Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.
Since insulin receptors and their downstream signaling molecules are organized in lipid rafts, proteomic analysis of adipocyte lipid rafts may provide new insights into the function of lipid rafts in adipogenesis and insulin signaling. To search for proteins involved in adipocyte differentiation and insulin signaling, we analyzed detergent-resistant lipid raft proteins from 3T3-L1 preadipocytes and adipocytes by 2-DE. Eleven raft proteins were identified from adipocytes. One of the adipocyte-specific proteins was globular C1q receptor (gC1qR), an acidic 32 kDa protein known as the receptor for the globular domain of complement C1q. The targeting of gC1qR into lipid rafts was significantly increased during adipogenesis, as determined by immunoblotting and immunofluorescence. Since the silencing of gC1qR by small RNA interference abolished adipogenesis and blocked insulin-induced activation of insulin receptor, insulin receptor substrate-1 (IRS-1), Akt, and Erk1/2, we can conclude that gC1qR is an essential molecule involved in adipogenesis and insulin signaling.
Serum-and glucocorticoid-inducible protein kinase 1 (SGK1) has been implicated in diverse cellular activities including the promotion of cell survival. The molecular mechanism of the role of SGK1 in protection against cellular stress has remained unclear, however. We have now shown that SGK1 inhibits the activation of SEK1 and thereby negatively regulates the JNK signaling pathway. SGK1 was found to physically associate with SEK1 in intact cells. Furthermore, activated SGK1 mediated the phosphorylation of SEK1 on serine 78, resulting in inhibition of the binding of SEK1 to JNK1, as well as to MEKK1. Replacement of serine 78 of SEK1 with alanine abolished SGK1-mediated SEK1 inhibition. Oxidative stress upregulated SGK1 expression, and depletion of SGK1 by RNA interference potentiated the activation of SEK1 induced by oxidative stress in Rat2 fibroblasts. Moreover, such SGK1 depletion prevented the dexamethasone-induced increase in SGK1 expression, as well as the inhibitory effects of dexamethasone on paclitaxel-induced SEK1-JNK signaling and apoptosis in MDA-MB-231 breast cancer cells. Together, our results suggest that SGK1 negatively regulates stressactivated signaling through inhibition of SEK1 function.
Using tunneling nanotubes (TNTs), various pathological molecules and viruses disseminate to adjacent cells intercellularly. Here, we show that the intracellular invasion of
Mycoplasma hyorhinis
induces the formation of actin- and tubulin-based TNTs in various mammalian cell lines.
M. hyorhinis
was found in TNTs generated by
M. hyorhinis
infection in NIH3T3 cells. Because mycoplasma-free recipient cells received mycoplasmas from
M. hyorhinis
-infected donor cells in a mixed co-culture system and not a spatially separated co-culture system, direct cell-to-cell contact via TNTs was necessary for the intracellular dissemination of
M. hyorhinis
. The activity of Rac1, which is a small GTP binding protein, was increased by the intracellular invasion of
M. hyorhinis
, and its pharmacological and genetic inhibition prevented
M. hyorhinis
infection-induced TNT generation in NIH3T3 cells. The pharmacological and genetic inhibition of Rac1 also reduced the cell-to-cell dissemination of
M. hyorhinis
. Based on these data, we conclude that intracellular invasion of
M. hyorhinis
induces the formation of TNTs, which are used for the cell-to-cell dissemination of
M. hyorhinis
.
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