
) and by caspase-mediated cleavage
(Endo et al., 2000
).
Although the corresponding evidence is in some cases still fragmentary and
often relies only on protein overexpression studies, PKD1 is thought to be
involved in the regulation of several cellular processes, including cell
proliferation (Rennecke et al.,
1999), cancer cell invasion of tissues
(Bowden et al., 1999
) and
apoptosis (Johannes et al.,
1998
). The best characterised function of PKD1 is its regulatory
role in the Golgi apparatus, where it is required for transport vesicle
formation and transport of proteins from the Golgi apparatus to the plasma
membrane (Jamora et al., 1999
;
Liljedahl et al., 2001
).
According to a current model, PKD1 is recruited to the Golgi apparatus by
binding of its first C1 domain to DAG in the Golgi membrane.
Together with effector proteins, PKD1 then forms a vesicle budding complex
that causes membrane deformation, formation of short tubules and finally
vesicle fission (for a review, see Van
Lint et al., 2002
). Binding of DAG to the first C1
domain of PKD1 is mainly involved in targeting and localisation of the kinase
(Maeda et al., 2001
;
Baron and Malhotra, 2002
). In
addition, the C1 domains of PKD1 may be involved in the regulation
of its kinase activity (Hausser et al.,
2002
).
RasGRPs
RasGRPs form a family of four isoforms that are characterised by an
N-terminal RasGEFN/RasGEF motif, an EF-hand motif and a C-terminal
C1 domain (Fig. 1)
(Ebinu et al., 1998;
Clyde-Smith et al., 2000
;
Rebhun et al., 2000
;
Lorenzo et al., 2001
;
Yang et al., 2002
).
Through their RasGEFN/RasGEF domains, all RasGRPs act as activators of Ras
and related small GTPases (Ebinu et al.,
1998; Clyde-Smith et al.,
2000
; Rebhun et al.,
2000
; Lorenzo et al.,
2001
; Yang et al.,
2002
) and thus stimulate the Ras/Raf/MEK/ERK pathway. Several
lines of evidence indicate that RasGRPs are involved in cell transformation
(Ebinu et al., 1998
;
Clyde-Smith et al., 2000
;
Dupuy et al., 2001
), T cell
receptor signalling and T cell differentiation
(Dower et al., 2000
;
Ebinu et al., 2000
), and
neuronal differentiation of PC12 cells
(Yamashita et al., 2000
). The
basis of these functional roles is in most cases the Ras-activating function
of RasGRPs and the resulting activation of the Ras/Raf/MEK/ERK pathway.
RasGRP1 and RasGRP3 have now been shown to be high-affinity
DAG/phorbol-ester receptors (Lorenzo et
al., 2000; Lorenzo et al.,
2001
), and RasGRP1 translocates to membrane compartments in
response to phorbol-ester treatment (Ebinu
et al., 1998
; Tognon et al.,
1998
). In intact cells, RasGRP1 couples muscarinic acetylcholine
receptors (Guo et al., 2001
)
and T cell receptors (Dower et al.,
2000
; Jones et al.,
2002
) to the Ras/Raf/MEK/ERK pathway independently of PKCs.
Indeed, in Ras signalling assays and cell proliferation assays, mutant
thymocytes that lack RasGRP1 are insensitive to phorbol esters and T cell
receptor activation (Dower et al.,
2000
). These genetic data, together with evidence from
complementary studies described above demonstrate beautifully that DAG-induced
induction of the Ras/Raf/MEK/ERK pathway at least in thymocytes
is entirely dependent on RasGRP1 and unlikely to involve PKCs. This
discovery is particularly striking because numerous studies in different cell
types have related the activation of the Ras/Raf/MEK/ERK pathway to PKC
activity in almost all cases relying on the conventionally used
pharmacological tools for PKC activation and inhibition, that is, phorbol
esters, indolocarbazoles, and bisindolylmaleimides (for reviews, see
Goekjian and Jirousek, 2001
;
Ventura and Maioli, 2001
).
The data obtained in RasGRP1 deleted mutant thymocytes provide the first
direct and convincing evidence for a cellular DAG signalling pathway that is
mediated by a non-PKC DAG/phorbol-ester receptor rather than by PKCs, as had
been thought previously. It is likely that thymocytes are not the only cell
type in which allegedly PKC-mediated effects on the Ras/Raf/MEK/ERK pathway
are in fact caused by RasGRPs.
