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Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34 Page 7 of 23

CCM2 PTB domain
Some PTB domains can bind to phospholipids and it was expected that such an affinity in CCM2 PTB domain
will transport CCM2 complex to the plasma membrane, however no consensus has been reached in that regard.
One study stated that CCM2 PTB domain is able to bind to phospholipids , while other studies suggested the
[85]
opposite [3,19] . CCM2 PTB domain has been shown to interact with two proteins, TrkA and Smurf1. TrkA is
present in neurons. The PTB domain of CCM2 binds to the cytoplasmic tail of TrkA . This complex binds with
[86]
CCM3 and STK25 to form a large complex. This may happen through a direct mechanism where CCM2 brings
CCM3 or an indirect mechanism. However, the formation of this complex leads to apoptosis. This pathway
is found to be especially important in the field of prognostic and therapeutic aspects of neuroblastoma and
medulloblastoma . CCM2 PTB domain was also reported to bind to Smurf1 HECT domain. This interaction
[87]
brings Smurf1 to the plasma membrane . This interaction has been related to RhoA degradation.The lack of
[88]
RhoA degradation results in uncurbed activation of Rho-associated coiled coil-forming kinase (ROCK) which
leads to stress fiber formation . One report stated that CCM2 protein in macrophages is found adjacent to
[89]
newly synthesized actin polymers. This localization of CCM2 is compatible with the predicted function of
regulating RhoA because RhoA is active in actively synthesizing actin polymers .
[22]
CCM2 C-terminal HH domain
A four nucleotide duplication of the last exon of CCM2 was introduced to create a mutation disrupting
the structure of the HH domain at the C-terminus. This mutated form of CCM2 protein was able to bind
to CCM1 and CCM3, however unable to bind to MEKK3, a Map3 kinase, suggesting that the HH domain
mediates the interaction between CCM2 and MEKK3 .
[90]

CCM2 KARET domain and LD motif
Some reports showed that CCM2 interacted with CCM3 through a KARET domain at the C-terminus
of CCM2 (residues 228-444), which overlaps with HH domain [16,17,63,86,87] . However, it was recently shown
through pull-down assays that the binding pocket was located in the middle of CCM2 (residues 223-238),
between the N-terminal PTB domain and C-terminal HH domain. This region was determined to contain
a LD motif, which binds to FAT-H domain on CCM3. The FAT-H domain of CCM3 contains a hydrophobic
pocket termed HP1 which is the binding site for CCM2. The LD motif adopts a 3.5 turn α-helix parallel
to α7-helix in CCM3. The interaction is largely made up of hydrophobic residues from CCM2 (T225, I226,
F228, L229, A232, I233, G236, and A237) binding to hydrophobic residues from CCM3 (I131, I134, A135,
I138, L142, V168, F174, L178, S171, K132, and K139). These hydrophobic resides are also extremely conserved
through evolution, indicating their significance . CCM2-CCM3 complex formation reciprocally protects
[63]
the proteins from degradation. Either CCM2 or CCM3 depletions result in concurrent decreases in the
reciprocal protein. Furthermore, CCM2 and CCM3 mutated cells grew slower than wild-type ones. This
mutant phenotype can be rescued with the addition of CCM2 or CCM3. Overexpression of CCM2 in CCM3-
depleted cells did not restore cellular proliferation, however overexpression of CCM3 in CCM2-depleted
cells did, indicating that the CCM3 may have a greater contribution to cell proliferation .
[91]

CCM2-Like protein
Recently CCM2-Like (Ccm2l) protein was identified in zebrafish. This protein bears large sequence similarity
to CCM2. It contains a PH domain as opposed to a PTB domain, found in CCM2. Our lab showed that
PTB domains and PH domains can have overlapping abilities . Despite the similarities, the function of
[19]
Ccm2l and CCM2 are not identical. The N-terminus of CCM1, which contains the NPxY motifs, can bind
to Ccm2l, potentially through the PH domain. Although, it is still unknown which of the three NPxY
motifs are used by Ccm2l for binding. Ccm2l, which is selectively expressed in EC , sequesters CCM1
[92]
by acting as a competitive inhibitor, however, it is unable to bind to CCM3. The ability to bind to CCM1
results in the inhibition of CCM2 mediated junctional stability. During embryogenesis, Ccm1 is expressed
in the notochord while Ccm2 is expressed heavily in tissue anterior to the notochord. Interestingly, Ccm2l is
found in both regions. Despite these differences, Ccm2l knockout zebrafish exhibits cardiac phenotype, i.e.,