From the
The protein kinase C family of enzymes transduces the myriad of signals promoting lipid hydrolysis. The prevalence of this enzyme family in signaling is exemplified by the diverse transduction mechanisms that result in the generation of protein kinase C's activator, diacylglycerol. Signals that stimulate members of the large families of G protein-coupled receptors, tyrosine kinase receptors, or non-receptor tyrosine kinases can cause diacylglycerol production, either rapidly by activation of specific phospholipase Cs or more slowly by activation of phospholipase D to yield phosphatidic acid and then diacylglycerol(1, 2, 3) . In addition, fatty acid generation by phospholipase A2 activation modulates protein kinase C activity(3) . Thus, multiple receptor pathways feeding into multiple lipid pathways have the common end result of activating protein kinase C by production of its second messenger.
Phorbol esters, potent tumor promoters, can substitute for diacylglycerol in activating protein kinase C(1, 2, 3) . Unlike diacylglycerol, phorbol esters are not readily metabolized, and treatment of cells with these molecules results in prolonged activation of protein kinase C. As a result, phorbol esters have proved invaluable in dissecting out protein kinase C-catalyzed phosphorylations in vivo.
In addition to regulation by diacylglycerol or phorbol
esters, all isozymes of protein kinase C require phosphatidylserine, an
acidic lipid located exclusively on the cytoplasmic face of membranes,
and some isozymes require Ca for optimal activity (4, 5, 6, 7) . This review discusses
the structure of the protein kinase C family, its enzymatic function,
and how structure and function are regulated by 1) cofactors and 2)
phosphorylation.
Members of the protein kinase C family are a single
polypeptide, comprised of an N-terminal regulatory region
(approximately 20-40 kDa) and a C-terminal catalytic region
(approximately 45 kDa) (Fig. 1). Cloning of the first isozymes
in the mid-1980s revealed four conserved domains:
C1-C4(8) . Each is a functional module, and many
unrelated proteins have one or the other(9) . The function of
each of these domains has been established by extensive biochemical and
mutational analysis; the C1 domain contains a Cys-rich motif,
duplicated in most isozymes, that forms the diacylglycerol/phorbol
ester binding site (Fig. 1, orange) (7) ; this
domain is immediately preceded by an autoinhibitory pseudosubstrate
sequence (Fig. 1, green)(10) ; the C2 domain
contains the recognition site for acidic lipids and, in some isozymes,
the Ca-binding site (Fig. 1, yellow) (9) . The C3 and C4 domains form the ATP- and substrate-binding
lobes of the kinase core (Fig. 1, pink and cyan)(11) . The regulatory and catalytic halves are
separated by a hinge region that becomes proteolytically labile when
the enzyme is membrane-bound(6) ; the proteolytically generated
kinase domain (protein kinase M), freed of inhibition by the
pseudosubstrate, is constitutively active (12) .
Figure 1: Schematic representation of the primary structure of conventional, novel, and atypical protein kinase Cs. Indicated are the pseudosubstrate domain (green), C1 domain comprising one or two Cys-rich motifs (orange), C2 domain (yellow) in the regulatory half, and the ATP-binding lobe (C3, pink) and substrate-binding lobe (C4, teal blue) of the catalytic region. The C2 domain of novel protein kinase Cs lacks amino acids involved in binding calcium but has key conserved residues involved in maintaining the C2 fold (hence its description as ``C2-like''). Atypical protein kinase Cs have only one Cys-rich motif, and phorbol ester binding has not been detected.
