Center for Experimental Therapeutics and Department of
Pharmacology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania
In recent years, there have been great advances in our understanding of
the pharmacology and biology of the receptors for the phorbol ester
tumor promoters and the second messenger diacylglycerol (DAG). The
traditional view of protein kinase C (PKC) as the sole receptor for the
phorbol esters has been challenged with the discovery of proteins
unrelated to PKC that bind phorbol esters with high affinity,
suggesting a high degree of complexity in the signaling pathways
activated by DAG. These novel "nonkinase" phorbol ester receptors
include chimaerins (a family of Rac GTPase activating proteins),
RasGRPs (exchange factors for Ras/Rap1), and Munc13 isoforms
(scaffolding proteins involved in exocytosis). In all cases, phorbol
ester binding occurs at the single C1 domain present in these proteins
and, as in PKC isozymes, ligand binding is a phospholipid-dependent
event. Moreover, the novel phorbol ester receptors are also subject to
subcellular redistribution or "translocation" by phorbol esters,
leading to their association to different effector and/or regulatory
molecules. Clearly, the use of phorbol esters as specific activators of
PKC in cellular models is questionable. Alternative pharmacological and
molecular approaches are therefore needed to dissect the involvement of
each receptor class as a mediator of phorbol ester/DAG responses.
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Introduction |
The
phorbol esters and related derivatives are the most widely used tumor
promoting agents in animal models of carcinogenesis. These diterpenes
have been extensively studied as ligands and activators of protein
kinase C (PKC), a family of serine-threonine kinases that transduce
signals upon activation of tyrosine kinase and G-protein coupled
receptors. It is well established that PKC is a key mediator of growth
factor, hormone, and neurotransmitter actions, and it has been
implicated in the control of numerous cellular functions, including
proliferation, differentiation, and apoptosis. Phorbol esters mimic the
actions of diacylglycerol (DAG), a lipid second messenger generated
directly by the action of phospholipase C isozymes or indirectly by the
phospholipase D/phosphatidic acid (PA) pathway. The higher potency and
stability of the phorbol esters compared with their corresponding DAG
analogs explains the widespread use of these compounds in cellular
studies (Blumberg, 1991
; Kazanietz, 2000).
It has long been known that PKC is the main receptor for the phorbol
ester tumor promoters. Binding of phorbol esters to PKC requires
phospholipids, and acidic phospholipids are the most efficient
cofactors for ligand binding. DAG or phorbol esters are required for
the reversible recruiting of PKC to membranes, a process referred to as
"PKC translocation". Specific modules in PKC isozymes are required
for these lipid interactions as well as for protein-protein
associations that regulate subcellular targeting. In this regard, the
conserved C1 and C2 domains in PKC isozymes play a key role in membrane
association. This review will focus on the molecular interactions
between the phorbol esters and their binding site, the C1 domain. The
main issue that will be discussed here is the novel concept that
phorbol esters and DAG can also mediate cellular responses through the
activation of proteins unrelated to PKC that possess a C1 domain.
Although popular models of DAG signaling and phorbol ester activation
describe PKC as their main receptor, the involvement of novel
"nonkinase" phorbol ester receptors has received considerably less attention.
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Phorbol Ester Responsive and Unresponsive C1 Domains |
Based on their structural and biochemical properties, PKC isozymes
can be categorized into three groups, of
which only two (cPKCs and nPKCs) bind phorbol esters and DAG. The
classical or conventional PKCs (cPKCs) include PKC
, -
I, -II,
and -
, which are physiologically regulated by calcium and DAG. The
novel PKCs (nPKCs) include PKC
, -
, -
, and -
. The nPKC
isozymes are calcium-independent but can be activated by DAG. The
atypical PKCs (PKC
and 
/
) are calcium-insensitive and phorbol
ester/DAG-unresponsive. A related kinase, PKCµ/PKD, can also be
regulated by phorbol esters, but the pattern of substrate specificity
is totally different from that of PKC isozymes
(Fig. 1). Details of the structural aspects of PKC isozymes and regulation of PKC function can be found in
many excellent reviews published in recent years (Hurley et al., 1997;
Newton and Johnson, 1998; Csukai and Mochly-Rosen, 1999; Dempsey et
al., 2000; Jaken and Parker, 2000; Parekh et al., 2000; Cho, 2001).
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Fig. 1.
Structure of PKC isozymes and novel phorbol ester
receptors. PS, pseudosubstrate domain; PH, PH domain; SH2, SH2 domain;
T, transmembrane domain; Rac-GAP, Rac GTPase-activating protein domain;
REM, Ras exchange motif; RasGEF, region with homology to the nucleotide
exchange factor domain of Sos; EF, EF hands. The C1 domain is
responsible for the high-affinity binding of phorbol esters and DAG.