Munc13s
Munc13 proteins constitute a family of three mammalian homologues of
Caenorhabditis elegans Unc-13 that are specifically localised to
presynaptic active zones, the transmitter secreting compartment of neurons
(Munc13-1, -2 and -3) (Brose et al.,
1995; Augustin et al.,
1999a
; Betz et al.,
1998
). They are characterised by an N-terminal C2
domain (in Munc13-1 and ubMunc13-2), a central C1/C2
tandem domain and a C-terminal C2 domain
(Fig. 1). In C.
elegans, Unc-13 is essential for coordinated movement
(Brenner, 1974
). Functional
analyses in deletion mutant mice, C. elegans and Drosophila
showed that Unc-13 and Munc13s are essential for synaptic vesicle priming
(Augustin et al., 1999b
;
Richmond et al., 1999
;
Aravamudan et al., 1999
;
Augustin et al., 2001
;
Varoqueaux et al., 2002
). At
the molecular level, Unc-13 and Munc13s act by unfolding and activating the
SNARE protein syntaxin and thereby promoting SNARE complex formation
(Betz et al., 1997
;
Brose et al., 2000
;
Richmond et al., 2001
). In the
absence of Unc-13/Munc13-mediated vesicle priming, synapses are completely
unable to secrete neurotransmitter
(Richmond et al., 1999
;
Aravamudan et al., 1999
;
Varoqueaux et al., 2002
).
All Munc13 isoforms bind phorbol esters and DAG with high affinity and
in common with PKCs translocate to the plasma membrane in
response to phorbol-ester binding (Betz et
al., 1998; Ashery et al.,
2000
). As is the case for PKC C1 domains
(Hommel et al., 1994
;
Quest et al., 1994
), mutation
of the first histidine residue in the Munc13-1 C1 motif to lysine
(H567K) abolishes DAG and phorbol-ester binding as well as
phorbol-ester-dependent membrane translocation
(Betz et al., 1998
). These
findings led to the hypothesis that Munc13 proteins are functional presynaptic
phorbol-ester receptors and targets of the DAG second messenger pathway that
act in parallel with PKCs to regulate transmitter release
(Betz et al., 1998
). This
hypothesis conflicted with numerous pharmacological studies that had
identified PKCs as the main mediators of phorbol-ester effects on transmitter
release from hippocampal neurons and that had established the concept that
PKCs are the only physiological DAG-dependent mediators of enhanced
neurotransmitter output that have a role in transient and long-term
potentiation of synaptic strength
(Stevens and Sullivan, 1998
)
(for a review, see Majewski and Iannazzo,
1998
).
The functional relevance of binding of DAG/phorbol esters to Munc13-1 in
vivo was determined in knockin mutant mice that express the
DAG/phorbol-ester-binding-deficient Munc13-1H567K mutant
instead of the wild-type Munc13-1 from the endogenous Munc13-1 locus
(Rhee et al., 2002).
Homozygous Munc13-1H567K mutant mice die immediately after birth,
demonstrating that an intact Munc13-1 C1 domain is essential for
survival. Hippocampal nerve cells from homozygous
Munc13-1H567K mutants are almost completely insensitive to
phorbol esters, whereas wild-type cells show robust increases in transmitter
release in response to phorbol-ester treatment
(Rhee et al., 2002
). The
residual phorbol-ester sensitivity in homozygous
Munc13-1H567K cells is due to the presence of small
amounts of Munc13-2, as demonstrated by the complete lack of phorbol-ester
responses in cells that express Munc13-1H567K in a
Munc13-2 deletion mutant background
(Fig. 3). Because expression
and function of PKCs is unaffected in Munc13-1H567K
mutants and Munc13-1 is not a substrate of phorbol-ester-activated PKCs
(Rhee et al., 2002
), these
genetic data indicate that the phorbol-ester-induced augmentation of
neurotransmitter release from hippocampal nerve cells is mediated exclusively
by Munc13 proteins and not by PKCs. PKC
, one of the prominent PKCs in
synapses, has been shown not to be involved in mediating phorbol-ester effects
on transmitter secretion (Goda et al.,
1996
). Thus, Munc13s rather than PKCs are the only functionally
relevant, phorbol-ester-and DAG-sensitive presynaptic regulators of
transmitter release.
|
In view of the Munc13-1H567K mutant phenotype in
hippocampal neurons, it is possible that other documented effects of phorbol
esters on regulated secretory processes are also mediated by Munc13s rather
than by PKCs. In this context, future genetic studies will have to determine
whether published phorbol-ester effects on the release of catecholamines
(chromaffin cells), insulin (ß cells), growth hormone (pituitary),
acetylcholine (neuromuscular junction) or dopamine (striatum) are mediated by
PKCs or Munc13s (Kanashiro and Khalil,
1998).