To date, 11
protein kinase C isozymes have been identified and classified into
three groups based on their structure and cofactor
regulation(3) . The best characterized and first discovered are
the conventional protein kinase Cs: , two alternatively spliced
variants
I and
II, and
. This class distinguishes itself
from the others in that function is regulated by Ca
;
its C2 domain contains a putative Ca
-binding site
(see below). The next well characterized are the novel protein kinase
Cs:
,
,
(L),
, and µ. These isozymes are
structurally similar to the conventional protein kinase Cs, except that
the C2 domain, while maintaining structural residues, does not have the
functional groups that appear to mediate Ca
binding
(see below). The least understood isozymes are the atypical protein
kinase Cs:
and
(I). These differ significantly in structure
from the other two classes; first, the C1 domain contains only one
Cys-rich motif (not two), and second, key residues that maintain the C2
fold do not appear to be present. Furthermore, these isozymes have been
reported not to respond to phorbol esters in vivo or in
vitro(3) . Perhaps adding to the three groups, two kinases
with a C2 domain similar to that of novel protein kinase Cs, but with
no C1 domain, have been identified(13, 14) .
The
crystal structure of the second Cys-rich repeat from the C1 domain of
protein kinase C was solved recently with (Fig. 2A) and without bound phorbol ester by Hurley and
co-workers(15) , as was the NMR structure of the corresponding
repeat from protein kinase C
, in the absence of
ligand(16) . Strikingly, this
sheet-rich domain undergoes
no conformational change upon ligand binding. Rather, binding of
phorbol ester plugs the hydrophilic binding site (a groove formed by
two unzipped
strands), so that the top third of the domain
displays a contiguous hydrophobic surface(15) .
Figure 2:
Structures of protein kinase C's
domains. A, C1 domain. The ribbon and surface diagram of amino
acids 231-280 in the second Cys-rich domain of protein kinase C
with bound phorbol ester (green) based on the
coordinates of Zhang et al.(15) is shown. Conserved
Cys (yellow) and His (purple) that coordinate the two
zinc atoms of each cysteine-rich repeat (78) (green
balls) are indicated. The arrow indicates the C12
position of the phorbol ester that is fatty acylated in bioactive
phorbol esters(51) . B, C2 domain. The ribbon diagram
of residues 167-240 from the C2 domain of synaptotagmin based on
the coordinates of Sutton et al.(17) is shown. The
five aspartates in the Ca
-binding site are indicated
in pink, the bulky hydrophobics on the back face in purple, and the adjacent two
strands that are positively
charged and likely constitute the acidic lipid-binding surface are in blue. Residues shown in orange are conserved in all
C2 domains(9) . C, catalytic (C3 and C4) domain. The
modeled structure of residues 340-632 of protein kinase C
II
with bound pseudosubstrate (residues 9-28) (20) is shown.
The upper lobe, involved primarily in nucleotide binding, is mainly
sheet (pink) and the lower lobe, containing the
substrate-binding cavity, is predominantly
helix (teal
blue). Indicated are ATP (cream), two Mn
atoms (red dots), and the pseudosubstrate (green) with the orange dot representing the alanine
at the phosphoacceptor position. The yellow loop at the
entrance to the catalytic site (below ATP) is the activation
loop(11) ; phosphorylation here aligns residues for
catalysis(75) . Reproduced from (20) .
The crystal
structure of the C2 domain of synaptotagmin, elucidated by Sprang and
co-workers(17) , reveals how the other half of the regulatory
region of protein kinase C folds. Fig. 2B shows the
core of this domain (``C2 key''): 5 aspartate residues form
the Ca-binding site (pink); on the back face
of this cleft are bulky aromatics (purple) adjacent to a basic
surface formed by two
strands (blue). Sossin and
Schwartz (18) noted that novel protein kinase Cs contain a C2
domain. The solved structure elucidates how these protein kinase Cs can
have this domain without being Ca
-regulated; the C2
domain of novel protein kinase Cs has the conserved residues that
maintain the fold of the domain (e.g.Fig. 2B, orange), but the coordinating oxygens in the
Ca
-binding site are mainly absent(9) .
A
modeled structure of the catalytic domain of protein kinase C II,
with bound pseudosubstrate, based on the crystal structure of protein
kinase A with bound inhibitory peptide (19) is shown in Fig. 2C(20) . The primary sequence of the
kinase core of conventional protein kinase Cs is approximately 40%
identical to that of protein kinase A's core. The N-terminal
residue of the model is just before the hinge region; the peptide chain
would continue on to the C2 and then C1 domains and then connect to the
pseudosubstrate. Modeling of the latter in the substrate binding cavity
reveals that it is held there, in part, by a cluster of acidic residues
that is unique to the protein kinase C family(20) . The
pseudosubstrate sequence was identified by House and Kemp (10) based on the ability of a synthetic peptide of this
sequence to inhibit protein kinase C.
