DRUG DEVELOPMENT RESEARCH 65:156–169 (2005)

DDR
Research Overview
Protein Kinase C Substrate Activators: Potential as Novel
Antidepressants
Miao-kun Sun1 and Daniel L. Alkon1,2
1
Blanchette Rockefeller Neurosciences Institute, Rockville, Maryland
2
Johns Hopkins University, Rockville, Maryland

Strategy, Management and Health Policy

Enabling Preclinical Development Clinical Development

Technology, Preclinical Toxicology, Formulation Phases I-III Postmarketing
Genomics, Research Drug Delivery, Regulatory, Quality, Phase IV
Proteomics Pharmacokinetics Manufacturing

ABSTRACT Depression and mood disorders are major public health concerns. Emerging evidence
suggests that abnormal functions of protein kinase C (PKC) isozymes, ubiquitous in the central nervous
system, may play a critical role in the pathogenesis of major depression and mood disorders. PKC activity
and expression in the brain regions that are involved in mood regulation are reduced in suicide victims and
are sensitive to stress-related damage. PKC isozyme dysfunction may contribute to mood dysfunction,
while PKC activators exhibit antidepressant pharmacology. Restoration of PKC activity thus represents an
important therapeutic goal in antidepressant therapy. PKC substrate activators, therefore, may have
important therapeutic value for the treatment of depression, especially when fine-tuning of selective
isoform activity can be effectively achieved pharmacologically. The success of antidepressant therapy with
bryostatin-1-like agents that act on PKC signaling cascades depends on whether such agents at their
effective doses would significantly disrupt or interfere with other vital functions that rely on a narrow range
of PKC activities. Drug Dev. Res. 65:156–169, 2005. c 2005 Wiley-Liss, Inc.

Key words: antidepressant; bryostatin; protein kinase C

INTRODUCTION many types of diseases, including mood disorders.

The protein kinase C (PKC) substrates and
signaling pathway play an important and regulatory
role in a wide range of vital biological functions and
processes [Gerecke et al., 2004; Poole et al., 2004;
Weinreb et al., 2004; Young et al., 2004; Cejas et al.,
2005], including proliferation, gene expression, synap-
tic plasticity, neuronal injury, differentiation, cell
growth and apoptosis, and oncogenesis, depending on
cell identity and states. The importance of a normal
functional control through PKC signaling cascades for
brain health and functions is well established. The
brain has, in fact, the highest concentration of PKC of
any organ in the body [Saito et al., 1988], especially in
vital neural structures that are involved in mood
regulation and cognition. Abnormal regulation and
activity of PKC and PKC substrates are implicated in
Evidence has been provided that decreased PKC
catalytic activity and expression are found in the
prefrontal cortex and the hippocampus in suicide
Abbreviations used: aPKC, atypical PKC; BDNF, brain-
derived neurotrophic factor; cPKC, classical PKC; GABA
g-aminobutyric acid; GAP, growth-associated protein; GluR,
glutamate receptor; HPA, hypothalamic-pituitary-adrenal; LTP,
long-term potentiation; mGluR, metabotropic glutamate receptor;
NMDA, N-methyl-D-aspartate; nPKC, novel PKC; PKC, protein
kinase C; RACK, receptor for activated C kinase.
Correspondence to: Miao-Kun Sun, Ph.D., Blanchette
Rockefeller Neurosciences, Institute, 9601 Medical Center Dr.,
Academic/Research Bldg., Room 319, Rockville, MD 20850.
E-mail: mksun@brni-jhu.org
Published online in Wiley InterScience (www.interscience.wiley.
com). DOI: 10.1002/ddr.20019

