Phospholipase C delta 1 regulates cell proliferation and cell-cycle progression from G1- to S-phase by control of cyclin E-CDK2 activity.
In the present study, we examined the role of PLC delta 1 (phospholipase C delta 1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLC delta 1, but not PLC beta 3 or PLC epsilon, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [(3)H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G(0)/G(1)-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G(1)-phase but delayed entry into S-phase. Consistent with these findings, G(1) cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G(1)-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E-CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLC delta 1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLC delta 1 in cell-cycle progression from G(1)-to-S-phase through regulation of cyclin E-CDK2 activity and p27 levels.
Kaproth-Joslin
Katherine A
KA
Department of Medicine, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
Li
Xiangquan
X
Reks
Sarah E
SE
Kelley
Grant G
GG
D000818:Animals; D049109:Cell Proliferation; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016193:G1 Phase; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D054803:Phospholipase C delta / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D034622:RNA Interference; D051381:Rats; D016196:S Phase
D019927:Cyclin E; D051357:Cyclin-Dependent Kinase 2; D054803:Phospholipase C delta