The fact that homozygous Munc13-1H567K mutant mice die
immediately after birth demonstrates that Munc13-1 in contrast to
individual PKC isoforms is an essential functional target of the DAG
second messenger pathway in the brain. Detailed physiological analyses showed
that the replacement of wild-type Munc13-1 with a DAG-binding-deficient
Munc13-1H567K mutant leads to striking functional changes
in hippocampal nerve cells. Munc13-1H567K mutant cells
exhibit a reduction in the number of fusion-competent vesicles, a stronger
depression of synaptic transmitter release during high-frequency action
potential trains, and a reduction in the activity-dependent refilling of the
fusion competent vesicle pool (Rhee et
al., 2002). These data indicate that DAG-dependent activation of
Munc13-1 allows nerve cells to adjust their vesicle priming machinery to
increases in activity levels. High-frequency stimulation and concomitant
Ca2+ influx or activation of presynaptic receptors appears to
activate PI-PLC isozymes (e.g. PI-PLC
and PI-PLCß) and thus lead
to transient increases in synaptic levels of DAG, which in turn binds to the
C1 domain of Munc13-1 and boosts its priming activity
(Rhee et al., 2002
;
Rosenmund et al., 2002
). The
fact that Munc13-1H567K mutant mice die immediately after
birth indicates that the C1-domain-dependent stimulation of
Munc13-1 activity and the resulting adaptation to high-activity levels is
important for neurons involved in essential body functions (e.g. rhythmically
active nerve cells in the respiratory system).
A molecular model of how Munc13-1 activation by DAG regulates synaptic
efficacy during periods of high synaptic activity can be inferred from the
mechanism of DAG-dependent membrane recruitment of PKCs
(Fig. 4). Munc13-1 is present
in a soluble pool and a pool that is tightly associated with the cytoskeletal
matrix of the presynaptic active zone by a proteinaceous linker
(Betz et al., 2001), and
soluble Munc13-1 can translocate to the plasma membrane in a phorbol-ester
dependent (and presumably also DAG-dependent) manner
(Betz et al., 1998
;
Ashery et al., 2000
). The
insoluble, active zone resident Munc13-1 is functionally integrated into the
release machinery of the active zone, has access to all necessary regulatory
proteins (Betz et al., 2001
)
and may define the basal pool of fusion competent vesicles that are
characterised by slow pool-refilling rates and high vesicular release
probability Pvr (Rhee et al.,
2002
). A second pool of fusion-competent vesicles that is
dependent on the Munc13-1 C1 domain and characterized by fast
refilling rates but low Pvr
(Rhee et al., 2002
) may be
generated by Munc13-1 molecules that have been recruited from the cytosol to
the presynaptic plasma membrane in a DAG-dependent manner. These recruited,
non-active-zone-resident Munc13-1 molecules could represent `ectopic' priming
sites that are partially functional but lack active-zone-specific regulatory
components, hence their fast refilling rate and low Pvr. In the
absence of stimulation, the number of `ectopic' priming sites would depend on
the resting DAG level in the presynaptic active zone plasma membrane.
Tonically present `ectopic' priming sites would be largely eliminated in
Munc13-1H567K neurons, leading to the observed reduction
in the size of the readily releasable vesicle pool
(Rhee et al., 2002
). The
number of `ectopic' sites would be increased by activity-dependent increases
in membrane DAG levels or by phorbol esters, which indeed cause increases in
the size of the readily releasable vesicle pool
(Stevens and Sullivan,
1998
).
|
Currently, the endogenous neurotransmitter systems and signal transduction
pathways that target Munc13s in intact mammalian neuronal networks are
unknown. Possible candidate mechanisms involve muscarinic and metabotropic
serotonergic systems, which appear to control the function of the C.
elegans Munc13 homologue UNC-13 via Gq/PI-PLCß and
G0
/DAG kinase, respectively
(Lackner et al., 1999
;
Miller et al., 1999
;
Nurrish et al., 1999
). Indeed,
presynaptic localisation of the soluble Unc-13 MR splice variant appears to be
regulated by DAG (via the G0
/DAG kinase pathway) in a manner
compatible with the postulated mechanism of DAG-dependent Munc13 recruitment
depicted in Fig. 4
(Nurrish et al., 1999
).
![]() |
Conclusions |
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Acknowledgments |
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References |
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