Identification of nuclear phosphatidylinositol 4,5-bisphosphate-interacting proteins by neomycin extraction.
Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X(n= 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.
PROBE Proteomics Platform, Department of Biomedicine, University of Bergen, Bergen, Norway.
Aurélia E
AE
Lewis
Lilly
L
Sommer
Magnus Ø
MØ
Arntzen
Yvan
Y
Strahm
Nicholas A
NA
Morrice
Nullin
N
Divecha
Clive S
CS
D'Santos
D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D009355:Neomycin / Q000494:pharmacology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D010716:Phosphatidylinositols / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D005982:Glutathione Transferase; D004264:DNA Topoisomerases, Type I; D009355:Neomycin