The Retinoblastoma Family Proteins Bind to and Activate Diacylglycerol Kinaseζ

Abstract

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...

Synergistic control of T cell development and tumor suppression by diacylglycerol kinase α and ζ

“…In addition to inhibiting RasGRP1 and Ras activation, DGK activity may also influence tumorigenesis through other mechanisms. For example, the retinoblastoma protein (pRB) binds to DGK and activates DGK to promote cell cycle arrest (43,44). In addition, DGK-derived PA could induce signaling events that suppress tumorigenesis.…”

Cytoplasmic localization of DGKζ exerts a protective effect against p53-mediated cytotoxicity

“…Both DGK activity and a functional NLS are necessary to block the cell cycle at G0/G1, suggesting that the cell cycle blockage results from DGKf-mediated metabolism of nuclear DG. In addition, DGKf interacts with the retinoblastoma protein (pRb) depending on its phosphorylation status (Los et al, 2006), and overexpression of DGKf is accompanied by decreased levels of pRb phosphorylated on Ser807/811 . Furthermore, in C2C12 mouse myoblasts, nuclear DGKf downregulates the expression of cyclin D through upregulation of TIS21/BTG2/PC3, a transcriptional regulator of cyclin D1 with a strong anti-proliferative function (Evangelisti et al, 2009).…”

Lipid Messenger, Diacylglycerol, and its Regulator, Diacylglycerol Kinase, in Cells, Organs, and Animals: History and Perspective

“…In addition, a nuclear PI-PLC activity is activated during the G2 phase and PI-PLC inhibitors lead to a decrease in nuclear PI-PLC activity and a cell cycle blockade at the G2 phase (Sun et al 1997). In this regard, DGKζ may bind to the retinoblastoma protein (pRB), a tumor suppressor and key regulator of the cell cycle, whose activity depends on the phosphorylation status of pRB (Los et al 2006). Furthermore, overexpression of a wild-type DGK ζ blocks the cell cycle at the G1 phase (Topham et al 1998) and decreases levels of pRB phosphorylation on Ser-807/811 (Evangelisti et al 2007b).…”