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Yongmei Pu Susan H. Garfield Noemi Kedei Peter M. Blumberg 《The Journal of biological chemistry》2009,284(2):1302-1312
Classic and novel protein kinase C (PKC) isozymes contain two zinc finger
motifs, designated “C1a” and “C1b” domains, which
constitute the recognition modules for the second messenger diacylglycerol
(DAG) or the phorbol esters. However, the individual contributions of these
tandem C1 domains to PKC function and, reciprocally, the influence of protein
context on their function remain uncertain. In the present study, we prepared
PKCδ constructs in which the individual C1a and C1b domains were
deleted, swapped, or substituted for one another to explore these issues. As
isolated fragments, both the δC1a and δC1b domains potently bound
phorbol esters, but the binding of [3H]phorbol 12,13-dibutyrate
([3H]PDBu) by the δC1a domain depended much more on the
presence of phosphatidylserine than did that of the δC1b domain. In
intact PKCδ, the δC1b domain played the dominant role in
[3H]PDBu binding, membrane translocation, and down-regulation. A
contribution from the δC1a domain was nonetheless evident, as shown by
retention of [3H]PDBu binding at reduced affinity, by increased
[3H]PDBu affinity upon expression of a second δC1a domain
substituting for the δC1b domain, and by loss of persistent plasma
membrane translocation for PKCδ expressing only the δC1b domain,
but its contribution was less than predicted from the activity of the isolated
domain. Switching the position of the δC1b domain to the normal position
of the δC1a domain (or vice versa) had no apparent effect on the
response to phorbol esters, suggesting that the specific position of the C1
domain within PKCδ was not the primary determinant of its activity.One of the essential steps for protein kinase C
(PKC)2 activation is
its translocation from the cytosol to the membranes. For conventional
(α, βI, βII, and γ) and novel (δ, ε, η,
and θ) PKCs, this translocation is driven by interaction with the
lipophilic second messenger sn-1,2-diacylglycerol (DAG), generated
from phosphatidylinositol 4,5-bisphosphate upon the activation of
receptor-coupled phospholipase C or indirectly from phosphatidylcholine via
phospholipase D (1). A pair of
zinc finger structures in the regulatory domain of the PKCs, the
“C1” domains, are responsible for the recognition of the DAG
signal. The DAG-C1 domain-membrane interaction is coupled to a conformational
change in PKC, both causing the release of the pseudosubstrate domain from the
catalytic site to activate the enzyme and triggering the translocation to the
membrane (2). By regulating
access to substrates, PKC translocation complements the intrinsic enzymatic
specificity of PKC to determine its substrate profile.The C1 domain is a highly conserved cysteine-rich motif (∼50 amino
acids), which was first identified in PKC as the interaction site for DAG or
phorbol esters (3). It
possesses a globular structure with a hydrophilic binding cleft at one end
surrounded by hydrophobic residues. Binding of DAG or phorbol esters to the C1
domain caps the hydrophilic cleft and forms a continuous hydrophobic surface
favoring the interaction or penetration of the C1 domain into the membrane
(4). In addition to the novel
and classic PKCs, six other families of proteins have also been identified,
some of whose members possess DAG/phorbol ester-responsive C1 domains. These
are the protein kinase D (5),
the chimaerin (6), the munc-13
(7), the RasGRP (guanyl
nucleotide exchange factors for Ras and Rap1)
(8), the DAG kinase
(9), and the recently
characterized MRCK (myotonic dystrophy kinase-related
Cdc42-binding kinase) families
(10). Of these C1
domain-containing proteins, the PKCs have been studied most extensively and
are important therapeutic targets
(11). Among the drug
candidates in clinical trials that target PKC, a number such as bryostatin 1
and PEP005 are directed at the C1 domains of PKC rather than at its catalytic
site.Both the classic and novel PKCs contain in their N-terminal regulatory
region tandem C1 domains, C1a and C1b, which bind DAG/phorbol ester
(12). Multiple studies have
sought to define the respective roles of these two C1 domains in PKC
regulation, but the issue remains unclear. Initial in vitro binding
measurements with conventional PKCs suggested that 1 mol of phorbol ester
bound per mole of PKC
(13-15).
On the other hand, Stubbs et al., using a fluorescent phorbol ester
analog, reported that PKCα bound two ligands per PKC
(16). Further, site-directed
mutagenesis of the C1a and C1b domains of intact PKCα indicated that the
C1a and C1b domains played equivalent roles for membrane translocation in
response to phorbol 12-myristate 13-acetate (PMA) and (-)octylindolactam V
(17). Likewise, deletion
studies indicated that the C1a and C1b domains of PKCγ bound PDBu
equally with high potency (3,
18). Using a functional assay
with PKCα expression in yeast, Shieh et al.
(19) deleted individual C1
domains and reported that C1a and C1b were both functional and equivalent upon
stimulation by PMA, with either deletion causing a similar reduction in
potency of response, whereas for mezerein the response depended essentially on
the C1a domain, with much weaker response if only the C1b domain was present.
Using isolated C1 domains, Irie et al.
