Ceramide Inactivates Cellular Protein Kinase Cα

Abstract

Ceramide mediates the effects of extracellular agents on cellular growth, differentiation and apoptosis. In this study, we explored the mechanisms by which ceramide induces its cellular effects. In Molt-4 cells, phorbol 12-myristate 13-acetate (PMA) induced retinoblastoma gene product (Rb) phosphorylation, and ceramide inhibited this effect, suggesting an inhibitory effect of ceramide on the protein kinase C (PKC) pathway, the primary target of PMA. Molt-4 cells contained primarily PKCalpha and betaII isoforms of PKC. To determine the effects of ceramide on PKC, we developed an immunoprecipitation assay for PKCalpha activity. Exposure of Molt-4 cells to C6-ceramide resulted in a concentration and time-dependent inhibition of immunoprecipitated protein kinase Calpha (PKCalpha). Initial inhibition was observed as early as 4.5 h after treatment of cells with C6-ceramide, and the activity was completely lost by 13 h. Inhibition of PKCalpha activity was seen at concentrations of ceramide as low as 5 microM with maximal effects occurring at a concentration of 15 microM. Both C2 and C6-ceramide were inhibitory, but C2 and C6 dihydroceramides were not. Ceramide did not directly inhibit PKCalpha in vitro or modulate the levels of PKCalpha protein, suggesting that ceramide acted indirectly. Moreover, ceramide did not inhibit PMA-induced translocation of PKCalpha. Taken together, these results suggested that ceramide caused inactivation of PKCalpha. Since PKC requires phosphorylation for activity, we determined the effects of ceramide on phosphorylation of PKCalpha. C6-ceramide inhibited basal and PMA-induced phosphorylation of PKCalpha. In addition, okadaic acid, a potent phosphatase inhibitor, slightly stimulated PKC activity and blocked the effects of ceramide on PKCalpha inhibition. These results demonstrate that ceramide causes inhibition/inactivation of PKCalpha and suggest these effects of ceramide may be mediated by a protein phosphatase.

Ceramide-Induced TCR Up-Regulation

“…Furthermore, these results also concur with the observation that inhibition of PP2A leads to down-regulation of TCR expression (9). It has been reported that ceramide inactivates cellular PKC␣, and it could be speculated that the observed TCR up-regulation could be caused by an inhibition of PKC activity (31). However, inhibition of PKC␣ was first observed following 4.5-h incubation with ceramide and could thus not explain the rapid TCR up-regulation observed in the present study.…”

Loss of protein kinase C function induces an apoptotic response

“…It is of particular interest that the apoptosis inducer ceramide can activate protein phosphatase 2A (Dobrowsky et al, 1993) and has been reported to induce the dephosphorylation and inactivation of PKCa (Lee et al, 1996). While it remains to be established in this context whether inactivation of PKCa is a necessary component of the ceramide induced apoptotic response, the data here suggest that this inactivation may indeed be critical.…”

Opposing effects of butyrate and bile acids on apoptosis of human colon adenoma cells: differential activation of PKC and MAP kinases

“…Interestingly, the literature suggests that PKC-a has a predominantly antiapoptotic role (Deacon et al, 1997). For example, PKC-a is inhibited during ceramide-induced apoptosis (Lee et al, 1996) and expression of a dominant-negative PKC-a induced apoptosis in CHO cells (Whelan and Parker, 1998). Thus, those bile acids known to promote cell proliferation and inhibit butyrate-induced apoptosis, that is, UDCA, CDCA and DCA (Mahmoud et al, 1999;McMillan et al, 2000), are likely to be mediated by the activation of PKC-a.…”

“…With respect to apoptosis, PKC-a and -bll appear to be antiapoptotic in most cells. PKC-a is inactivated by proapoptotic factors including ceramide (Lee et al, 1996) and is known to phosphorylate bcl-2, potentiating its antiapoptotic function in mitochondria (Ruvolo et al, 1998). PKC-bll is a mitotic lamin kinase (Goss et al, 1994) involved in the regulation of proliferation.…”