c

2005 Wiley-Liss, Inc.
 PROTEIN KINASE C SUBSTRATE ACTIVATORS
victims [Pacheco et al., 1996; Pandey et al., 1997, 2003,
2004], and reduced PKC activity in fibroblasts cultured
from skin biopsies from patients with melancholic
depression [Akin et al., 2005]. PKC isozymes, there-
fore, may represent an important therapeutic target for
antidepressive and anti-suicidal therapy. In this short
article, we intend to review the role of PKC isozymes in
mood regulation and pathogensis of mood disorders,
especially depression, and indicate the potential of
developing agents acting on the PKC systems as future
antidepressants.
PROTEIN KINASE C ISOZYMES
PKC activity, with the active site usually located
at the C-terminus to phosphorylate serine and
threonine residues, is mediated by a family of at least
twelve PKC isozymes [Toker, 1998; Rabah et al., 2001;
Oster et al., 2004]. Each isoform of the twelve
PKC isozymes that have been identified so far is
encoded by a separate gene, with the exception of the
bI and bII isoforms, which are splice variants. The
twelve PKC isoforms can be further divided into three
subgroups, classical PKC (cPKC), novel PKC (nPKC),
and atypical PKC (aPKC), based on their molecular
structures and thus co-factor requirements for activa-
tion.
The structural features of PKC include two main
regulatory domains (the activator-binding C1 and the
Ca21 -binding C2 domains) at the NH2-terminal that
mediate membrane association and activation, a C-
terminal active site that functions as a serine/threonine
kinase, and a pseudosubstrate sequence adjacent to the
C1 domain. The C1 and C2 stand for conserved
domain 1 and 2, respectively. The C1 domains of PKC
represent zinc finger structures that bind diacylglycerol
and other non-Ca21 PKC activators. For instance, the
regulatory domains of nPKC contain two C1 sub-
domains (C1A and C1B), each of which interacts with
sn-1,2-diacylglycerol and phorbol esters [Hurley et al.,
1997; Irie et al., 2002]. The C1A and C1B domains are,
however, not equivalent, with their relative affinities
depending on the isozymes. The C1A domain of PKCd,
for example, has high affinity for diacylglycerol and is
critical for the diacylglycerol-induced membrane bind-
ing and PKCd activation, while the C1B domain
exhibits high affinity for phorbol esters [Stahelin
et al., 2004]. These differences indicate potential for
developing isozyme-selective PKC activators. The
activator-binding sites within the C1 domain of PKC
are also able to bind alcohols and anesthetics [Slater
et al., 2004]. The C2 domain also contains binding sites
for lipids and proteins. The catalytic domain of PKC
isozymes has the C3 and C4 domains. The former
possesses the binding site for ATP, the phosphate donor
157
for phosphotransferase activity. The latter has the
binding site for substrates.
The cPKC contains the activator-binding C1
domain and the C2 region corresponding to the Ca21
binding site [Nishizuka, 1988]. The cPKC requires
Ca21 as a co-factor for activation and consists of a, bI,
bII, and g isoforms. The nPKC isozymes, including d, e,
e’, Z, y, and m isoforms, on the other hand, are calcium-
independent [Nishizuka, 1988]. These isoforms do not
have the Ca21 binding C2 region [Nishizuka, 1988;
Gszchwendt et al., 1992; Ways et al., 1992] and do not
require Ca21 as a co-factor for activation. They contain
a C2-like structure, which may be involved in
interaction with proteins and phospholipids. The C2
domain of PKCd, for instance, directly binds to
phosphotyrosine peptides [Benes et al., 2005], mediat-
ing PKCd interaction with CDCP1, a Src binding
glycoprotein and thus functioning as a phosphotyrosine
binding domain. A common feature of the cPKC and
nPKC is that they contain both C1A and C1B sub-
domains and can all be activated by diacylglycerol,
phorbol esters, and bryostatins. The aPKC isozymes,
the third subgroup, include z and l/i isoforms. The
aPKC contains a single C1 domain and does not bind
diacylglycerol, phorbol esters, or bryostatins. It does
not contain the Ca21 binding C2 region and one of the
repeated cysteine-rich zinc finger binding motifs within
the C1 domain [Nishizuka, 1992; Yang and Kazanietz,
2003]. It is not clear how aPKC activity is regulated
in vivo.
The various PKC isoforms are usually co-ex-
pressed in the same neurons. The biological signifi-
cance of the heterogeneity of the PKC family has not
been really established. In most cases, we do not know
which particular PKC isozyme(s) actually mediates the
observed effects. Obviously, information that is carried
by intensity levels of calcium activity and waves
requires signal mediators that can detect and transduce
such information. Signal cascades that are not triggered
by calcium waves are better relayed by the calcium-
independent pathways. aPKC appears to have higher
structural restriction for activators than the other
subgroups, although the biological meaning of such
difference remains to be studied. Nevertheless, evi-
dence has been provided that atypical PKCz isozymes,
whose expression levels are correlated with nucleotide
excision repair activity and protein levels, probably
regulate the expression of nucleotide repair proteins
[Louat et al., 2004]. Unfortunately, the PKC pharma-
cology has been limited by the availability of water-
soluble, specific inhibitors of PKC isozymes for an in
vivo administration. The pharmacological antagonism
and isozyme involvement, therefore, remain to be
