Neuregulin-dependent protein synthesis in C2C12 myotubes and rat diaphragm muscle

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [³H]phenylalanine into the protein pool) were investigated in differentiated C₂C₁₂ myotubes and rat diaphragm muscle (DIAm). In C₂C₁₂ myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased 30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [³H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K. Akt; ErbB; heregulin; protein biosynthesis; skeletal muscle     

Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells

Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.     

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.     

Alcohol regulates eukaryotic elongation factor 2 phosphorylation via an AMP-activated protein kinase-dependent mechanism in C2C12 skeletal myocytes

Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.     

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts

Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43 °C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43 °C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P < 0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.     

Protein synthesis during oxygen conformance and severe hypoxia in the mouse muscle cell line C2C12

Oxygen conformance can be described as the ability to reduce energy demand, and hence oxygen consumption, in response to a decline in oxygen availability without a decrease in the concentration of ATP. It has been proposed that oxygen conformance may enhance cellular survival at low oxygen concentrations. We demonstrate that non-contracting C2C12 cells, a mouse skeletal muscle cell line, are capable of oxygen conformance. Typically, we found oxygen consumption to decline by 30-40% as the concentration of oxygen was reduced from 100 microM to 10 microM. Unexpectedly, the rate of protein synthesis, a major energy consumer in the cell, did not decrease significantly during oxygen conformance. Unlike oxygen conformance, severe hypoxia (<0.5 microM) caused a 36% decline in the concentration of PCr, and under these conditions of energy stress, the rate of protein synthesis declined by 43%. We conclude that there are two distinct metabolic responses to declines in oxygen concentration in non-contracting C2C12 cells.     

Endotoxin transiently inhibits protein synthesis through Akt and MAPK mediating pathways in C2C12 myotubes

In this study, the effect of lipopolysaccharide (LPS) on protein synthesis (PS) and intracellular signaling factors that regulate it have been investigated in C2C12 murine-derived myotubes. In particular, the role of Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinases (MAPKs) [p38 and extracelluar regulated protein kinase (ERK1/2)] have been examined. The direct effect of LPS on PS was measured at 3 and 18 h. LPS significantly decreased PS at 3 h but not at the 18-h time point. This effect was preceded by decreased Akt phosphorylation at 5 and 30 min after LPS administration. The mTOR phosphorylation exhibited a long time dose-dependent increase at all the time points. Similarly, the activity-related phosphorylation of p38 and ERK1/2 significantly increased in a time- and dose-dependent manner at all the time points. Polymyxin B abolished the LPS-induced decrease in PS rate. The phosphatidylinositol 3-kinase inhibitor LY-0294002 in combination with LPS significantly decreased the rate of PS by 81% and alone by 66%, respectively, for the 3- and 18-h time points, whereas p38 and ERK inhibitors in combination with LPS significantly decreased the rate PS rate at the 18-h time point by 41% and 59%, respectively, compared with control cells. In conclusion, LPS alone transiently decreased the rate of PS by 50% at 3 h; this effect is most likely mediated via the Toll-like receptor 4 (TLR4)-Akt/mTOR pathway, and both p38 and ERK when inhibited in the presence of LPS at 3 h have a similar effect in preventing the LPS-induced reduction in PS.     

Mechanism of mitochondrial stress-induced resistance to apoptosis in mitochondrial DNA-depleted C2C12 myocytes

In this study, we show that partial mitochondrial DNA (mtDNA) depletion (mitochondrial stress) induces resistance to staurosporine (STP)-mediated apoptosis in C2C12 myoblasts. MtDNA-depleted cells show a 3-4-fold increased proapoptotic proteins (Bax, BAD and Bid), markedly increased antiapoptotic Bcl-2, and reduced processing of p21 Bid to active tBid. The protein levels and also the ability to undergo STP-mediated apoptosis were restored in reverted cells containing near-normal mtDNA levels and restored mitochondrial transmembrane potential. Inhibition of apoptosis closely correlated with sequestration of Bax, Bid and BAD in the mitochondrial inner membrane, increased Bcl-2 and Bcl-X(L), and inability to process p21 Bid. These factors, together with the reduced activation of caspases 3, 9 and 8 are possible causes of mitochondrial stress-induced resistance to apoptosis. Our results suggest that a highly proliferative and invasive behavior of mtDNA-depleted C2C12 cells is related to their resistance to apoptosis.     

Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways

Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T^202/Y²⁰⁴P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y⁷⁰⁵P-STAT3) and MEK (T^202/Y²⁰⁴P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S⁴⁷³) and GSK-3β (S⁹) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S⁹P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y²¹⁶P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost.     

