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Page 2 of 10 Antwi-Adjei et al. Vessel Plus 2021;5:35 https://dx.doi.org/10.20517/2574-1209.2021.48

Conclusion: CCM3 and GCKIII act upstream of Furry-Tricornered, suggesting the conservation from yeast of a
Hippo-like signaling pathway that regulates morphogenesis. We speculate that some combination of
Furry/Furrylike and STK38/38L are therefore likely to act downstream of CCM3 in endothelial cells.

Keywords: CCM3, GCKIII, NDR, STK38/38L, tube morphogenesis, planar cell polarity

INTRODUCTION
Cerebral cavernous malformations (CCMs) are common vascular defects found in the capillaries of the
[1]
central nervous system (reviewed in Lampugnani et al. ). Familial CCM shows an autosomal dominant
manner of inheritance, with variable expression and incomplete penetrance (reviewed in Riolo et al. ).
[2]
Somatic loss of a second (Knudsonian 2 hit mechanism) CCM allele is believed to result in localized
[3]
vascular lesions in which grossly dilated, thin-walled capillaries lacking surrounding support cells undergo
repeated hemorrhage leading to headaches, neurological deficits and stroke . Lesions are believed to be
[4]
largely clonally derived, containing few wild type endothelial cells (although also see ). Mutations in three
[5]
[6]
different genes cause disease and account for at least 80% of cases of familial CCM. These genes - Krev
interaction trapped protein 1, Malcavernin, and Programmed Cell Death 10 - are also known as CCM1,
CCM2 and CCM3, respectively. Generally, the CCM proteins have been thought to act as scaffolding for
regulators of cytoskeletal remodeling and/or signaling pathways. The CCM proteins can physically interact,
forming a ternary complex bridged by CCM2. The complex may be recruited to the plasma membrane
through interaction with a putative transmembrane receptor protein, Heart of Glass , or by interaction
[7]
with the Vascular endothelial growth factor receptor 2 . The precise functions of the individual proteins,
[8,9]
and of the various complexes, remain key subjects of inquiry. The role of CCM3 has been particularly
controversial, as CCM3 is also found in other complexes and has been suggested to operate in a CCM1-
[10]
and CCM2-independent manner [11-15] .

Consistent with the idea that CCM3 may act independently of CCM1 and CCM2, mutations in CCM3 cause
an earlier lethality in mice, and in humans, patients with CCM3 mutations show a much earlier age of onset
and a more severe course of disease [12,16-18] . Likewise, patients with mutations in CCM3, but not CCM1 or
CCM2, develop multiple meningiomas at a high frequency . These differences may reflect a requirement
[17]
[19]
for CCM3 in epithelia , or may hint at a distinct molecular function for CCM3 even in endothelial cells.
We have turned to the simple Drosophila melanogaster model system to dissect CCM3 function.
Importantly, CCM3 is well conserved, but neither CCM1 nor CCM2 orthologs have been reported in flies.
In prior work, we identified a requirement for CCM3 and GCKIII in tracheal terminal cell tube
morphogenesis , and have since gone on to show that GCKIII directly activates the Nuclear Dbf2-related
[20]
(NDR) kinase, Tricornered (NDR1, 2 in vertebrates, also known as STK38 and STK38L) by
phosphorylation . Altogether, our work shows that CCM3 is a novel component of an ancient kinase
[21]
cascade conserved from yeast: the RAM (Regulation of Ace2 and Morphogenesis) pathway in budding
yeast, and the MOR (Morphogenesis Orb6 network) pathway in Schizosacharomyces pombe .
[22]

In yeast and flies, NDR kinase signaling typically requires co-factors including Mob (mps one binder) and
Furry family members . Four Mob family members have been identified (Mats, Mob2, Mob3 and Mob4)
[23]
in flies, and single Furry protein (Tao3p in budding yeast and Mor2p in fission yeast). So far, work on the
invertebrate fry genes indicates essential roles in Trc signaling, with known requirements in the generation
of actin-based cellular protrusions (wing hairs and bristles [24-30] ), in the control of dendritric branching and
tiling in dendritic arborization neurons [31-33] , and in follicle cell polarity . The vertebrate orthologs of Fry
[34]