The aPKCs (not included in the figure) possess a single C1 domain that
is unable to bind DAG or phorbol esters.
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Through a series of deletional studies and site-directed mutagenesis,
it was established that the C1 domain, a motif of 50 or 51 amino acids
located in the N-terminal regulatory region of PKC, is the minimum
domain required for phorbol ester/DAG binding (Ono et al., 1989;
Kazanietz et al., 1994, 1995a; Quest et al., 1994). This domain is
duplicated in tandem (C1a and C1b) in cPKCs, nPKCs, and in
PKCµ/PKD. The C1 domains are rich in cysteine and possess the motif
HX12CX2CX13/14CX2CX4HX2CX7C,
where H is histidine, C is cysteine, and X is any other amino acid. The
two histidines and five of the cysteines coordinate two
Zn2+ ions in each C1 domain. Mutation of any of
the essential histidines or cysteines affects the structural integrity
of the domain and consequently disrupts ligand binding (Kazanietz et
al., 1995c). Important features have been revealed when the structure
of the PKC C1b domain in complex with phorbol ester was elucidated
by X-ray crystallography (Zhang et al., 1995). The domain consists of
two small sheets and a C-terminal -helix. Phorbol esters insert
lengthwise into a narrow groove between two pulled-apart strands at
one tip of the domain, and in this way form a contiguous hydrophobic
surface. The acyl chain of phorbol esters is involved in the insertion
of the C1 domain into the membrane. It is also recognized that
hydrophobic residues at the rim of the binding cleft that are
positioned toward the membrane are critical for ligand and lipid
interactions (Medkova and Cho, 1999; Wang et al., 2001). Ligand binding
to the C1 domain leads to a large-scale conformational change in PKC
that results in the allosteric activation of the enzyme and stimulation
of its phosphotransferase activity (Orr et al., 1992; Newton, 1997;
Dutil and Newton, 2000).
Although many proteins that have C1 domains are found in databases, in
most cases, these C1 domains lack essential features for phorbol
ester/DAG recognition, such as in the case of PKC, Raf-1, DAG
kinases, or Vav (this last protein was mistakenly defined as a phorbol
ester receptor in earlier articles). Interestingly, a similar overall
topology was observed for the phorbol ester-sensitive and phorbol
ester-insensitive C1 domains, as revealed by structural studies of the
Raf-1 C1 domain. However, residues that are critical for ligand binding
are not present in these phorbol ester-insensitive C1 domains. For
example, a loop between two strands is absent in the Raf-1 C1
domain, and some of the essential hydrophobic residues are not present.
Nevertheless, the Raf-1 C1 domain binds acidic phospholipids and is
probably involved in the interaction with Ras (Mott et al., 1996),
suggesting that phorbol ester-unresponsive C1 domains are still
implicated in lipid- and/or protein-protein associations.
One of the important novel concepts that emerged in the past few years
is that C1 domains of proteins unrelated to the PKC isozymes are
capable of binding phorbol esters with high affinity (Ron and
Kazanietz, 1999; Kazanietz, 2000). A key finding was the discovery of
n-chimaerin, a protein unrelated to PKCs that has a phorbol
ester-responsive C1 domain (Hall et al., 1990). When expressed in
Escherichia coli as a TrpE- or GST-fusion protein, recombinant n-chimaerin bind
[3H]phorbol 12,13-dibutyrate (PDBu) after
renaturation in the presence of Zn2+. Although
the initial binding study revealed a dissociation constant (Kd) for
[3H]PDBu higher than those reported for PKC
isozymes (Ahmed et al., 1990), a subsequent characterization of this
protein revealed affinities for phorbol esters and DAG that were
indistinguishable from those of PKCs (Areces et al., 1994). Several
additional proteins possessing a single C1 domain were later defined as
phorbol ester receptors: Caenorhabditis elegans Unc-13 and
its mammalian homologs, the Munc13s (Maruyama and Brenner, 1991; Brose
et al., 1995) and mammalian RasGRP (Ebinu et al., 1998).
Pharmacological characterization of these proteins indicates that they
all have the structural elements required for phorbol ester binding
within the C1 domain. The alignment of phorbol ester-responsive C1
domains is shown in Fig. 2. A unique
feature of these novel phorbol ester receptors is that, unlike PKC
isozymes, they do not have a kinase domain in their structure. These
findings raised the hypothesis that DAG signaling may proceed through
alternative, PKC-independent pathways.
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Fig. 2.
Alignment of the C1 domains of novel phorbol ester
receptors. The C1 domain of RasGRP2 has been included even if there is
no evidence yet that it binds phorbol esters. The PKC C1b domain is
included for comparison. Conserved residues are shown in bold.  ,
aromatic amino acid;  , hydrophobic amino acid (I, L, V, M);  ,
basic amino acid (K, R);  , acidic amino acid (D, E).
h, human; r, rat.
