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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 DGK, but not DGK␣ or DGK, interacts with pRB in vitro and in vivo. Binding of DGK to pRB is dependent on the phosphorylation status of pRB, since only hypophosphorylated pRB interacts with DGK. DGK also binds to the pRB-related pocket proteins p107 and p130 in vitro and in cells. Although DGK did not affect the ability of pRB to regulate E2F-mediated transcription, we found that pRB, p107, and p130 potently stimulate DGK activity in vitro. Finally, overexpression of DGK in pRB-null fibroblasts reconstitutes a cell cycle arrest induced by ␥-irradiation. These results suggest that DGK may act in vivo as a downstream effector of pRB to regulate nuclear levels of diacylglycerol and phosphatidic acid. Diacylglycerol (DAG)3 regulates many cellular processes, including proliferation, differentiation, and cell migration, by modulating the activity of several proteins, such as protein kinase C (PKC), Ras guanyl nucleotidereleasing proteins, chimaerins, and Munc 13 (1). DAG can be produced by the action of several different signal transduction pathways, including phospholipase C-mediated hydrolysis of phosphoinositides or phosphatidylcholine and phospholipase D-mediated hydrolysis of phosphatidylcholine followed by dephosphorylation of phosphatidic acid (PA), and during de novo synthesis of phospholipids (2).DAG is not only produced at the plasma membrane but at other intracellular sites as well, including the nucleus. Nuclear DAG levels are increased in liver as a consequence of two-thirds partial hepatectomy (3) and in cell cultures treated with insulin-like growth factor 1, which stimulates proliferation (4, 5). This suggests that nuclear DAG levels are intimately linked with cell cycle progression, but a causal relationship has not been firmly established. An attractive hypothesis is that nuclear DAG stimulates cell cycle progression via a DAG-binding protein such as PKC (1, 6). Indeed, DAG in the nucleus recruits and activates PKC in response to insulin-like growth factor 1 stimulation of Swiss 3T3 cells, which is required for G 1 to S phase transition (4, 7). However, the role of PKC in regulating the cell cycle is complex, with different PKC isoforms inducing a cell cycle arrest or stimulation of cell cycle progression. Furthermore, the same PKC isoform is able to induce both an arrest and progression through the cell cycle when expressed in different cell types (8).In the nucleus, DAG kinase (DGK) controls the levels of DAG generated from PI-phospholipase C-mediated hydrolysis of PI(4,5)P 2 (9), and nuclear DGK activity can be sti...

Inositide signaling at the plasma membrane has been implicated in the regulation of numerous cellular processes including cytoskeletal dynamics, vesicle trafficking, and gene transcription. Studies have also shown that a distinct inositide pathway exists in nuclei, where it may regulate nuclear processes such as mRNA export, cell cycle progression, gene transcription, and DNA repair. We previously demonstrated that nuclear PtdIns(4,5)P(2) synthesis is stimulated during progression from G1 through S phase, although mechanistic details of how cell cycle progression impinges on the regulation of nuclear inositides is unknown. In this study, we demonstrate that pRB, which regulates progression of cells from G1 through S phase interacts both in vitro and in vivo with Type I PIPkinases, the enzymes responsible for nuclear PtdIns(4,5)P(2) synthesis. Moreover, this interaction stimulates the activity of Type Ialpha PIPkinase in an in vitro assay. Using murine erythroleukamia (MEL) cells expressing a temperature-sensitive mutant of large T antigen (LTA), we demonstrate changes in vivo in nuclear PtdIns(4,5)P(2) levels that are consistent with the ability of LTA to disrupt pRB/Type I interactions. This study, for the first time, provides a potential mechanism for how cell cycle progression could regulate the levels of nuclear inositides.

We previously showed that the retinoblastoma protein (pRB), a key regulator of G1 to S-phase transition of the cell cycle, binds to and stimulates diacylglycerol kinase-zeta (DGKzeta) to phosphorylate the lipid second messenger diacylglycerol into phosphatidic acid. pRB binds to the MARCKS phosphorylation-site domain of DGKzeta that can be phosphorylated by protein kinase C (PKC). Here, we report that activation of PKC by phorbol ester inhibits DGKzeta binding to pRB. Ro 31-8220, a specific inhibitor of PKC, alleviated this inhibition of binding. Mimicking of PKC phosphorylation of serine residues (by S/D but not S/N mutations) within the DGKzeta-MARCKS phosphorylation-site domain also prevented DGKzeta binding to pRB, suggesting that PKC phosphorylation of these residues negatively regulates the interaction between DGKzeta and pRB. In PKC overexpression studies, it appeared that activation of particularly the (wild-type) PKCalpha isoform inhibits DGKzeta binding to pRB, whereas dominant-negative PKCalpha neutralized this inhibition. PKCalpha activation thus prevents DGKzeta regulation by pRB, which may have implications for nuclear diacylglycerol and phosphatidic acid levels during the cell cycle.

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