studied in detail in the future.
158 SUN AND ALKON
MOLECULAR TARGETS
PKC and PKC substrates are important signal
molecules in the regulation of mood and other vital
functions of the brain, both as molecular targets of
signal cascades and as mediators targeting molecular
pathways downstream. Activation of PKC leads to
changes in such vital responses as enhancing calcium
action potentials, increasing neurotransmitter release,
and decreasing voltage-gated Na1 currents [Carr et al.,
2002, 2003; Chen et al., 2005] and voltage-dependent
K1 currents [Alkon et al., 1986; 1998; Farley and
Auerbach, 1986] as well as calcium-activated potassium
current in hippocampus, all relevant to information
processing and evaluation in the brain.
PKC is activated by synaptic inputs and intracel-
lular signals that are involved in information processing
and mood regulation, including glutamatergic inputs
[Hasham et al., 1997], cholinergic inputs [Chen et al.,
2005], serotonergic inputs [Carr et al., 2002, 2003],
dopaminergic inputs [Maurice et al., 2001], intracel-
lular calcium and diacylglycerol elevations, and other
hormones [Sato et al., 2004]. Growth and other stimuli
cause a transient increase in levels of cellular
diacylglycerol, which activates PKC as well as produces
some other effects that appear to be PKC-independent
[Andoh et al., 2004]. PKC signaling triggered by local
cell–cell contact appears to be a mechanism through
which astrocytes regulate neuronal development. This
mechanism in brief involves local contact with astro-
cytes via integrin receptors that elicits PKC activation
and excitatory synaptogenesis in neurons, via the
arachidonic acid cascade [Hama et al., 2004]. The
cascade represents an underlying mechanism for global
neuronal maturation following local astrocyte adhesion,
probably including neurogenesis in adults, as matured
hippocampal astrocytes regulate neurogenesis by
instructing stem cells to adopt a neuronal fate
[Song et al., 2002a,b]. It is not clear, however, whether
the integrin-PKC-cascade plays an essential role in
the regulation of mood in the adult brains. Never-
theless, active neurogenesis/neural remodeling may
play an essential role in maintaining a normal mood.
PKCs interact with a large diverse family of PKC-
interacting proteins [Ron and Kazanietz, 1999; New-
ton, 2001; Poole et al., 2004], including the receptors
for activated C-kinase (RACKs). These phosphopro-
teins are the PKC substrates, through which PKC
regulates cellular functions. One of the phosphopro-
teins that are heavily phosphorylated by PKC is
localized to the presynaptic terminal. It was first
identified by Gispen and his colleagues [Dokas et al.,
1984] in the Netherlands and was named B50 by them
for the brain protein with an approximate molecular
weight of 50 kDa. It was then named F1 [Routtenberg
et al., 2000] and GAP-43 for a growth-associated
protein with an approximate molecular weight of 43
kDa [Skene et al., 1986]. GAP-43 is found exclusively
in the brain, is localized to the membranes of axonal
processes, and is not found in membranes of dendritic
processes ([Jacobson et al., 1986; Benowitz et al., 1988;
Goslin et al., 1988], except in the olfactory system
[Verhaagen et al., 1989]).
The separate localization of PKC and its mem-
brane-associated substrates, such as GAP-43, in the
cells dictates an essential step in the PKC activation:
translocation. Such activation is indeed associated with
a well-characterized learning-induced PKC transloca-
tion from the cytosol to the membrane fraction [Olds
et al., 1989; McPhie et al., 1993], an essential step
leading to substrate activation. The PKC translocation
from the cytoplasm to the membrane component of the
cells is associated with phosphorylation of GAP-43
[Akers and Routtenberg, 1987]. The phenomenon of
PKC translocation occurs both post- and presynapti-
cally [Akers et al., 1986], as in the case of phosphoryla-
tion of GAP-43 [Akers and Routtenberg, 1987], as well
as postsynaptically [Olds et al., 1989].
CELLULAR SIGNALING
PKC mediates various extracellular signals into
the cell and intracellular signal events [Tanaka and
Nishizuka, 1994]. PKC can ‘‘detect’’ the frequency
and amplitudes of neural spikes as well as the pattern
and frequency of synaptic inputs, in unique patterns
and cycles of calcium-induced translocation and
diacylglycerol-mediated kinase activation. For instance,
receptor stimuli that trigger repetitive calcium spikes
induce PKCg in a way in which PKCg responds to
persistent diacylglycerol increases combined with high-
but not low-frequency calcium spikes [Oancea and
Meyer, 1998]. Only high-frequency calcium spikes and
persistent calcium increases can lead to an integration
of cPKC activity, inducing a much higher amplitude of,
and long-lasting, kinase activity. While cPKC is not
activated when calcium spikes are absent, other PKC
isoforms are well positioned to respond to signal events
without associated calcium spikes. Protein kinase C
also regulates the nuclear localization of diacylglycerol
kinase-z [Topham et al., 1998], which terminates
signaling from diacylglycerol by converting it to
phosphatidic acid. The nuclear-localization signal of
diacylglycerol kinase-z is localized in a region that is
homologous to the phosphorylation-site domain of the
MARCKS protein, a major target for PKC. Reducing
nuclear diacylglycerol levels by diacylglycerol kinase-z
attenuates cell growth.
 PROTEIN KINASE C SUBSTRATE ACTIVATORS