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Ligand Binding Properties of the Novel Phorbol Ester Receptors |
The structure of the novel "non-PKC" phorbol ester receptors
is shown in Fig. 1. Based on the experimental evidence collected in the
last years, it is now clear that all phorbol ester receptors bind their
ligands with high affinity, although marked differences in
structure-activity and lipid-cofactor requirements exist among them. In
the following sections, a detailed characterization of the novel
"nonkinase" phorbol ester receptors is presented.
Pharmacological Properties of Chimaerins.
This novel family of
phorbol ester receptors resembles a "chimaera" between the
regulatory region of PKC isozymes and BCR, the breakpoint cluster
region protein involved in the translocation of Philadelphia chromosome
in chronic myelogenous leukemia. n-Chimaerin (later renamed
1-chimaerin) was originally isolated as a 34-kDa protein highly
expressed in brain (Hall et al., 1990). Three additional isoforms
(2-, 1-, and 2-chimaerin) were isolated later, all of which
had a single C1 domain highly homologous to C1 domains in PKC isozymes
(see Fig. 2). These proteins are alternative spliced products from the
- and -chimaerin genes. Because splicing occurs upstream of the
C1 domain, products from each gene have identical C1 domains (Hall et
al., 1993; Leung et al., 1993, 1994). The C1 domains of - and
-chimaerins are almost identical (94% identity). The C-terminal
breakpoint cluster region homology domain of n-chimaerin has
GTPase-activating protein (GAP) activity for Rac and therefore promotes
the hydrolysis of GTP to GDP from this small GTP-binding protein
(Diekmann et al., 1991). The main structural difference between the
spliced variants is an SH2 domain located at the N terminus of 2-
and 2-chimaerins (Fig. 1).
A thorough characterization of 2-chimaerin as a phorbol ester
receptor showed important similarities with PKC isozymes and also
striking differences. Scatchard plot analysis revealed that 2-chimaerin binds [3H]PDBu with high
affinity in the presence of phosphatidylserine (PS) vesicles. The
Kd value is approximately 1 nM (Caloca
et al., 1997), which is in the same range as the
Kd values of cPKCs and nPKCs for this
radioligand (Kazanietz et al., 1993). Contrasting results were observed
when structure-activity relationship was studied. The most remarkable
difference was found for the ligand thymeleatoxin, an analog of the
second-stage tumor promoter mezerein. This ligand showed a marked
preference for PKC relative to 2-chimaerin (approximately
60-fold). This difference in ligand binding affinity is the greatest
observed so far for different phorbol ester receptor classes using in
vitro assays (Caloca et al., 1997). On the other hand, several DAG
analogs show a slight preference for 2-chimaerin relative to PKC
(Caloca et al., 1999). Studies of cofactor dependence show that PS is
the most effective phospholipid for supporting [3H]PDBu binding. Unlike PKC, PS dependence
and ligand binding affinity were not affected by calcium; in this
regard, 2-chimaerin resembles the nPKCs (Caloca et al., 1997). These
results lead to several important conclusions. First, whereas ligands
can spatially accommodate into the binding groove of the 2-chimaerin
C1 domain, it is likely that unique interactions with specific residues
take place within each C1 domain. Subtle structural differences between C1 domains might exist to explain differences in ligand recognition. Second, differences observed in cofactor-dependence, namely calcium and/or lipid requirement, may confer unique regulatory properties to
each phorbol ester receptor in a cellular context and probably contribute to their differential intracellular targeting. Lastly, differences in binding properties may have important implications for
the selective pharmacological manipulation of each receptor class.
Pharmacological Properties of Unc-13 and Munc13 Isoforms.
Unc-13 was identified in a search for genes responsible for defects in
coordinated movement in C. elegans and encodes a 1734-amino acid protein with sequence similarity to the regulatory region of PKC
(Maruyama and Brenner, 1991). The central region of Unc-13 has a single
C1 domain and a C2 domain located immediately downstream. A second C2
domain is located at the carboxyl terminus. As in the nPKCs, the C2
domains in Unc-13 are involved in phospholipid recognition in a
calcium-independent manner. Although the initial characterization of
this protein shows that it binds [3H]PDBu in a
phospholipid-independent manner, subsequent reports revealed that, as
expected, ligand binding was phospholipid-dependent (Ahmed et al.,
1992, Kazanietz et al., 1995b). Scatchard plot analysis using
[3H]PDBu showed a low nanomolar affinity for
the C1 domain of Unc-13 expressed in E. coli, and only
modest differences in ligand recognition were observed compared with
PKC (Kazanietz et al., 1995b).