Activation of MAPKs in thrombin-stimulated ventricular myocytes is dependent on Ca2+-independent PLA2

Thrombin stimulation of isolated rabbit ventricular myocytes activates a membrane-associated, Ca²⁺-independent PLA₂ (iPLA₂) that selectively hydrolyzes plasmalogen phospholipids and results in increased production of arachidonic acid and lysoplasmenylcholine. To determine whether MAPK regulates myocardial iPLA₂ activity, we isolated ventricular myocytes from rabbit heart by collagenase digestion and pretreated them with MAPK inhibitors before stimulating them with thrombin. Pretreatment with PD-98059 to inhibit p42/44 MAPK or SB-203580 to inhibit p38 MAPK had no significant effect on thrombin-stimulated, membrane-associated iPLA₂ activity. Thrombin stimulation resulted in significant increases in both p42/44 and p38 MAPK activity after 2 min. Pretreatment with the iPLA₂-selective inhibitor bromoenol lactone completely inhibited thrombin-stimulated MAPK activity, suggesting that activation of MAPKs was dependent on iPLA₂ activation. Ventricular myocyte MAPK activity was increased by incubation of the myocytes with lysoplasmenylcholine, a metabolite produced by iPLA₂-catalyzed membrane plasmalogen phospholipid hydrolysis. Altogether, these data suggest that activation of MAPKs occurs downstream of and is dependent on iPLA₂ activation in thrombin-stimulated rabbit ventricular myocytes. lysoplasmenylcholine; cell signaling; protease-activated receptors     

Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR

The role of the AMP-activated kinase (AMPK) as a metabolic sensor in skeletal muscle has been far better characterized for glucose and fat metabolism than for protein metabolism. Therefore, the studies presented here were designed to examine the effects of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR)-induced AMPK signaling on effector mechanisms of mRNA translation and protein synthesis in cultures of C(2)C(12) myotubes. The findings show that, following AICAR (2 mM) treatment, AMPK phosphorylation was increased within 15 min and remained elevated throughout a 60-min time course. In association with the increase in AMPK phosphorylation, global rates of protein synthesis declined to 90, 70, and 63% of the control values at the 15-, 30-, and 60-min time points, respectively. By 60 min, polysomes disaggregated into free ribosomal subunits, suggesting an inhibition of initiation of mRNA translation. However, phosphorylation of eukaryotic elongation factor 2 was increased at 15 and 30 min but then declined to control values by 60 min, suggesting a transient inhibition of translation elongation. The decline in protein synthesis and changes in mRNA translation were associated with a repression of the mammalian target of rapamycin (mTOR) signaling pathway, as indicated by increased association of Hamartin with Tuberin, increased association of regulatory associated protein of mTOR with mTOR, and dephosphorylation of the downstream targets ribosomal protein S6 kinase-1 and eukaryotic initiation factor 4E-binding protein-1. They were also associated with activation of the MAPK signaling pathway, as indicated by increased phosphorylation of MEK1/2 and ERK1/2 and the downstream target eIF4E. Overall, the data support the conclusion that AICAR-induced AMPK activation suppresses protein synthesis through concurrent repression of mTOR signaling and activation of MAPK signaling, the combination of which modulates transient changes in the initiation and elongation phases of mRNA translation.     

C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression

Insulin regulates the uptake of glucose into skeletal muscle and adipocytes by redistributing the tissue-specific glucose transporter GLUT4 from intracellular vesicles to the cell surface. To date, GLUT4 is the only protein involved in insulin-regulated vesicular traffic that has this tissue distribution, thus raising the possibility that its expression alone may allow formation of an insulin-responsive vesicular compartment. We show here that treatment of differentiating C2C12 myoblasts with dexamethasone, acting via the glucocorticoid receptor, causes a >or=10-fold increase in GLUT4 expression but results in no significant change in insulin-stimulated glucose transport. Signaling from the insulin receptor to its target, Akt2, and expression of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor, or SNARE, proteins syntaxin 4 and vesicle-associated membrane protein are normal in dexamethasone-treated C2C12 cells. However, these cells show no insulin-dependent trafficking of the insulin-responsive aminopeptidase or the transferrin receptor, respective markers for intracellular GLUT4-rich compartments and endosomes that are insulin responsive in mature muscle and adipose cells. Therefore, these data support the hypothesis that GLUT4 expression by itself is insufficient to establish an insulin-sensitive vesicular compartment.     

Lopinavir impairs protein synthesis and induces eEF2 phosphorylation via the activation of AMP-activated protein kinase

HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K.     

Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts

Summary Our previous studies have demonstrated that expression of growth-associated genes is regulated by the adhesive state of the cell. To understand the role of cell adhesion in regulating the switch from growth to differentiation, we are studying the differentiation of mouse myoblasts into multinucleated contractile myotubes. In this report, we describe a novel means of culturing C₂C₁₂ myoblasts that permits an analysis of the role of cell adhesion in regulating the sequential induction of muscle-specific genes that control myogenesis. Suspension of an asynchronous, proliferating population of myoblasts in a viscous gel of methylcellulose dissolved in medium containing 20% serum induces growth arrest in G₀ phase of the cell cycle without a concomitant induction of muscle-specific genes. Reattachment to a solid substratum in 20% serum, 0.5nM bFGF, or 10 nM IGF-1 rapidly activates entry of the quiescent cells into G₁ followed by a synchronous progression of the cell population through into S phase. bFGF or IGF-1 added separately facilitate only one passage through the cell cycle, whereas 20% serum or the two growth factors added together support multiple cell divisions. Adhesion of suspended cells in DMEM alone or with 3 nM IGF-1 induces myogenesis as evidenced by the synthesis of myogenin and myosin heavy chain (MHC) proteins followed by fusion into myotubes. bFGF completely inhibits this differentiation process even in the presence of myogenic doses of IGF-1. Addition of 3 nM IGF-1 to quiescent myoblasts maintained in suspension culture in serum-free conditions does not induce myogenin or MHC expression. Thus, adhesion is a requirement for the induction of muscle gene expression in mouse myoblasts. The development of a muscle cell culture environment in which proliferating myoblasts can be growth arrested in G₀ without activating muscle-specific gene expression provides a means of analyzing the synchronous activation of either the myogenic or growth programs and how adhesion affects each process, respectively. Supported by training grant T32-HL07035     

PUGNAc induces protein ubiquitination in C2C12 myotube cells

O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates many cellular processes including the cell cycle, cell signaling, and protein trafficking. Dysregulation of O‐GlcNAcylation may be involved in the development of insulin resistance and type 2 diabetes. Therefore, it is necessary to identify cellular proteins that are induced by elevated O‐GlcNAcylation. Here, using adenosine 5′‐triphosphate affinity chromatography, we employed a proteomic approach in order to identify differentially expressed proteins in response to treatment with the O‐GlcNAcase inhibitor, O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), in mouse C2C12 myotube cells. Among 205 selected genes, we identified 68 nucleotide‐binding proteins, 14 proteins that have adenosinetriphosphatase activity, and 10 proteins with ligase activity. Upregulation of proteins, including ubiquitin‐activating enzyme E1, proteasome subunit 20S, cullin‐associated NEDD8‐dissociated protein 1, ezrin, and downregulation of the protein nucleoside diphosphate kinase B, were confirmed by western blot analysis. In particular, we found that the protein ubiquitination level in C2C12 cells was increased by PUGNAc treatment. This is the first report of quantitative proteomic profiles of myotube cells after treatment with PUGNAc, and our results demonstrate the potential to enhance understanding of the relationship between insulin resistance, O‐GlcNAc, and PUGNAc in the future. Copyright © 2015 John Wiley & Sons, Ltd.     

The subcellular localization and protein stability of mouse alpha-actinin 2 is controlled by its nuclear receptor binding motif in C2C12 cells

Alpha actinin (ACTN) has emerged as a multitasking protein, whose roles range from bundling actin filaments to functioning as a versatile protein interaction platform for proteins involved in structural or signaling aspects. We report here that ACTN2, one of the four ACTN isoforms, may shuttle between the cytoplasm and nucleus where the nuclear exportation takes place in a CRM1-dependent manner. The majority of ACTN2 was found to localize in the cytoplasm and exhibit a lower stability which was demonstrated using either mutants carrying mutated nuclear receptor binding motif or inhibitors against the ubiquitin- and calpain-dependent degradation pathways. Horse serum induced differentiation of C2C12 cells also caused the redistribution of nuclear ACTN2 to the cytoplasm, which subcellular compartment the ACTN2 behaves as an unstable protein. Our data indicated that the model in which ACTN2 functions as a multi-talented coregulator may be controlled by the differential protein stability modulated via nucleo-cytoplasmic trafficking in C2C12 cells.     

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-zeta employing C2C12 mouse myoblasts. Immunocytochemistry coupled to confocal laser scanning microscopy showed that both endogenous and green fluorescent protein-tagged overexpressed DGK-zeta localized mostly to the nucleus. In contrast, overexpressed DGK-alpha, -beta, -delta, and -iota did not migrate to the nucleus. DGK-zeta was present in the nuclear speckle domains, as also revealed by immuno-electron microscopy analysis. Moreover, DGK-zeta co-localized and interacted with phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1), that is involved in inositide-dependent signaling pathways important for the regulation of cell proliferation and differentiation. Furthermore, we report that DGK-zeta 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-zeta increased during myogenic differentiation of C2C12 cells, while DGK-zeta 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-zeta might play some fundamental role during myogenic differentiation of C2C12 cells.