Altered PKC signal activity affects many receptor/
channel states and activity, membrane electrical
properties, and thus signal transfer between neurons.
These include the potassium channels, glutamatergic
cationic channels, voltage-gated Na1 channels, and g-
aminobutyric acid (GABA)A receptor channels. GABAA
receptors associate directly with both PKCb II and the
receptor for activated C-kinase (RACK-1) in neurons
[Brandon et al., 1999, 2000, 2002a–c]. The calcium-
and phospholipid-dependent PKCs control membrane
electrical properties [Hoffman and Johnston, 1998;
Manseau et al., 1998] and modulate neurotransmitter
receptor function [Roche et al., 1996; Macek et al.,
1998; Suen et al., 1998]. For instance, activation of
PKC by phorbol esters and synthetic diacylglyceroles
has been shown to attenuate voltage-gated potassium
currents in a number of cell types, including the CA1
pyramidal cells in the hippocampus [Alkon et al., 1986;
Grega et al., 1987; Doerner et al., 1988; Linden et al.,
1992; Mao et al., 2004]. PKCe phosphorylates the
voltage-gated Na1 channels in hippocampal neurons
and is responsible for PKC-mediated reduction of Na1
currents in the neurons [Chen et al., 2005]. A reduction
in the voltage-gated Na1 currents is expected to
increase spike threshold and reduce spike train
duration, as shown in prefrontal cortex neurons [Carr
et al., 2002, 2003]. Reduction in potassium channel
conductance, on the other hand, represents an
important change that is associated with potentiated
synaptic transmission [Lo Turco et al., 1988].
PKC also modulates the glutamatergic system,
including the ionotropic receptors and metabotropic
receptors. PKC activation, for example, with 12-O-
tetradecanoylphorbol modifies both a-amino-3-hydroxy-
5-methyl-4-isoxazoleproprionate receptor and N-
methyl-D-aspartate (NMDA) receptor’s mobility,
increasing their extrasynaptic and synaptic diffusion
[Groc et al., 2004]. In addition, PKC activation has
been found to result in phosphorylation of NR1
subunit of the NMDA receptor at its C1 domain (at
serines 890 and 896). The NR1 C1 domain contains a
third serine at 897, which is phosphorylated by PKA
[Tingley et al., 1997]. Activation of type I mGluRs
appears to activate PKCg, but not PKCa or PKCb
[Sánchez-Pérez and Felipo, 2005]. PKCg activation
preferentially phosphorylates serine 890, while PKCa
activation leads to a preferential phosphorylation at
serine 896 [Sánchez-Pérez and Felipo, 2005]. PKC
activation in the hippocampal CA1 pyramidal cells has
also been reported to produce an inhibition of the slow
after-hyperpolarization, thus resulting in an enhanced
excitability. Evidence has also been provided that
mGluR6-containing kainate receptors are probably
the trigger for PKC-mediated inhibition of slow after-
159
hyperpolarization in the hippocampal CA1 pyramidal
neurons [Melyan et al., 2002]. Glutamate also desensi-
tizes mGluR5a and mGluR5b, through PKC-mediated
phosphorylation of mGluR5 at multiple sites [Gereau
and Heinemann, 1998].
In the brain at large and in the hippocampus
particularly, the GABA system plays an important role
in controlling neuronal activity and information trans-
fer through the neural network related to mood
regulation and cognition. PKC also appears to mediate
brain-derived neurotrophic factor (BDNF)-induced
modulation of GABAA receptor phosphorylation and
activity [Jovanovic et al., 2004]. The impact of BDNF-
PKC-GABA transmission cascade in the hippocampus
and related brain structures on mood remains to be
evaluated in the future.
In addition to its modulating receptor/channel
states and activity and membrane electrical properties,
PKC plays an important role in the regulation of
synaptic transmission in the nervous system. First, PKC
regulates the synthesis of many neurotransmitters.
Choline acetyltransferase synthesizes acetylcholine in
the cholinergic neurons. The enzyme is differentially
phosphorylated by PKC isoforms on four serines (Ser-
440, -346, -347, -476) and one threonine (Thr-255),
regulating its catalytic activity [Dobransky et al., 2004].
PKC activity plays an important regulatory role in
GABAergic synaptic transmission [Okada et al., 2004].
In the glutamate system, phorbol esters have been
shown to increase the size of the readily releasable pool
and the rate at which the pool refills at glutamatergic
hippocampal synapses. This effect is produced by a
PKC-dependent mechanism [Stevens and Sullivan,
1998]. The calcium- and phospholipid-dependent
PKCs also regulate neurotransmitter release [Nicholls,
1998; Stevens and Sullivan, 1998] and regulate gene
expression in mature neurons [Roberson et al., 1999].
However, others report that phorbol ester- and
diacylglycerol-induced augmentation of transmitter
release is mediated by Munc13s, a presynaptic protein
that contains diacylglycerol/phorbol ester binding C1
domain, but not by PKCs [Rhee et al., 2002].
PKC AGENTS AND MOOD PHARMACOLOGY
PKC and Mood Regulation
Activation of PKC leads to the phosphorylation of
numerous proteins, including the NR1 subunit of the
NMDA receptor. In the neural networks, PKC
activation generally facilitates synaptic transmission,
including such responses as an enhancement of
calcium influx, an increase in neurotransmitter release,
and a decrease in a calcium-activated current in the
hippocampus. In cultured hippocampal neurons, for
160 SUN AND ALKON
example, activation of PKC induces rapid morphologi-
cal plasticity in dendrites and spines [Pilpel and Segal,