Homologs of Unc-13 in mammalian (Munc13) and Drosophila
melanogaster (Dunc13) have been isolated (Brose et
al., 1995; Aravamudan et al., 1999; Song et al., 1999). Three mammalian
isoforms exist: Munc13-1, Munc13-2, and Munc13-3. These are large,
brain-specific proteins with divergent N termini and conserved C
termini containing C1 and C2 domains. A third C2 domain is present only
at the N-terminal region of Munc13-1, suggesting potential differences
in phospholipid regulation between Munc13 isoforms (Fig. 1).
Experiments using a GST-fused C1 domain of Munc13-1 revealed that it
binds [3H]PDBu with high affinity
(Kd = 5 nM using liposomes containing 20% of PS), and mutation of one of the essential histidines within the
C1 domain abolished ligand binding. As observed for phorbol ester
responsive PKCs, DAG displaces [3H]PDBu from
the Munc13-1 binding site (Betz et al., 1998). There is not yet any
evidence that the D. melanogaster homolog binds phorbol esters.
Pharmacological Properties of RasGRPs.
RasGRP1 (originally
named RasGRP) is the prototype of a novel family of guanine nucleotide
exchange factors (GEFs), enzymes that catalyze the exchange of GDP by
GTP in GTP-binding proteins and thereby promote their activation.
RasGRP1 was identified by Stone and coworkers using a fibroblast
transformation assay in a search for proteins that could complement a
transformation-defective allele of Ras (Ebinu et al., 1998). This
protein is highly expressed in brain and thymus and is also found in
bone marrow, spleen, and kidney (Ebinu et al., 1998; Kawasaki et al.,
1998; Tognon et al., 1998; Yamashita et al., 2000). Sequence analysis
shows a single C1 domain located at the C-terminal region. RasGRP1 also possesses a pair of atypical EF hands that bind calcium; a proline-rich motif; and the domains responsible for nucleotide exchange, the CDC25
box and the Ras exchange motifs (Ebinu et al., 1998; Tognon et al.,
1998).
[3H]PDBu binds to the RasGRP1 C1 domain with an
affinity of 0.6 nM in the presence of PS vesicles. Structure-activity
analysis reveals only minor differences in ligand recognition compared with PKCs. However, RasGRP1 has distinct lipid cofactor dependence, as
described recently by Lorenzo et al. (2000). Indeed, the C1 domain plus
the EF hand motif was markedly less dependent on acidic phospholipids
than PKC. Despite the presence of the atypical EF hands, phorbol
ester binding was not affected by calcium.
Related RasGRPs have been recently isolated (Fig. 1). CalDAG-GEF-I
(also called GRP2 or HCDC25L) is a GEF for Rap1 (Kawasaki et al., 1998;
Rebhun et al., 2000). Its alternatively spliced variant, RasGRP2, has
GEF activity for Rap1, N-Ras, and K-Ras (Clyde-Smith et al., 2000). So
far, there is no evidence that RasGRP2 variants bind phorbol esters or
DAG. A third member of the group is RasGRP3, a Ras exchange factor
(Rebhun et al., 2000; Yamashita et al., 2000). Recent evidence from the
Blumberg's lab shows that RasGRP3 is also a high affinity phorbol
ester receptor in the presence of anionic phospholipids. The
Kd value of
[3H]PDBu for RasGRP3 in the presence of PS
vesicles is 1.5 nM (Lorenzo et al., 2001).
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Regulation and Function of the Novel Phorbol Ester Receptors |
The accepted model for the regulation of PKC activity involves a
conformational change and allosteric activation upon DAG/phorbol ester
binding. One of the hallmarks for the activation of PKC isozymes by
phorbol esters is their translocation or change in intracellular
localization, a complex process that depends on lipid-binding modules.
After engagement of the C1 and C2 domains to the membrane, the
autoinhibitory pseudosubstrate in PKC is removed from the
substrate-binding site in the catalytic region, as demonstrated in a
series of elegant studies by the Newton lab (Orr et al., 1992; Orr and
Newton, 1994; Dutil and Newton, 2000). PKC translocation also involves
a series of protein-protein interactions that play an important role in
determining intracellular localization as well as integration with
other signaling pathways, thereby conferring function specificity for
each PKC isozyme (Mochly-Rosen and Gordon, 1998; Ron and Kazanietz,
1999; Jaken and Parker, 2000).
Considering that the novel phorbol ester receptors have only a single
C1 domain and in most cases lack other phospholipid-interacting motifs
present in PKCs, a key question was whether they are able to
redistribute in response to phorbol ester stimulation. In this regard,
strong experimental evidence indicates that chimaerins, Munc13s and
RasGRPs redistribute in response to phorbol esters. The differential
localization of each novel receptor and the functional consequences of
such redistribution will be discussed in the next sections.
