Defining the Functional Domain of Programmed Cell Death 10 through Its Interactions with Phosphatidylinositol-3,4,5-Trisphosphate

Cerebral cavernous malformations (CCM) are vascular abnormalities of the central nervous system predisposing blood vessels to leakage, leading to hemorrhagic stroke. Three genes, Krit1 (CCM1), OSM (CCM2), and PDCD10 (CCM3) are involved in CCM development. PDCD10 binds specifically to PtdIns(3,4,5) P3 and OSM. Using threading analysis and multi-template modeling, we constructed a three-dimensional model of PDCD10. PDCD10 appears to be a six-helical-bundle protein formed by two heptad-repeat-hairpin structures (alpha 1-3 and alpha 4-6) sharing the closest 3D homology with the bacterial phosphate transporter, PhoU. We identified a stretch of five lysines forming an amphipathic helix, a potential PtdIns(3,4,5) P3 binding site, in the alpha 5 helix. We generated a recombinant wild-type (WT) and three PDCD10 mutants that have two (Delta 2KA), three (Delta 3KA), and five (Delta 5KA) K to A mutations. Delta 2KA and Delta 3KA mutants hypothetically lack binding residues to PtdIns(3,4,5) P3 at the beginning and the end of predicted helix, while Delta 5KA completely lacks all predicted binding residues. The WT, Delta 2KA, and Delta 3KA mutants maintain their binding to PtdIns(3,4,5) P3. Only the Delta 5KA abolishes binding to PtdIns(3,4,5) P3. Both Delta 5KA and WT show similar secondary and tertiary structures; however, Delta 5KA does not bind to OSM. When WT and Delta 5KA are co-expressed with membrane-bound constitutively-active PI3 kinase (p110-CAAX), the majority of the WT is co-localized with p110-CAAX at the plasma membrane where PtdIns(3,4,5) P3 is presumably abundant. In contrast, the Delta 5KA remains in the cytoplasm and is not present in the plasma membrane. Combining computational modeling and biological data, we propose that the CCM protein complex functions in the PI3K signaling pathway through the interaction between PDCD10 and PtdIns(3,4,5) P3.