Diacylglycerol Signaling

3 Phorbol Esters and Diacylglycerol: The PKC Activators
of the corresponding residues in the C1b domain of PKC delta to arginine progressively
led to loss of ligand-binding activity. Conversely, mutation of the arginine
residues in the isolated C1 domains of PKC zeta and iota to the corresponding
residues in the C1b domain of PKC delta restored phorbol ester responsiveness.
Whether one can design ligands that will exploit the structural peculiarities of
such atypical C1 domains that retain the binding cleft geometry remains to be
determined.
The DAG/phorbol ester responsiveness of the C1 domains of the Vav isoforms
is unclear. The Vav family members function as guanyl exchange factors for the
small GTPase Rho (Hornstein et al. 2004; Swat and Fujikawa 2005). We showed
that Vav1 bound neither [ 3 H]PDBu nor [ 3 H]bryostatin under conditions in which
very weak binding affinity should still have been detectable (Kazanietz et al. 1994).
On the other hand, modeling suggests that the C1 domain of Vav2 is very similar
to that for PKC (Heo et al. 2005), and crystallographic analysis of a Vav1 fragment
including the C1 domain together with the DH and PH domains likewise indicates
that the binding cleft in the C1 domain is preserved (Rapley et al. 2008). This cleft
is located in apposition to the DH domain, which might both prevent access by
ligand and prevent association with the lipid bilayer, which provides one of the
important elements of the overall pharmacophore. Nonetheless, the retention of
the binding site geometry raises the exciting possibility that appropriately modified
ligands could access this binding site and disrupt the activating function of Vav
proteins for Rho family members.
The C1 domain of RasGRP2 has a seemingly homologous sequence to that of
the other members of the RasGRP family. Although it was able to bind to anionic
phospholipid vesicles as did the C1 domains of the other family members, it did not
respond to either the addition of exogenous DAG or of phorbol ester with translocation
when expressed in cells or with enhanced association with phospholipid vesicles
in vitro (Johnson et al. 2007). In light of its close sequence homology, the basis for
its lack of ligand recognition should be of considerable interest.
3.18 C1 Domains with Reduced Affinity
Intermediate between C1 domains with high affinity for DAG/phorbol ester and
those without measureable affinity (as yet) are those C1 domains with reduced
activity, but where the reduced affinity raises the question of whether these C1
domains are still capable of recognizing physiological levels of endogenous DAG.
For example, we have shown that the C1 domains of MRCK alpha and beta indeed
bind PDBu but with 60–90-fold weaker affinities than the C1 domain of PKC delta
(Choi et al. 2008). Since it is not likely that there would be such differences in the
concentration of endogenous DAG, a probable explanation is that other coregulators
in the case of MRCK contribute to the membrane association, complementing
the contributions from the liganded C1 domain itself.
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