Activation of Chimaerins by Phorbol Esters: Rac Regulation and an
Important GAP.
The first novel phorbol ester receptor shown to
translocate in response to phorbol esters was 2-chimaerin. Using
subcellular fractionation techniques in COS-1 cells, Caloca et al.
(1997) found that PMA redistributes this Rac-GAP protein from the
soluble (cytosolic) to a particulate fraction. However, remarkable
differences in the kinetics of translocation and dose-dependence exist
between 2-chimaerin and PKC. The looser membrane association
found for 2-chimaerin may indeed reflect the absence of some of the
essential structural motifs present in PKC isozymes. In addition,
molecular modeling studies revealed that a positively charged amino
acid in the 2-chimaerin C1 domain (arginine in position 9 of the
motif) makes the surface less hydrophobic and thus intrinsically less capable of membrane association (Fig. 3).
Structure-activity analysis for translocation shows that thymeleatoxin,
a poor ligand for chimaerins, failed to translocate 2-chimaerin even
though it potently redistributes PKC. Reduced hydrogen bonding
interactions with the hydrophobic core in the 2-chimaerin C1 domain
may contribute to the inability of this ligand to redistribute
2-chimaerin (Caloca et al., 2001).
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Fig. 3.
Modeling of the PKC C1b and 2-chimaerin C1
domains. Phorbol ester and related analogs bind at the tip of the
domain and create a contiguous hydrophobic surface that promotes the
insertion of the domain in the lipid bilayer. The figure shows the
docking of thymeleatoxin to each C1 domain. [Reprinted from Caloca MJ,
Wang HB, Delemos A, Wang S, and Kazanietz MG (2001) Phorbol esters and
related analogs regulate the subcellular localization of
2-chimaerin, a non-protein kinase C phorbol ester receptor.
J Biol Chem 276:18303-18312. Copyright
© 2001 American Society for Biochemistry and Molecular Biology.
Used with permission.]
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Phorbol ester-induced translocation of 2-chimaerin was found to be
independent of PKC activation, because it still occurs in the presence
of a PKC inhibitor. Moreover, disruption of the 2-chimaerin C1
domain by mutation of essential cysteine 246 (the third cysteine in the
motif) abolished translocation (Caloca et al., 1999). The requirement
of the 2-chimaerin C1 domain for translocation was confirmed by
deletional analysis (Caloca et al., 2001). More importantly, these
results support the concept that a single C1 domain is sufficient for
translocation, as described previously in experiments using isolated
PKC C1 domains (Oancea et al., 1998) and mutated PKC isozymes (Szallasi
et al., 1996; Bogi et al., 1998; Lorenzo et al., 1999).
Studies using GFP-2-chimaerin revealed a cytoplasmic staining in the
absence of phorbol ester stimulation, and a significant translocation
both to the plasma membrane and to the perinucleus after phorbol ester
treatment (Fig. 4). Interestingly,
colocalization of 2-chimaerin with a Golgi marker was observed
(Caloca et al., 2001; Wang and Kazanietz, 2002). Early experiments
using PKC mutants show that the C1 domain probably plays a role in
Golgi targeting (Lehel et al., 1995, 1996). More recently, Maeda et al.
(2001) reported that the C1a domain of PKCµ/PKD recruits this PKC-related kinase to the Golgi. In a search for
chimaerin-interacting proteins that may be involved in perinuclear
targeting, we have recently isolated Tmp21-I, a cis-Golgi
protein. Tmp21-I is a member of the p24 family of transmembrane
proteins involved in sorting/trafficking in the early secretory
pathway. Deletion of the C1 domain in either 1-chimaerin or
2-chimaerin impairs the interaction, thereby implying a novel
function for this domain in protein-protein associations in addition to
its role in lipid and phorbol ester binding. A remarkable finding is
that PMA is capable of promoting the association of 2-chimaerin with
Tmp21-I at the Golgi in a PKC-independent manner, which supports a
functional role of Tmp21-I as a chimaerin anchoring protein (Wang and
Kazanietz, 2002). Therefore, in analogy to PKC isozymes,
association with specific interacting proteins may also play a role in
the intracellular targeting of chimaerins. Although very little
information is available on the regulation of Golgi function and
intracellular transport mechanisms by phorbol ester receptors, a role
for DAG in protein transport from the Golgi to the cell surface has
been described previously (Huijbregts et al., 2000). Interestingly,
1-chimaerin regulates Golgi stability during interphase (Alonso et
al., 1998).
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Fig. 4.