2004]. Although we still lack a detailed knowledge of
how these molecular and synaptic changes are involved
in mood regulation and depressogenesis, evidence
indicates that the hippocampus in mammals is involved
in mood regulation [Sheline et al., 1996; Bremner et al.,
2000; Cryan et al., 2002]. It is a highly plastic and
vulnerable brain structure that is damaged in major
depression that changes PKC signaling cascades. PKC
isozymes have also been reported to mediate or
partially mediate effects of various agents in producing
cell protective effects [Weinreb et al., 2004; Toyomoto
et al., 2005].
The involvement of PKC in mood regulation is
implicated by several factors. Brain, especially the
hippocampus and amygdala, which play critical roles in
mood regulation, express the highest levels of PKC
in the body. Abnormality of PKC signal transduction
is involved in depressed mood [Akin et al., 2005],
suggesting an abnormality of factors controlling the
expression or degradation of PKC isozymes. PKC
cascade activity and functions in the hippocampus and
associated structures are impaired by stress and
depressogenic events. Furthermore, reduced PKC
activity is associated with suicide victims [Pacheco
et al., 1996; Pandey et al., 1997, 2003, 2004].
PKC is directly involved in the molecular events
that are known to play a critical role in depression.
PKC is involved in regulation of 5-HT receptor activity
[Rahimian and Hrdina, 1995], down-regulation of 5-
HT reuptake [Ramamoorthy et al., 1998], and regula-
tion of norepinephrine transporter [Apparsundaram
et al., 1998a,b]. Phorbol esters selectively diminish
norepinephrine transporter capacity (Vmax) and its
cell-surface expression, but not its Km, a reaction that
is sensitive to PKC inhibitors [Apparsundaram et al.,
1998b]. These observations are consistent with the role
of catecholamines in mood regulation and catechola-
mine pharmacology in antidepressive treatment
[Klimek et al., 1997; Cryan et al., 2002; Sun and
Alkon, 2002; Hahn et al., 2005]. An over-activated
hypothalamic-pituitary-adrenal (HPA) axis underlies
stress reaction, depression, and a suicidal mood [Lopez
et al., 1992]. Activation of the HPA axis causes down-
regulation of certain PKC isozymes in rat brain
[Pandey and Dwivedi, 2000]. One of the important
links of PKC in mood regulation is its signaling
connection with the phosphoinositide signaling system
[Gould and Manji, 2002], which mediates intracellular
responses to activity of several monoaminergic systems.
Lithium, a known mood-stabilizer frequently
used as first-line treatment of bipolar affective
disorders, acutely increases PKC-a activity approxi-
mately twofold in HEK293 cells and PC12 cells and in
mouse hippocampus [Li and Jope, 1995; Manji and
Chen, 2002; Kirshenboim et al., 2004]. The activation
can be blocked with PKC inhibitors such as
GF109203X, Ro31-8425, or GÖ6976, a cPKC inhibitor,
and leads to serine phosphorylation and inhibition of
glycogen synthase kinase-3b [Kirshenboim et al., 2004].
However, its inhibitory action on synthase kinase-3b is
rather weak, requiring a relative high concentration
(IC50 5 2 mM [Klein and Melton, 1996; Stambolic
et al., 1996], which is above its therapeutic concentra-
tion levels, and thus may not contribute to its
therapeutic effects. Chronic treatment with lithium,
on the other hand, decreases the expression and
activity of PKC both in cultured cells and in the rat
hippocampus [Casebolt and Jope, 1991; Bitran et al.,
1995; Watson and Lenox, 1996; Jensen and Mork,
1997; Wang et al., 2001]. Lithium is, of course, not an
antidepressant. Imipramine, a tricyclic antidepressant,
though not citalopram, has been shown to increase
PKC levels in primary rat neuronal cultures [Pákáski
et al., 2005].
The consistent findings for the roles of PKC in
mood regulation and mood disorders are that a
dysfunction of PKC signal cascade may underlie a
depressed mood. Restoring the depressed PKC and
PKC substrate activity, through the use of PKC
activators, may have antidepressant activity.
PKC Activators
The main focus of agents that act on PKC
isoforms and cascades as potential mood therapeutics
is PKC activators. PKC is activated by Ca21, phospho-
lipids and diacylglycerol, phorbol-esters or other PKC
activators [Inoue et al., 1997]. PKC isozymes are
cytosolic in the inactive state. In the inactive con-
formation, a pseudosubstrate sequence interacts with
the substrate-binding region, keeping the enzyme in
the inactive state. PKC isozymes become activated as a
result of their association with membrane containing
acid phospholipids. This association is triggered and
strongly facilitated by PKC activators.
There are several types of PKC activators
currently available for pharmacological study and
development. PKC activators, such as diacylglycerol,
arachidonic acid, phorbol esters, bryostatins, aplysia-
toxins, and teleocidins, bind to a hydrophilic cleft in a
largely hydrophobic surface of the C1 domains,
resulting in an enhanced hydrophobicity of the surface
and promoting the interaction between the C1 domain
and the phospholipid bilayer of the cell membranes
and driving removal of the pseudosubstrate region
from the catalytic site of the enzyme.
 PROTEIN KINASE C SUBSTRATE ACTIVATORS
Diacylglycerol (DAG) is a non-tumor-promoting
endogenous PKC activator and is released by the
receptor-mediated hydrolysis of membrane-resident
phosphatidylinositol 4,5-diphosphate or by hydrolysis