Protein kinase C typically phosphorylates serine or threonine
residues in basic sequences but displays significantly less specificity
than protein kinase A(29) . First, unlike protein kinase
A(29) , no clear requirements for positive charge at specific
positions are apparent from analysis of sequences around
phosphorylation sites (30, 31, 32) or from
analysis of synthetic peptide
substrates(23, 26, 33) . Second, protein
kinase C displays lower stereospecificity than protein kinase A (25) , phosphorylating both D- and L-stereoisomers of configurational isomers of a number
of alcohols(24) . Lawrence and co-workers (24) have
suggested that protein kinase C's lack of stereospecificity could
reflect substrate binding in either direction (i.e. C to N or
N to C) in the substrate-binding cavity(24) .
Protein kinase C also autophosphorylates in vitro(34, 35) by an intramolecular mechanism (36) at the N terminus, hinge, and C terminus(37) ; the latter site is a poor in vitro site because it is almost quantitatively phosphorylated in vivo (see below).
In addition to
catalyzing phosphorylation reactions, protein kinase C has ATPase and
phosphatase activity. The enzyme catalyzes a cofactor-dependent and
substrate-stimulated hydrolysis of ATP(38) , and it can work
backwards (i.e. as a phosphatase) in the presence of excess
ADP. ()
A key regulator of protein kinase C function in vivo is likely to be subcellular distribution of both the enzyme and substrate (40) . Protein kinase C isozymes are distributed differentially throughout the cell (and differently among many cell types)(39) , and a number of targetting proteins have been described(40) .
Deciphering the specific functions of isozymes likely awaits the development of isozyme-specific inhibitors(41) . The application of combinatorial chemistry toward this goal has provided the first isozyme-specific inhibitor(42) ; similar specificity using antisense DNA has been demonstrated for in situ studies(43) .
The function of protein kinase C is regulated by two equally important mechanisms. First, the enzyme is rendered catalytically competent by phosphorylations that correctly align residues for catalysis and localize protein kinase C to the cytosol. Second, binding of ligands or, in some cases, substrate activates the enzyme by removing the pseudosubstrate from the substrate-binding site.
Biochemical experiments have established that, as
predicted(10) , activation of protein kinase C is accompanied
by removal of its pseudosubstrate from the kinase
core(20, 44) . Specifically, the basic pseudosubstrate
is protected from proteolysis when the enzyme is not catalytically
active but becomes highly sensitive to proteolysis by trypsin or
endoproteinase Arg-C upon activation(44) . Importantly, the
pseudosubstrate is unmasked whether protein kinase C is activated by
conventional (phosphatidylserine, diacylglycerol, and
Ca), non-conventional (e.g. short chained
phosphatidylcholines(45) ), or cofactor-independent substrates (e.g. protamine(46) )(20) . Consistent with
this, incubation of protein kinase C with an antibody directed against
the pseudosubstrate was shown to activate the enzyme, presumably by
removing the pseudosubstrate from the active site(47) .
Diacylglycerol and phorbol
esters serve as hydrophobic anchors to recruit protein kinase C to the
membrane; they cause a dramatic increase in the enzyme's membrane
affinity ()that is linearly related to the mol fraction C1
ligand in the bilayer, is reversible, and can occur in the absence of
acidic lipids and C2 domain
interactions(
)(55, 57, 58, 59, 60, 61) .
Differences in the biological action of these two classes of ligands
are accounted for by the 2 orders of magnitude increased potency of
phorbol esters compared with diacylglycerol (58) and the long
life of phorbol esters in cells. The phorbol ester domain structure
suggests how the membrane anchor works; by capping the hydrophilic
ligand groove, phorbol ester binding alters the surface hydrophobicity
of the domain, thus promoting the membrane interaction in the absence
of conformational changes(15) .
