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 (alpha1-3 and alpha4-6) 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 alpha5 helix. We generated a recombinant wild-type (WT) and three PDCD10 mutants that have two (Delta2KA), three (Delta3KA), and five (Delta5KA) K to A mutations. Delta2KA and Delta3KA mutants hypothetically lack binding residues to PtdIns(3,4,5)P3 at the beginning and the end of predicted helix, while Delta5KA completely lacks all predicted binding residues. The WT, Delta2KA, and Delta3KA mutants maintain their binding to PtdIns(3,4,5)P3. Only the Delta5KA abolishes binding to PtdIns(3,4,5)P3. Both Delta5KA and WT show similar secondary and tertiary structures; however, Delta5KA does not bind to OSM. When WT and Delta5KA 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 Delta5KA 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.
Dibble
Christopher F
CF
Department of Pharmacology, School of Medicine, and the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America.
Horst
Jeremy A
JA
Malone
Michael H
MH
Park
Kun
K
Temple
Brenda
B
Cheeseman
Holly
H
Barbaro
Justin R
JR
Johnson
Gary L
GL
Bencharit
Sompop
S
D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D053683:Oncostatin M / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D051017:Apoptosis Regulatory Proteins; D008565:Membrane Proteins; C495390:PDCD10 protein, human; D018129:Phosphatidylinositol Phosphates; D011518:Proto-Oncogene Proteins; D053683:Oncostatin M