The retinoblastoma family proteins bind to and activate diacylglycerol kinase zeta.
The retinoblastoma protein (pRB) is a tumor suppressor and key regulator of the cell cycle. We have previously shown that pRB interacts with phosphatidylinositol-4-phosphate 5-kinases, lipid kinases that can regulate phosphatidylinositol 4,5-bisphosphate levels in the nucleus. Here, we investigated pRB binding to another lipid kinase in the phosphoinositide cycle, diacylglycerol kinase (DGK) that phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. We found that DGKzeta, but not DGKalpha or DGK, interacts with pRB in vitro and in vivo. Binding of DGKzeta to pRB is dependent on the phosphorylation status of pRB, since only hypophosphorylated pRB interacts with DGKzeta. DGKzeta also binds to the pRB-related pocket proteins p107 and p130 in vitro and in cells. Although DGKzeta did not affect the ability of pRB to regulate E2F-mediated transcription, we found that pRB, p107, and p130 potently stimulate DGKzeta activity in vitro. Finally, overexpression of DGKzeta in pRB-null fibroblasts reconstitutes a cell cycle arrest induced by gamma-irradiation. These results suggest that DGKzeta may act in vivo as a downstream effector of pRB to regulate nuclear levels of diacylglycerol and phosphatidic acid.
Los
Alrik P
AP
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
Vinke
Fabian P
FP
de Widt
John
J
Topham
Matthew K
MK
van Blitterswijk
Wim J
WJ
Divecha
Nullin
N
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D004249:DNA Damage; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000378:metabolism; D050684:E2F Transcription Factors / Q000737:chemistry; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D005720:Gamma Rays; D015972:Gene Expression Regulation, Neoplastic; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000378:metabolism; D050720:Retinoblastoma-Like Protein p107 / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130 / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D050684:E2F Transcription Factors; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D008156:Luciferases; D005982:Glutathione Transferase; D019852:Diacylglycerol Kinase