Translocation of 2-chimaerin. Phorbol esters
promote a dual translocation of 2-chimaerin to the plasma membrane
and the perinuclear region. At the plasma membrane, 2-chimaerin
associates with Rac and promotes the hydrolysis of GTP from this
GTPase, leading to its inactivation. At the perinuclear site,
2-chimaerin and other chimaerin isoforms bind to Tmp21-I, which
probably serves as a chimaerin-anchoring protein. Top, colocalization
(yellow) by fluorescent microscopy of 2-chimaerin
(red) and Tmp21-I (green) in the
perinuclear region of COS-1 cells. Bottom, colocalization
(yellow) of 2-chimaerin (green) and
RacV12 (red) in the plasma membrane of COS-1 cells,
predominantly in membrane ruffles, as indicated by the
arrows.
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As described above, chimaerins have a RacGAP domain and therefore
accelerate the hydrolysis of GTP from Rac, leading to its inactivation.
Given the high-affinity binding of phorbol esters for chimaerins and
their effects on chimaerin translocation, a question arises: can
phorbol esters regulate chimaerin GAP activity? This hypothesis was
initially explored for 1-chimaerin using Rac-GTP hydrolysis assays,
which revealed low albeit significant increases in Rac-GAP activity by
PMA (Ahmed et al., 1993). Similar experiments performed with
2-chimaerin show no significant changes in Rac-GAP activity in the
presence of PMA (M. J. Caloca, H. Wang, and M. G. Kazanietz, in
preparation). On the other hand, acidic phospholipids such as PS
or PA markedly increase chimaerin GAP activity (Ahmed et al., 1993;
Caloca et al., 2001). PMA promotes the association of 2-chimaerin
with RacV12 (an activated form of Rac) in COS-1 cells, as judged by
coprecipitation assays (Caloca et al., 2001). Taken together, these
results support a "positional" model in which phorbol esters (and
probably DAG) primarily redistribute 2-chimaerin to membranes where
it binds Rac, and allosteric activation is triggered by membrane
phospholipids. It remains to be explored how redistribution of
chimaerins to the perinuclear region relates to Rac signaling.
Importantly, a large pool of Rac in its inactive, GDP-bound form is
located in the perinuclear region (Kraynov et al., 2000). Therefore, it
is tempting to speculate that 2-chimaerin and/or other chimaerin
isoforms also play a role in the maintenance of the perinuclear Rac in
an inactive state before this GTPase moves to the plasma membrane (Fig.
4).
There is strong experimental evidence that chimaerins inhibit
Rac-mediated effects (Table 1). Among
other functions, Rac is involved in actin cytoskeleton reorganization,
adhesion, migration, and cell cycle control (Coso et al., 1995; Ridley,
1996; Kjoller and Hall, 1999; Schmitz et al., 2000). Interestingly,
ectopic expression of 1-chimaerin alters cytoskeletal and adhesive
properties of NIH 3T3 fibroblasts. The assembly of integrin receptors,
the organization of actin stress fibers and the formation of focal adhesions is also impaired (Herrera and Shivers, 1994). Expression of
the 1-chimaerin GAP domain in leukocytes inhibits cytoskeletal responses to FMLP and CSF-1, and blocks phagocytosis, as also observed
with a dominant negative (N17) Rac mutant (Cox et al., 1997).
2-Chimaerin is involved in neuritogenesis, and a role for the SH2
domain in 2-chimaerin has been proposed, suggesting that interaction
with phosphotyrosine proteins yet to be identified may be critical
(Hall et al., 2001). We have preliminary evidence that overexpression
of 2-chimaerin impairs EGF signaling and cell proliferation, and it
inhibits the metastatic potential of breast cancer cells
(Lorenzano-Menna et al., submitted; M. J. Caloca, H. Wang,
and M. G. Kazanietz, in preparation). Interestingly, a marked
reduction in 2-chimaerin expression was observed in high-grade
astrocytomas, suggesting a dysregulation of Rac signaling in tumor
progression (Yuan et al., 1995).
Munc13 Isozymes: Phorbol Ester Activation and Exocytosis.
With
the exception of a Munc13-2 splice variant, which is ubiquitously
expressed, Munc13 proteins are mainly expressed in brain (Augustin et
al., 1999; Koch et al., 2000). Evidence for phorbol ester-induced
translocation of Munc13 isozymes has been reported in human embryonic
kidney 293 cells transiently transfected with GFP-fused Munc13
constructs. These experiments show that all three Munc13 isoforms
translocate to the plasma membrane in response to PMA. The involvement
of the C1 domain was confirmed in experiments showing that a mutant
insensitive to phorbol esters does not redistribute. On the other hand,
a truncated mutant of Munc13-1 comprising only its C1 domain
translocated in response to PMA (Betz et al., 1998). Despite some
evidence of translocation of Munc13 to the Golgi in response to phorbol
esters, the biological relevance of this redistribution has yet to be
determined (Song et al., 1999).
