Nuclear diacylglycerol kinase-zeta is a negative regulator of cell cycle progression in C2C12 mouse myoblasts.
The nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting diacylglycerol (DG) into phosphatidic acid, terminate DG-dependent events. It has been demonstrated that nuclear DGK-zeta interferes with cell cycle progression. We previously reported that nuclear DGK-zeta expression increased during myogenic differentiation, whereas its down-regulation impaired differentiation. Here, we evaluated the possible involvement of nuclear DGK-zeta in cell cycle progression of C2C12 myoblasts. Overexpression of a wild-type DGK-zeta, which mainly localized to the nucleus (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked the cells in the G1 phase of the cell cycle, as demonstrated by in situ analysis of biotinylated-16-dUTP incorporated into newly synthesized DNA and by flow cytometry. In contrast, down-regulation of endogenous DGK-zeta by short interfering RNA (siRNA) increased the number of cells in both the S and G2/M phases of the cell cycle. Cell cycle arrest of cells overexpressing wild-type DGK-zeta was accompanied by decreased levels of retinoblastoma protein phosphorylated on Ser-807/811. Down-regulation of endogenous DGK-zeta, using siRNA, prevented the cell cycle block characterizing C2C12 cell myogenic differentiation. Overall, our results identify nuclear DGK-zeta as a key determinant of cell cycle progression and differentiation of C2C12 cells.
Evangelisti
Camilla
C
Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Cell Signalling Laboratory, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy.
Tazzari
Pier Luigi
PL
Riccio
Massimo
M
Fiume
Roberta
R
Hozumi
Yasukazu
Y
Falà
Federica
F
Goto
Kaoru
K
Manzoli
Lucia
L
Cocco
Lucio
L
Martelli
Alberto M
AM
D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D004261:DNA Replication; D019852:Diacylglycerol Kinase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D032446:Myoblasts / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D007527:Isoenzymes; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D019852:Diacylglycerol Kinase; C512480:diacylglycerol kinase zeta, mouse