(20) suggested that the C1a
domain of PKCα but not those of PKCβ or PKCγ bound
[3H]PDBu preferentially; different ligands showed a generally
similar pattern but with different extents of selectivity. Using synthesized
dimeric bisphorbols, Newton''s group reported
(21) that, although both C1
domains of PKCβII are oriented for potential membrane interaction, only
one C1 domain bound ligand in a physiological context.In the case of novel PKCs, many studies have been performed on PKCδ
to study the equivalency of the twin C1 domains. The P11G point mutation of
the C1a domain, which caused a 300-fold loss of binding potency in the
isolated domain (22), had
little effect on the phorbol ester-dependent translocation of PKCδ in
NIH3T3 cells, whereas the same mutation of the C1b caused a 20-fold shift in
phorbol ester potency for inducing translocation, suggesting a major role of
the C1b domain for phorbol ester binding
(23). A secondary role for the
C1a domain was suggested, however, because mutation in the C1a domain as well
as the C1b domain caused a further 7-fold shift in potency. Using the same
mutations in the C1a and C1b domains, Bögi et al.
(24) found that the binding
selectivity for the C1a and C1b domains of PKCδ appeared to be
ligand-dependent. Whereas PMA and the indole alkaloids indolactam and
octylindolactam were selectively dependent on the C1b domain, selectivity was
not observed for mezerein, the 12-deoxyphorbol 13-monoesters prostratin and
12-deoxyphorbol 13-phenylacetate, and the macrocyclic lactone bryostatin 1
(24). In in vitro
studies using isolated C1a and C1b domains of PKCδ, Cho''s group
(25) described that the two C1
domains had opposite affinities for DAG and phorbol ester; i.e. the
C1a domain showed high affinity for DAG and the C1b domain showed high
affinity for phorbol ester. No such difference in selectivity was observed by
Irie et al. (20).PKC has emerged as a promising therapeutic target both for cancer and for
other conditions, such as diabetic retinopathy or macular degeneration
(26-30).
Kinase inhibitors represent one promising approach for targeting PKC, and
enzastaurin, an inhibitor with moderate selectivity for PKCβ relative to
other PKC isoforms (but still with activity on some other non-PKC kinases) is
currently in multiple clinical trials. An alternative strategy for drug
development has been to target the regulatory C1 domains of PKC. Strong proof
of principle for this approach is provided by multiple natural products,
e.g. bryostatin 1 and PEP005, which are likewise in clinical trials
and which are directed at the C1 domains. A potential advantage of this
approach is the lesser number of homologous targets, <30 DAG-sensitive C1
domains compared with over 500 kinases, as well as further opportunities for
specificity provided by the diversity of lipid environments, which form a
half-site for ligand binding to the C1 domain. Because different PKC isoforms
may induce antagonistic activities, inhibition of one isoform may be
functionally equivalent to activation of an antagonistic isoform
(31).Along with the benzolactams
(20,
32), the DAG lactones have
provided a powerful synthetic platform for manipulating ligand: C1 domain
interactions (31). For
example, the DAG lactone derivative 130C037 displayed marked selectivity among
the recombinant C1a and C1b domains of PKCα and PKCδ as well as
substantial selectivity for RasGRP relative to PKCα
(33). Likewise, we have shown
that a modified DAG lactone (dioxolanones) can afford an additional point of
contact in ligand binding to the C1b domain of PKCδ
(34). Such studies provide
clear examples that ligand-C1 domain interactions can be manipulated to yield
novel patterns of recognition. Further selectivity might be gained with
bivalent compounds, exploiting the spacing and individual characteristics of
the C1a and C1b domains (35).
A better understanding of the differential roles of the two C1 domains in PKC
regulation is critical for the rational development of such compounds. In this
study, by molecularly manipulating the C1a or C1b domains in intact
PKCδ, we find that both the C1a and C1b domains play important roles in
PKCδ regulation. The C1b domain is predominant for ligand binding and
for membrane translocation of the whole PKCδ molecule. The C1a domain of
intact PKCδ plays only a secondary role in ligand binding but stabilizes
the PKCδ molecule at the plasma membrane for downstream signaling. In
addition, we show that the effect of the individual C1 domains of PKCδ
does not critically depend on their position within the regulatory domain. 相似文献
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C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a > 100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains. 相似文献
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We report the assignment of the 110 amino acid second calponin homology domain of human α-actinin-4. The two calponin homology
domains of α-actinin combine to regulate F-actin binding. 相似文献
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Kazuo Yamasaki Taro Tamaki Sumiko Uzawa Ushio Sankawa Shoji Shibata 《Phytochemistry》1973,12(12):2877-2882
Feeding of benzoic acid-[7-14C], benzaldehyde-[7-14C] and cinnamic acid-[3-14C] to Ephedra distachya resulted in efficient incorporations of 14C into the α-carbon atom of the side chain of l-ephedrine. Thus ephedrine was shown to be biosynthesized by the condensation of a C6C1 portion which is derived from phenylalanine via cinnamate and an unidentified C2-N fragment. 相似文献
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