In addition to increasing protein kinase C's membrane affinity, C1 ligands may also stabilize the active conformation of protein kinase C. Diacylglycerol doubles the catalytic efficiency of enzyme that is bound to phosphatidylserine(57, 59, 62, 63) ; it also stimulates the activation promoted by fatty acids (3) and short chained phosphatidylcholines(45) .
C1
ligands markedly reduce the concentration of Ca required for the phosphatidylserine-dependent activation of
protein kinase C (64) . The molecular basis for this does not
arise from allosteric interactions between the C1 and C2 domain sites;
Ca
has no effect on protein kinase C's affinity
for either C1 ligand
(59, 65) . Rather, the
apparent synergy between these two activators arises because each, by
separate mechanisms, increases the affinity of protein kinase C for
membranes. Consistent with no allosteric interactions, the structure of
the phorbol ester-binding domain is unchanged by phorbol ester binding (15) .
In the absence of C1 ligands,
protein kinase C binds acidic lipids with little selectivity for the
headgroup beyond the requirement for negative charge (59) (this
interaction is Ca-regulated for conventional protein
kinase Cs; see next section). This binding is of relatively low
affinity, is sensitive to ionic strength, is accompanied by a
conformational change that exposes the hinge region to proteolysis, and
is typically not accompanied by much activation or pseudosubstrate
exposure (Fig. 3, top
middle)(44, 59, 60) . Note that the
hinge exposure is independent of the active state of the kinase,
reflecting rather the ``membrane-bound conformation'' of the
enzyme (67) .
Figure 3:
Model for the regulation of protein kinase
C by 1) phosphorylation and 2) membrane binding and pseudosubstrate
release. Newly synthesized protein kinase C (PKC) associates
with the detergent-insoluble fraction of cells (72) (bottom
left). It is processed to the mature, cytosolic form by three
functionally distinct phosphorylations: transphosphorylation at the
activation loop to render the kinase catalytically competent (Thr-500
in II); an autophosphorylation at the C terminus (Thr-641 in
II) that stabilizes the catalytically competent
conformation(73) ; and a second autophosphorylation at the C
terminus (Ser-660 in
II) that releases protein kinase C into the
cytosol(73) . This triple phosphorylated mature form is
inactive because the pseudosubstrate occupies the substrate-binding
cavity (middle). Generation of diacylglycerol (DG)
causes the affinity of protein kinase C for membranes to increase
dramatically. Membrane translocation is mediated by diacylglycerol
binding to the C1 domain and phosphatidylserine (PS) binding
to the C2 domain (top right). The affinity for acidic lipids
is increased by Ca
for conventional protein kinase
Cs, likely by structuring the lipid-binding surface, but not for novel
protein kinase Cs, whose lipid-binding surface may already be
structured. Protein kinase C can bind to membranes with low affinity
with either C1 domain ligands (not shown) or with C2 domain ligands (top middle). However, it is the high affinity binding (top left) mediated by both domains that results in
pseudosubstrate release and maximal activation. Asterisks indicate the exposed hinge, which becomes proteolytically labile
upon membrane binding (independently of pseudosubstrate
release(67) ), and the exposed pseudosubstrate, which becomes
proteolytically labile upon activation (independently of membrane
binding(20) ).
The presence of diacylglycerol causes a striking and selective increase in conventional and novel protein kinase C's affinity for phosphatidylserine that is accompanied by activation and pseudosubstrate release (Fig. 3, top left)(6) . This high affinity interaction is 1 order of magnitude stronger for surfaces containing phosphatidyl-L-serine compared with other acidic lipids such as phosphatidyl-D-serine (59) . Thus, specific structural elements of the L-serine headgroup are required for the high affinity binding of protein kinase C to membranes containing C1 ligands. Because phosphatidylserine promotes the binding of phorbol esters to a single recombinant Cys-rich domain(55) , the specificity may arise from additional interactions of the L-serine headgroup with the C1 domain or with new surfaces created at the C1-C2 interface. Kinetic studies suggest that protein kinase C interacts cooperatively with multiple phosphatidylserine molecules(6, 7) .
