Stress-induced ER to Golgi translocation of ceramide synthase 1 is dependent on proteasomal processing.

The ceramide synthase (CerS) enzymes are key regulators of ceramide homeostasis. CerS1 is central to regulating C18 ceramide which has been shown to be important in cancer and the response to chemotherapeutic drugs. Previous work indicated that some drugs induced a novel and specific translocation of CerS1 from the endoplasmic reticulum to the Golgi apparatus. We now show that diverse stresses such as UV light, DTT, as well as drugs with different mechanisms of action induce CerS1 translocation. The stresses cause a specific cleavage of the CerS1 enzyme, and the cleavage is dependent on the action of the proteasome. Inhibition of proteasome function inhibits stress-induced CerS1 translocation, indicating that this proteolytic cleavage precedes the translocation. Modulation of protein kinase C activity shows that it plays a central role in regulating CerS1 translocation. Analysis of the C-terminus of the CerS1 protein shows that several KxKxx motifs are not involved in regulating stress induced translocation. The study suggests that diverse stresses initiate responses through different signaling pathways, which ultimately converge to regulate CerS1 localization. The data provide an increasingly detailed understanding of the regulation of this important enzyme in normal and stressed cells and support the idea that it is uniquely regulated with respect to the other CerS enzymes.

(Dihydro)ceramide synthase 1 regulated sensitivity to cisplatin is associated with the activation of p38 mitogen-activated protein kinase and is abrogated by sphingosine kinase 1.

Resistance to chemotherapeutic drugs often limits their clinical efficacy. Previous studies have implicated the bioactive sphingolipid sphingosine-1-phosphate (S-1-P) in regulating sensitivity to cisplatin [cis-diamminedichloroplatinum(II)] and showed that modulating the S-1-P lyase can alter cisplatin sensitivity. Here, we show that the members of the sphingosine kinase (SphK1 and SphK2) and dihydroceramide synthase (LASS1/CerS1, LASS4/CerS4, and LASS5/CerS5) enzyme families each have a unique role in regulating sensitivity to cisplatin and other drugs. Thus, expression of SphK1 decreases sensitivity to cisplatin, carboplatin, doxorubicin, and vincristine, whereas expression of SphK2 increases sensitivity. Expression of LASS1/CerS1 increases the sensitivity to all the drugs tested, whereas LASS5/CerS5 only increases sensitivity to doxorubicin and vincristine. LASS4/CerS4 expression has no effect on the sensitivity to any drug tested. Reflecting this, we show that the activation of the p38 mitogen-activated protein (MAP) kinase is increased only by LASS1/CerS1, and not by LASS4/CerS4 or LASS5/CerS5. Cisplatin was shown to cause a specific translocation of LASS1/CerS1, but not LASS4/CerS4 or LASS5/CerS5, from the endoplasmic reticulum (ER) to the Golgi apparatus. Supporting the hypothesis that this translocation is mechanistically involved in the response to cisplatin, we showed that expression of SphK1, but not SphK2, abrogates both the increased cisplatin sensitivity in cells stably expressing LASS1/CerS and the translocation of the LASS1/CerS1. The data suggest that the enzymes of the sphingolipid metabolic pathway can be manipulated to improve sensitivity to the widely used drug cisplatin.

A new functional motif in Hox domain-containing ceramide synthases: identification of a novel region flanking the Hox and TLC domains essential for activity.

Ceramide is synthesized in mammals by a family of ceramide synthases (CerS) each of which uses a relatively restricted set of fatty acyl-CoAs for N-acylation of the sphingoid long chain base (Pewzner-Jung, Y., Ben-Dor, S., and Futerman, A. H. (2006) J. Biol. Chem. 281, 25001-25005). CerS are characterized by two functional domains, the Tram-Lag-CLN8 (TLC) domain and the homeobox (Hox) domain, which is found in all mammalian CerS except CerS1. We now demonstrate that the majority of the Hox domain is not required for CerS activity since its deletion in CerS5 does not affect activity. Subsequently, we define a highly conserved new motif of 12 amino acid residues that flanks the Hox and TLC domains but is not part of the TLC domain, which is essential for CerS5 and CerS6 activity. Two positively charged residues in this domain, one of which is conserved in all putative CerS in all organisms, are essential for activity since site-directed mutagenesis of either (Lys-134 and Lys-140 in CerS5) results in an approximately 50% loss of activity, whereas mutation of both leads to a complete loss of activity. Because this region is conserved across species, we propose that it plays a previously unidentified and essential role in CerS activity and can be used as a new motif to define Hox domain-containing CerS.

Enhanced intracellular mobility and nuclear accumulation of DNA plasmids associated with a karyophilic protein.

The use of synthetic gene delivery systems in human gene transfer is hampered by poor transfection efficiencies, largely because of the inability of DNA to translocate across the nuclear pore complex. A means to overcome this barrier is to bind the DNA to nuclear localization signals (NLSs), which are recognized by shuttling receptors of the nuclear import machinery. Here, we studied the intracellular transport of plasmid DNA microinjected into HeLa cell cytoplasm, alone or as a complex with intact or NLS-deleted NFkappaB p50, using confocal microscopy imaging. We found that association of NLS-carrying p50 with DNA facilitated not only nuclear entry of the DNA but also its migration through the cytoplasm toward the nucleus. Facilitated transport of p50-DNA complexes in the cytoplasm proceeded along microtubules in a dynein-dependent manner and is mediated by the heterodimeric nuclear transport receptor that recognizes the p50-born NLS.