of phosphatidyl choline. It binds to the C1 domain of
cPKC and nPKC, with binding affinity at mM levels (for
displacing bound [20 3H]phorbol 12,13-dibutyrate
from [Kang et al., 2004]). The DAG hydrocarbon
chains are to facilitate partitioning into the lipid-rich
membrane environment (Fig. 1). One carbonyl and one
hydroxyl groups interact with PKC C1 domains,
forming hydrogen bonds (Fig. 1). The other carbonyl
group is involved in the interaction with the lipid
bilayer. Its lower affinity than that of the phorbol esters
for binding to PKC isozymes is at least partially due to
its conformational flexibility. The interaction between
PKC and DAG may serve to develop new PKC
activators with higher affinity, such as DAG-lactones
(Fig. 1) [Baba et al., 2004; Lee et al., 2004; Pu et al., 2005].
Phorbol esters, such as phorbol 12.13-dibutyrate
and phorbol 12-myristate 13-acetate (Fig. 1), also bind
to the C1 domain of cPKC and nPKC, with binding
affinities over 2 orders of magnitude greater than those
of diacylglycerol. The phorbol esters have been
reported to be potent PKC stimulators. However, their
potential as therapeutic drugs is limited by their action
as tumor promoters [Blumberg, 1988]. Their higher
potencies are derived from a conformationally rigid
orientation of hydrophilic pharmacophores. Phorbol
esters differ only in their hydrophobic side chains at
positions 12 and 13. These side chains of phorbols are
believed to change binding affinities only, not to alter
the binding mode of phorbol esters to PKC. The
hydrogen bond binding of phorbol esters includes C3
(C 5 O) and C20 (OH) (Fig. 1). Any modification to
these key functional groups abrogates or reduces the
activity of the phorbol esters. The side chain of Gln27
of the C1B domain plays an important role in the
binding site geometry to PKC activators. Mutations
Gln27 to Gly or Gly27 to Trp led to a complete loss of
the binding of phorbol 12.13-dibutyrate to the C1B
domain [Pak et al., 2001]. Its carbonyl group and
backbone NH group form an intraresidual hydrogen
bond (68%). These alternating hydrogen bonds effec-
tively connect two loops that form the binding site. The
NH2 of its side chain forms a hydrogen bond with the
carbonyl of Tyr8 (43%) and/or the carbonyl of Ser9
(28%) [Hritz et al., 2004]. According to Hritz et al.
[2004], the bind of phorbol 12-myristate 13-acetate to
the C1B domain of PKC-g isozyme involves hydro-
phobic interactions of Pro11 with the seven-member
ring of phorbol 12-myristate 13-acetate and/or Tyr22
with the five-member ring of phorbol 12-myristate 13-
acetate, and four hydrogen bonds between phorbol
161
12-myristate 13-acetate and the C1B domain: the C3
carbonyl group with the NH group of Gly23 (84%), the
hydroxyl group at C20 acts as the hydrogen bond donor
to the carbonyl oxygen of Leu 21 (98%) and as the
hydrogen bond acceptor with the NH group of Thr 12
(93%), and a weaker hydrogen bond between the
carbonyl oxygen of the ester at C13 and the hydroxyl
group of Ser 10 (37%). The C4 hydroxyl group of
phorbol esters is not necessary for PKC binding
[Tanaka et al., 2001], while the C9 hydroxyl group is
highly involved in the interaction with the lipid bilayer
[Hritz et al., 2004]. There are reports, however, that
some observed effects that are produced with phorbol
esters and viewed as PKC-mediated may be mediated by
other signaling molecules, not by PKCs [Rhee et al., 2002].
Bryostatin-1 [Trenn et al., 1988; Pettit, 1991], a
macrocyclic lactone with a chemical structure unre-
lated to phorbol esters, is an antineoplastic agent that
potently activates PKC. It is isolated from the marine
Bugula neritina [Pettit, 1991]. It possesses some
favorable pharmacological profiles as an anticancer
agent, including inhibiting cancer growth, acting
synergistically with other antineoplastic agents, stimu-
lating the immune system, and reversing multidrug
resistance. Bryostatin-1, acting as a partial agonist, can
selectively modulate PKC isozymes, such as PKCa,
PKCd, and PKCe [Isakov et al., 1993; Galron et al.,
1994; Zhang et al., 1996; Yoo et al., 2001], at very low
concentrations (at nM or sub-nM concentrations) but
prevent tumor cell growth, probably through a
selective down-regulation of PKCa in several cancer
cell lines [Jalava et al., 1993; Stanwell et al., 1994] and
preventing certain PKCd from undergoing down-
regulation [Szallasi et al., 1994]. Bryostatin-1 is distinct
from the phorbol esters in several properties. For
instance, unlike the phorbol esters, it lacks tumor-
promoting capabilities and actually counteracts tumor
promotion induced by phorbol esters [Hennings et al.,
1987]. In clinical trials as an anti-tumor agent,
bryostatin-1 is reasonably well tolerated, having a
maximum tolerated dose of 25 m g m 2 week 1 [Clamp
et al., 2003]. Its main dose-limiting toxicity is myalgia.
These profiles make it an especially attractive lead for
the design of new PKC probes. The binding of
bryostatin-1 to PKC results in PKC activation, autop-
hosphorylation, and translocation to the cell mem-
brane. Bryostatin-1-bound PKC is then down-
regulated by ubiquitination and degradation in protea-
somes. Short exposures to bryostatin-1 activate PKC,
while prolonged exposures are followed by down-
regulation of PKC. Down-regulation is most significant
when PKC is exposed to high and/or prolonged high
concentrations of bryostatin-1 [Isakov et al., 1993;
Galron et al., 1994; Song et al., 2001]. Studies of
162 SUN AND ALKON

