Subnuclear localization and differentiation-dependent increased expression of DGK-zeta in C2C12 mouse myoblasts.

Diacylglycerol kinases (DGKs) catalyze phosphorylation of diacylglycerol (DG) to yield phosphatidic acid (PA). Previous evidence has shown that the nucleus contains several DGK isoforms. In this study, we have analyzed the expression and subnuclear localization of DGK-ζ employing C2C12 mouse myoblasts. Immunocytochemistry coupled to confocal laser scanning microscopy showed that both endogenous and green fluorescent protein-tagged overexpressed DGK-ζ localized mostly to the nucleus. In contrast, overexpressed DGK-α, -β, -δ, and-ι did not migrate to the nucleus. DGK-ζ was present in the nuclear speckle domains, as also revealed by immuno-electron microscopy analysis. Moreover, DGK-ζ co-localized and interacted with phosphoinositide-specific phospholipase Cβ1 (PLCβ1), that is involved in inositide-dependent signaling pathways important for the regulation of cell proliferation and differentiation. Furthermore, we report that DGK-ζ associated with nuclear matrix, the fundamental organizing principle of the nucleus where many cell functions take place, including DNA replication, gene expression, and protein phosphorylation. Nuclear DGK-ζ increased during myogenic differentiation of C2C12 cells, while DGK-ζ down-regulation by siRNA markedly impaired differentiation. Overall, our findings further support the importance of speckles and nuclear matrix in lipid-dependent signaling and suggest that nuclear DGK-ζ might play some fundamental role during myogenic differentiation of C2C12 cells