The C2 domain
structure provides tantalizing insight into how Ca might increase the affinity of conventional, but not novel,
protein kinase Cs for acidic lipids. For conventional protein kinase
Cs, binding of Ca
to the aspartate-lined
``mouth'' (Fig. 2B) might clamp together the
upper and lower lobes, thus orienting the bulky aromatics on the back
face of the mouth to interact with the membrane and orienting the basic
face of the
sheet behind the site to interact with lipid
headgroups. For novel protein kinase Cs (such as
), the presence
of an Arg instead of an Asp at one of the positions in the site (9) might cause the mouth to adopt the closed conformation, so
that the domain is already structured to bind acidic lipids. C1 ligands
would then target novel protein kinase Cs to membranes, with the
reduction in dimensionality promoting the binding of the C2 domain to
acidic lipids. Consistent with this, the lipid regulation of novel
protein kinase Cs is the same as that for conventional protein kinase
Cs, except that it occurs in the absence of
Ca
(70) .
Regulation by Phosphorylation in Vivo
Pulse-chase experiments by Fabbro and co-workers (72) provided the first evidence that protein kinase C is
phosphorylated in vivo. Specifically, they showed that protein
kinase C is first synthesized as an inactive, dephosphorylated
precursor with an apparent M of 74 kDa; this was
chased to a transient 77-kDa phospho-form and then to the final 80-kDa
mature form. Mass spectrometry has recently revealed that protein
kinase C is modified by three phosphorylations in vivo (73,
74). The differential dephosphorylation of these sites by protein
phosphatases 1 and 2A(75) , as well as analysis of
phosphorylation site mutants(76, 77) , has allowed the
function of each phosphorylation to be identified (73) .
A
model consistent with biochemical data is presented in the lower
half of Fig. 3. Newly synthesized protein kinase C
associates with a detergent-insoluble cell fraction(72) ; it is
rendered catalytically competent upon phosphorylation by a putative
protein kinase C kinase on its activation loop (Fig. 2C). Negative charge on this loop at the entrance
to the active site correctly aligns residues involved in catalysis in
diverse kinases (11) ; replacement of the phosphorylated
residue (Thr-500) with Glu in protein kinase C II results in
activatable enzyme (77) whereas replacement with neutral
non-phosphorylatable residues in this isozyme (77) or protein
kinase C
(76) results in kinase that cannot be activated.
The first consequence of the transphosphorylation appears to be
autophosphorylation at the C terminus of the kinase; this residue is
Thr-641 in protein kinase C
II (9 residues removed from the C
terminus of the model in Fig. 2C)(73, 74) . Phosphorylation
here likely stabilizes the catalytically competent conformation of the
kinase as it replaces the requirement for negative charge at the
activation loop(73) ; this phosphorylation causes the first
detectable shift in electrophoretic mobility(73, 75) .
Last, the enzyme autophosphorylates further along the C terminus
(Ser-660 in protein kinase C
II) in a motif shared by several
other kinases(73) ; this phosphorylation causes the final shift
in electrophoretic mobility and releases the mature enzyme into the
cytosol(73, 75) . It is this 80-kDa form, localized to
the detergent-soluble fraction, that has been extensively purified and
studied in vitro. Curiously, it is only half-phosphorylated at
the activation loop (but quantitatively phosphorylated at the two
C-terminal sites)(73) , suggesting that
dephosphorylation/transphosphorylation at this position may regulate
the kinase in response to stimuli. The mature form then translocates to
the membrane and undergoes the pseudosubstrate regulation discussed
above.
Protein kinase C is regulated by two distinct mechanisms: by phosphorylation which regulates the active site and subcellular localization of the enzyme, and by second messengers which promote protein kinase C's membrane association and resulting pseudosubstrate exposure. Regulation by two independent mechanisms may provide exquisite fine-tuning for this family of enzymes, ensuring low basal activity in the midst of complex intracellular signaling pathways.