O
O

R2
R2 O O

2
2 1 3
1 3
R1 O OH
R1 O OH (S )-1,2-Diacyl-
sn -glycerol
DAG-Lactone
(DAG) O
O
O O

O O O O
O O
H H
12 13 13
12
H H
9 9
H H
OH OH
4 4 20
20
3 3
OH OH OH OH
O O

Phorbol 12,13-dibutyrate

O
Phorbol 12-myristate 13-acetate

11 O O
19
HO O

HO O O C

A H O

3 O

AcO OH
26
1 O OH Bryostatin-1
O

Fig. 1. Chemical structures of some PKC activators. Some likely H-bonds formed when binding to the PKC isozymes are indicated with
red circles, as H-bonding acceptors; and green rectangles, as H-bonding donors of the activators.

structure-activity relations of bryostatin-1 for PKC B-ring exocyclic olefin can be deleted from the 20-
isoforms [Wender et al., 1999, 2002] indicate that the membered structure without inducing much change in
intact 20-membered macrolactone ring is needed for PKC-binding affinity. The C(26) free hydroxyl is
good PKC binding activity while the A-ring and the essential for a good interaction with PKC isozymes,
 PROTEIN KINASE C SUBSTRATE ACTIVATORS
while the C1 carbonyl group is also important for a
high affinity [Wender and Baryza, 2005]. The C19
hydroxyl group may be involved in the interaction with
the lipid bilayer during binding, while the C3 hydroxyl
group plays an important role in the conformation of
the molecule [Wender et al., 1999].
Consistent with the observations of a reduced
function of PKC and PKC substrates in the depressed
and suicide victims, we recently found that in an animal
model of depressive behavior, an administration of
small doses of bryostatin-1 produced a powerful
antidepressant activity, a response that is sensitive to
a PKC inhibitor [Sun and Alkon, 2005].
CONCLUSIONS AND FUTURE DIRECTIONS
There is no doubt that PKCs play an important
role in mood regulation and that agents acting on the
PKC and PKC substrates have promising therapeutic
values in antidepressive therapy. Several issues, how-
ever, need to be addressed before bryostatin-1 and its
analogues might be developed as therapeutic drugs.
The single most important issue is that PKC is
involved in a variety of functions and neurological
disorders so that the impact of changing its activity
is likely wide and severe. It thus remains to be
demonstrated whether a relatively non-selective acti-
vator of this pathway would be clinically useful for the
treatment of the psychiatric illness. This issue is likely
solved through defining the underlying isozyme(s) and
developing isozyme-specific agents. PKC is activated
by glutamate [Hasham et al., 1997]. PKC isozyme
activation plays an important pathological role in anoxic
LTP of the glutamatergic synaptic responses in the CA1
pyramidal cells of the hippocampus [Hammond et al.,
1994; Hsu and Huang, 1997, 1998; Bickler et al., 2004],
glutamate excitotoxicity in cultured rat hippocampal
neurons [Hasham et al., 1997], tau phosphorylation
[Ekinci and Shea, 1997], caspases-mediated apoptosis
[Yashida et al., 2003], and amyloid neurotoxicity
[Kuperstein et al., 2001]. Global ischemia increases
PKCd mRNA and protein levels in the cortex and
hippocampus and these levels are also increased in the
compromised peri-infarct region after local cerebral
ischemia [Miettinen et al., 1996; Koponen et al., 2000;
Perez-Pinzon et al., 2005]. The increased PKCd
expression in the penumbral area may be responsible
for the delayed neuronal damage [Phan et al., 2002].
PKCd is an essential for reperfusion injury after stroke
and its specific inhibitors may prove useful in
attenuating the reperfusion injury [Chou and Messing,
2005]. PKC inhibitors have been shown to decrease
infarct size in vivo in rats with transient middle cerebral
artery occlusion when administered at the onset, at 1 h,
or at 6 h of reperfusion [Bright et al., 2004].
163
PKC activators are known to stimulate cardio-
myocyte hypertrophy [Vega et al., 2004], with an
elevated risk for the development of heart failure. The
particular isoforms involved include PKCa [Braz et al.,
2002; Simonis et al., 2002; Wang et al., 2003], PKCb
[Bowman et al., 1997; Wang et al., 2003], PKCd
[Simonis et al., 2002], and PKCe [Mochly-Rosen et al.,
2000; Wang et al., 2003]. Activation of PKCd and
mitochondrial translocation occur at the onset of heart
reperfusion after cardiac ischemia and mediate apop-
tosis by facilitating the accumulation and dephosphor-
