Cerebral cavernous malformations (CCMs) are vascular malformations that affect the central nervous system and result in cerebral hemorrhage, seizure and stroke. CCM arises from loss-of-function mutations in one of three genes: CCM1, CCM2 and CCM3 (PDCD10). CCM3 mutations in human often result in a more severe form of the disease, and CCM3 knockout mice show severe phenotypes with yet-to-be defined mechanisms. We have recently reported that CCM3 regulates UNC13 family-mediated exocytosis. Here we investigate endothelial cells (EC) exocytosis in CCM disease progression. We find that CCM3 suppresses UNC13B/VAMP3-dependent exocytosis of angiopoietin-2 (ANGPT2) in brain endothelial cells. CCM3 ablation in EC augments exocytosis and secretion of ANGPT2, correlating with destabilized EC junctions, enlarged lumen formation, and endothelial cell-pericyte dissociations. UNC13B deficiency that blunts ANGPT2 secretion from EC or an ANGPT2 neutralization antibody normalizes the defects caused by CCM3 deficiency. More importantly, ANGPT2 neutralization antibody treatment or UNC13B deficiency blunts the CCM lesion phenotypes, including disruption of EC junctions, vessel dilation and pericyte dissociation, in the brains and retinas caused by endothelial cell-specific CCM3 inactivation. Our study reveals that enhanced secretion of ANGPT2 in endothelial cells contributes to the progression of the CCM disease, providing a novel therapeutic approach to treat this devastating pathology.

Type I phosphatidylinositol phosphate kinase (PIP5K1) phosphorylates the head group of phosphatidylinositol 4-phosphate (PtdIns4P) to generate PtdIns4,5P2, which plays important roles in a wide range of cellular functions including Wnt signaling. However, the lack of its structural information has hindered the understanding of its regulation. Here we report the crystal structure of the catalytic domain of zebrafish PIP5K1A at 3.3 Å resolution. This molecule forms a side-to-side dimer. Mutagenesis study of PIP5K1A reveals two adjacent interfaces for the dimerization and interaction with the DIX domain of the Wnt signaling molecule dishevelled (DVL). Although these interfaces are located distally to the catalytic/substrate binding site, binding to these interfaces either through dimerization or the interaction with DIX stimulates PIP5K1 catalytic activity. DIX binding additionally enhances PIP5K1 substrate binding. Thus, this study elucidates regulatory mechanisms for this lipid kinase and provides a paradigm for the understanding of PIP5K1 regulation by their interacting molecules.

SUMMARY Polarized vesicle transport plays an important role in cell polarization, but the mechanisms underlying this process and its role in innate immune responses are not well understood. Here, we describe a phosphorylation-regulated polarization mechanism that is important for neutrophil adhesion to endothelial cells during inflammatory responses. We show that the protein kinase PKN1 phosphorylates RPH3A, which enhances binding of RPH3A to GTP-bound RAB21. These interactions are important for polarized localization of RAB21 and RPH3A in neutrophils, which leads to PIP5K1C90 polarization. Consistent with the roles of PIP5K1C90 polarization, the lack of PKN1 or RPH3A impairs neutrophil integrin activation, adhesion to endothelial cells, and infiltration in inflammatory models. Furthermore, myeloid-specific loss of PKN1 decreases tissue injury in a renal ischemia-reperfusion model. Thus, this study characterizes a mechanism for protein polarization in neutrophils and identifies a potential protein kinase target for therapeutic intervention in reperfusion-related tissue injury.

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Highlights d Cell attachment is required for chemical-induced cytoskeleton polarization d Cell attachment induces local PM curvature increase to recruit and polarize SRGAP2 d Polarized SRGAP2 induces sequential polarization of PtdIns4P, PIP5K1C90, and pMLC d This SRGAP2 polarization signaling axis regulates neutrophil firm adhesion