Munc13 isoforms act as scaffolding proteins that interact with elements
of the exocytotic machinery, such as syntaxin, Doc2, RIM1, and spectrin
(Betz et al., 1997, 2001; Orita et al., 1997; Sakaguchi et al., 1998;
Duncan et al., 1999), and therefore play an essential role in
exocytosis (Table 1). Munc13-1 acts as a factor that transfers unprimed
vesicles to a pool of release-competent, primed vesicles. Phorbol
esters promote a transient interaction of Munc13-1 with the
calcium-binding protein DOC2. This association was independent of PKC
activation and required an intact Munc13 C1 domain (Orita et al., 1997;
Duncan et al., 1999). This event may be important in the regulation of
vesicular trafficking and is probably a key step in phorbol
ester-dependent enhancement of exocytosis from presynaptic terminals.
The importance of phorbol ester-regulated exocytosis via Munc13 was
confirmed in experiments using microinjection of Munc13-1 mRNA into
Xenopus laevis embryos. The gain-of-function effect of
Munc13-1 occurs only when its C1 domain is intact (Betz et al., 1998).
RasGRPs: Phorbol Ester Activation of Ras Independent of PKC.
The activation of the Ras cascade by phorbol esters has been
extensively investigated in the last decade. Multiple points of cross
talk between the PKC and Ras pathways, both upstream and downstream of
Ras, have been reported (Cai et al., 1997; El-Shemerly et al., 1997;
Marais et al., 1998; Schonwasser et al., 1998). The discovery of
RasGRP1, a nucleotide exchange factor for Ras with transforming
potential, uncovered a novel link between receptor-mediated stimulation
of DAG signaling and Ras activation. It is well established that the C1
domain in RasGRP1 is critical for its activation in cellular models.
Binding of PMA to the RasGRP1 C1 domain promotes its translocation from
the cytosol to membrane fractions. In NIH 3T3 cells stably expressing
RasGRP1, serum or PMA translocates RasGRP1 to cellular structures
around the nucleus and to the cell periphery (Ebinu et al., 1998;
Tognon et al., 1998). A recent interesting study by Lorenzo et al.
(2001) showed a biphasic translocation of RasGRP3: at lower PMA
concentrations, it translocates predominantly to the plasma membrane,
and at higher concentrations, a perinuclear and nuclear membrane
distribution was observed. Colocalization with a Golgi marker was
detected in the perinucleus.
RasGRP1 may serve as a direct link between receptors coupled to DAG
generation and Ras activation at the plasma membrane. A schematic model
of RasGRP1 regulation is depicted in Fig.
5. Expression of RasGRP1 increases the
GTP loading of Ras, an effect that is further increased by PMA (Ebinu
et al., 1998). Although the involvement of phorbol ester-responsive
PKCs has not been ruled out, similar experiments using RasGRP3 showed
that the increase in Ras-GTP loading by PMA cannot be blocked by a PKC
inhibitor (Lorenzo et al., 2001). It seems that recruitment to the
plasma membrane is sufficient to activate RasGRPs, as judged by the
ability of a prenylated form of RasGRP1 to activate the Ras-dependent mitogen-activated protein kinase -ERK cascade (Tognon et al., 1998). A
mutated RasGRP1 lacking the C1 domain failed to activate the ERK
cascade and lost its characteristic transforming potential. Thus, the
phorbol ester/DAG binding site has a dominant role in RasGRP1
activation. Further support for a link between DAG signaling and RasGRP
has been recently provided in a study showing a direct association of
RasGRP1 with DAG kinases (Topham and Prescott, 2001).
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Fig. 5.
Schematic of RasGRP1 activation. DAG binds to the C1
domain of RasGRP1 in the plasma membrane. Consequently, RasGRP1
promotes GDP/GTP exchange on Ras, leading to the activation of the
downstream Raf-ERK cascade.
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Unlike RasGRP1 and RasGRP3, the regulation of RasGRP2 by phorbol esters
or DAG remains undefined. RasGRP2 has dual Ras/Rap1 GEF activity and is
localized to the plasma membrane by post-translation modifications
(palmitoylation and myristoylation). Its spliced variant CalDAG-GEFI,
on the other hand, lacks the N-terminal consensus sequence for lipid
modification and is confined to the cytosol. Despite the presence of a
C1 domain, RasGRP2 fails to redistribute after phorbol ester treatment.
Nevertheless, a substantial proportion of CalDAG-GEFI translocates to
particulate fractions in cells treated with PMA for at least 15 min
(Clyde-Smith et al., 2000). Although PMA enhances the Rap1GEF and
RasGEF activities of RasGRP2 variants in COS cells (Clyde-Smith et al.,
2000), evidence for a direct phorbol ester interaction using binding
assays is still needed for RasGRP2.