ylation of the pro-apoptotic BAD (Bcl-2 associated
death promoter), dephosphorylation of Akt, cyto-
chrome c release, PARP cleavage, and DNA laddering
[Murriel et al., 2004]. Evidence has also been provided
that inhibition of PKCd is able to provide the
protection of the heart from ischemia and reperfu-
sion-induced damage [Inagaki et al., 2003a,b]. Inhibi-
tion of PKC with ruboxistaurin (LY333531), probably
mostly through PKCb and PKCa, effectively reduces
left ventricular fibrosis and dysfunction following
myocardial infarction [Boyle et al., 2005]. In addition,
enhanced activation of PKC is correlated with the
vascular complications of diabetes mellitus [Ishii et al.,
1996]. Inhibition of PKCd and activation of PKCe
appear to protect against myocardial ischemia, but a
low level of PKCd activation is essential to maintain
normal cardiomyocyte cytoskeletal integrity [Hahn
et al., 2002].
Another issue is the oxidant-sensitivity of all the
forms of PKC isozymes [Jung et al., 2004; Li and
Takemoto, 2005]. PKCe is, for instance, sensitive to
oxidative stress, with its activation leading to neuronal
death [Jung et al., 2004]. Oxidative activation of PKCg
has been shown to be independent of cellular
diacylglycerol levels and insensitive to okadaic acid, a
phosphatase inhibitor [Li and Takemoto, 2005]. Thus,
dephosphorylation is not involved.
On the other hand, PKC activation may act to
protect neurons from mitochondrial destruction during
apoptosis [Ko et al., 2005]. One function of PKC
activation by oxidative stress is to redistribute it into
lipid rafts, disassemble gap junction plaques, and thus
inhibit gap junctional communication. This mechanism
may underlie hypoxic preconditioning, protecting cells
from further exposure to damaged signals and pre-
venting passage of apoptotic signals to adjacent cells [Li
and Takemoto, 2005]. PKCe may thereby be involved
in ischemic preconditioning [Chou and Messing, 2005].
PKCd attenuates kainite-induced cell death of mouse
cortical neurons in culture [Jung et al., 2005].
Bryostatin-1 activates a-secretase and thus reduces
the production and accumulation of neurotoxic amy-
loid. PKCd is also capable of activating the extracellular
164 SUN AND ALKON
signal-regulated kinase signaling pathway through Ras-
dependent and -independent cascades, regulating cell
proliferation and enhancing cell survival [Yang and
Kazanietz, 2003], opposite to its pro-apoptotic action.
These concerns, including impact on cardiovas-
cular function, stroke/ischmeia, and neuropathic noci-
ception [Yajima et al., 2005], may be addressed when
the PKC isoform involvement in particular actions is
defined in details and when more selective PKC
isoform activators and inhibitors are developed.
Currently, the pharmacological advantages as antide-
pressants possessed by the bryostatins are exciting.
However, their selectivity for PKC isoforms is still
rather limited. In addition, the C1 domain structures
[Yang and Kazanietz, 2003] also exist in other
molecules, such as PKD kinases [Van Lint et al.,
2002], chimaerin Rac GTPase-activating proteins
[Caloca et al., 2003], Ras guanyl nucleotide-releasing
protein [Ebinu et al., 1998], Ras and Rap1 exchange
factors, MUNC13 scaffolding proteins [Rhee et al.,
2002], and diacylglycerol kinases b and g [Shindo et al.,
2001]. Some of the effects that have been attributed to
PKC isozymes in response to phorbol esters and other
activators may, therefore, be mediated by PKC-
independent pathways [Yang and Kazanietz, 2003].
All these can only be answered by further intensive
studies. It appears that the ligand-binding sites of the
PKC isozymes possess some different profiles so that
highly potent isozyme-selective PKC activators may be
developed in the near future [Nakagawa et al., 2003;
Sridhar et al., 2003] and that PKC activators have
powerful antidementic and antidepressive activities in
animal models [Sun, 1999; Sun et al., 2002; Sun and
Alkon, 2001, 2002, 2003a,b, 2004a,b, 2005]. With a full
understanding of the intracellular targets of individual
PKC isozymes in different cell types [Hopf et al.,
2005], the use of pharmacological or molecular
therapeutic approaches based on modulation of PKC
activity/cascades may be justified clinically in the
future.
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