Identification of Cyclin A2 as the Downstream Effector of the Nuclear Phosphatidylinositol 4,5-Bisphosphate Signaling Network

Ka-Kei Ho,Erika Rosivatz,David J Mann,Alexandra A Anderson,Rüdiger Woscholski,Eric W.-F Lam

doi:10.1074/jbc.m706623200

Abstract

In addition to the well characterized phosphoinositide second messengers derived from the plasma membrane, increasing evidence supports the existence of a nuclear phosphoinositide signaling network. The aim of this investigation was to dissect the role played by nuclear phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in cell cycle progression and to determine the cell cycle regulatory component(s) that are involved. A number of cytosolic/nuclear PtdIns(4,5)P2-deficient Swiss 3T3 cell lines were established, and their G 0/G 1/S cell cycle phase transitions induced by defined mitogens were examined. Our results demonstrate that nuclear PtdIns(4,5)P2 down-regulation caused a delay in phorbol ester-induced S phase entry and that this was at least in part channeled through cyclin A2 at the transcriptional level. In summary, these data identify cyclin A2 as a downstream effector of the nuclear PtdIns(4,5)P2 signaling network and highlight the importance of nuclear PtdIns(4,5)P2 in the regulation of mammalian mitogenesis.

Highlights

Activation of diverse signal transduction pathways can cause quiescent cells to re-enter the cell cycle
Our results demonstrate that nuclear PtdIns(4,5)P2 down-regulation caused a delay in phorbol ester-induced S phase entry and that this was at least in part channeled through cyclin A2 at the transcriptional level
Our results show that in cytosolic PtdIns(4,5)P2-deficient cells, the delay in bombesin-induced S phase entry was channeled through cyclin D1, whereas in nuclear PtdIns(4,5)P2-deficient cells, the delay in phorbol ester-induced S phase entry was channeled through cyclin A2

Summary

EXPERIMENTAL PROCEDURES

Antibodies, and Cells—The antibodies used for immunoblotting were as follows: anti-cyclin D1 (72-13G), antip (C-19G), anti-Cdk (C-163), anti-Cdk (C-22), anti-Cdk (C-21), anti-p70S6K (C-18), and anti-Erk-1 (C-16) (Santa Cruz Biotechnology, Inc.); anti-␣-tubulin (TAT-1), anti-cyclin A (E23.1), and anti-cyclin D3 (DCS28) (Cancer Research UK); anti-phospho-Akt (Ser-473; 587F11) (Cell Signaling Biotechnology); and anti-pRb (G3-245) (Pharmingen). Dishes of confluent and quiescent cells were incubated in Dulbecco’s modified Eagle’s medium containing various supplements at the following concentrations: NCS (10%), insulin 100 ng/ml GST-PLC␦1 PH domain in 3% fatty acid-free BSA/PBS was applied to the cells and incubated for 30 min. (For the competition experiment, the GSTPLC␦1 PH domain/BSA/PBS solution was preincubated with 100 ng of Ins(1,4,5)P3 (Sigma) for 30 min.) After the incubation, coverslips were washed (3 ϫ 10 min) with PBS with gentle rocking and incubated for 60 min with anti-GST antibody (Novagen) in 3% fatty acid-free BSA/PBS (1:10,000 dilution), followed by PBS washes as before. Cells were incubated with TRITC-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) in 3% fatty acid-free BSA/PBS (1:100 dilution) for 60 min. All immunofluorescent images shown were acquired with the same exposure settings

RESULTS

RNA was extracted and analyzed

DISCUSSION