Inherited mutations in three genes lead to the familial form of Cerebral Cavernous
Malformations (CCM). These vascular dysplasias most commonly occur in the brain, and
manifest as dilated, mulberry-shaped lesions with a single endothelial layer. The
consequences of these lesions can be leakage and sequelae such as focal neurological
deficits, epilepsy, or hemorrhagic stroke. Until recently, however, the molecular
basis for the acquisition of CCM disease was unknown. The three genes associated with
CCM disease encode the proteins KRIT1/CCM1 (Krev interaction trapped 1/cerebral cavernous
malformations 1), CCM2/malcavernin/OSM (cerebral cavernous malformations 2, osmosensing
scaffold for MEKK3), and CCM3/PDCD10 (cerebral cavernous malformations 3/programmed
cell death 10). Maintenance of a normal vasculature requires expression of all three
proteins. Almost all of the discovered mutations in these genes result in truncations
of their protein products, although some rare missense mutations have been found to
encode misfolded protein [1]. The three proteins implicated in the disease were predicted
to be distinct from one another in structure, but their molecular level architecture
and many details of their normal function and protein-protein interaction networks
were unknown. Therefore, to better understand the signaling processes that are affected
by CCM disease it was necessary to address these questions from the ground up, starting
at the atomic level [2].
The formation of a ‘CCM complex’ between KRIT1, CCM2 and CCM3 was previously suggested,
but without targeted disruption of the interactions and selective probing of the functional
consequences of disruption, the specific role(s) of heterotrimerization have been
hard to define. We tackled these questions using a structure-directed approach. Our
studies revealed the molecular basis of a preferred interaction site between KRIT1
and CCM2 [1] and the molecular basis for the interaction of CCM2 with CCM3 [3], thus
providing the atomic-level framework for the CCM complex. When we crystallized CCM3,
we found that it is of an unexpected fold encompassing two domains [4], the C-terminal
of which directly interacts with a conserved motif in CCM2 [3]. Targeted disruption
of the interaction between CCM2 and CCM3 has a number of functional consequences.
CCM2 and CCM3 reciprocally stabilize one another; knockdown of either CCM2 or CCM3
results in severely reduced stability of the other protein. Importantly, the decreased
stability can be rescued by re-expression of the wild-type protein but not protein
that has been mutated at the binding site. Loss of either expressed protein also deleteriously
impacts proliferation and network formation in endothelial cells. Interestingly, CCM3
expression can rescue proliferation in CCM2 depleted cells, but CCM2 cannot rescue
expression in CCM3 depleted cells and the interaction surface between CCM2 and CCM3
must be preserved to rescue proliferation. Conversely, CCM2 is better able to rescue
endothelial network formation than CCM3. An important role of the CCM complex in endothelial
cells therefore seems to be stabilization of the CCM proteins, allowing them to achieve
their overlapping but distinct roles.
The CCM proteins also interact with other signaling proteins. We, and others, have
shown the molecular basis for interactions of KRIT1 with the Rap1 small GTPase [5]
and with the suppressor of integrin activation, ICAP1 [6]. We have also very recently
discovered how CCM2 interacts with the MAP kinase kinase kinase, MEKK3. A previously
uncharacterized N-terminal helical region of MEKK3 directly binds the C-terminal HHD
(harmonin homology domain) of CCM2 [7]. Targeted disruption of this interaction was
not observed to have an impact on MEKK3 catalytic activity, but did alter MEKK3 sub-cellular
localization. Disruption of the CCM2:MEKK3 interaction also increases Rho/ROCK signaling,
potentially implying that this upregulation may be related to the ability of CCM2
and MEKK3 to interact. In vivo, targeted disruption of the CCM2:MEKK3 interaction
increased the permeability of the neurovasculature. This study therefore provides
a link between CCM2, MEKK3, and the dysregulation of Rho/ROCK signaling that has previously
been observed in CCM disease.
Taken together, our recent studies, and those from other groups not mentioned here
due to space and formatting constraints, suggest that the proteins of the CCM complex
not only require one another for reciprocal stabilization, but also act as a platform
for signal transduction. A more intricate understanding of how the CCM complex signaling
platform is formed, how its formation is regulated, and how it interacts with binding
partners are clearly required. Nonetheless, the molecular level reasons why CCM disease
is so distinctly associated with loss of KRIT1, CCM2 and CCM3 are now becoming clearer.