It is conceivable that RasGRPs have the potential to contribute to the
mitogenic and tumor promoting effects of the phorbol esters. In
addition to its transforming potential in fibroblast models, RasGRP1
regulates thymocyte differentiation and T-cell activation.
Overexpression of RasGRP1 in T cells enhances TCR-Ras-ERK signaling in
response to calcium/PMA. In addition, RasGRP1 is differentially
associated with membranes after TCR stimulation (Ebinu et al., 2000).
Recent experiments illustrated that RasGRP1-null mutant mice have a
significant reduction in the number of mature thymocytes. Remarkably,
thymocytes from RasGRP1-deficient mice have a defective proliferative
response and an impaired activation of the Ras-ERK cascade in response
to PMA or anti-CD3 (Dower et al., 2000). Thus, RasGRP1 provides a
nonredundant link between TCR ligation and activation of Ras signaling
in thymocytes (Table 1).
| |
Pharmacological Considerations: Should We Rethink Our View of
Phorbol Esters as Selective PKC Activators? |
The discovery of chimaerins, and more recently additional novel
phorbol ester receptors, strongly supports the concept that a high
degree of complexity exists in the pathways downstream of DAG
generation. Many effects of phorbol esters that have been initially
attributed to PKC isozymes may probably involve other targets and
therefore require reevaluation. A similar concept applies to PKC
inhibitors that target the phorbol ester binding site, such as
calphostin C. It is clear now that this so-called "selective" PKC
inhibitor blocks [3H]PDBu binding not only to
PKC isozymes but also to chimaerins, RasGRPs, and Unc-13 (Areces et
al., 1994; Kazanietz et al., 1995b; Lorenzo et al., 2000). Thus, the
use of calphostin C can lead to misleading conclusions, and great care
should be taken in the interpretation of results. In light of the
similar sensitivity of C1 domains to the archetypical phorbol esters
and DAG, new strategies should be developed to achieve selective
regulation of each pathway. The discrimination of targets by the analog
thymeleatoxin is a good example of how subtle differences in ligand
recognition exist between different C1 domains, and a careful
examination of these structural variables through modeling studies is
probably the best approach for the rational design of C1 domain ligands.
An important emerging concept is that differential activation of
phorbol ester receptors can be achieved by selective intracellular targeting. Proof-of-principle has been established for PKC isozymes using selective peptides targeted to protein-protein interaction sites
(Csukai and Mochly-Rosen, 1999). Interesting studies by Wang et al.
(1999, 2000) have recently revealed that selective translocation of PKC
isozymes in cellular models can be achieved using different classes of
analogs (such as bryostatin 1 or the novel DAG lactones) or by varying
the ligand lipophilicity. Furthermore, we have recently demonstrated
the selective activation of PKC in prostate cancer cells by a
rationally designed DAG analog (a DAG lactone). Despite its similar
potency for PKC and PKC in binding and kinase assays, this
compound translocates each PKC to different intracellular compartments
(Garcia-Bermejo et al., 2002). An important lesson from these studies
is that marked discrepancies exist between in vitro and cellular
effects of C1 domain-directed ligands and, more importantly, that in
vitro pharmacological screenings may underestimate the selectivity
observed in cellular assays. This novel pharmacological principle may
prove to be useful in the design of selective analogs for each phorbol
ester receptor class and in this way help to dissect DAG-mediated
pathways as well as to elucidate the cellular functions of the novel
phorbol ester receptors. It has been shown recently that DAG kinase binds [3H]PDBu with high affinity through its C1A domain
(Shindo et al., 2001). This is the first evidence that a DAG kinase is
a specific phorbol ester receptor.
I am grateful to the members of my laboratory for comments on
the manuscript. I am indebted to Drs. Maria Jose Caloca and HongBin
Wang for their outstanding contributions to the understanding of
chimaerin regulation and function. Dr. Patricia Lorenzo has provided
insightful contributions to the sections describing RasGRP regulation.
This work was supported by grants from the Department of
Defense, the American Cancer Society, and the National Institutes of Health.
PKC, protein kinase C;
DAG, diacylglycerol;
PA, phosphatidic acid;
cPKC, classic/conventional PKC;
nPKC, novel protein
kinase C;
PDBu, phorbol 12,13-dibutyrate;
GAP, GTPase-activating
protein;
PS, phosphatidylserine;
GEF, guanine nucleotide exchange
factor;
PMA, phorbol 12-myristate 13-acetate;
ERK, extracellular
signal-regulated kinase;
TCR, T cell receptor.
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Abstract
Introduction
