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Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells

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Abstract

Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase’s translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation—where PKCθ plays a critical role.

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    Authors and Affiliations

    1. Department of Biology, Faculty of Sciences, Lebanese University, Hadath, Lebanon

      Rouba Hage-Sleiman

    2. Department of Biomedical Science, Faculty of Health Sciences, Global University, Batrakiyye, Beirut, Lebanon

      Asmaa B. Hamze

    3. Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon

      Aimée F. El-Hed, Randa Attieh, Lina Kozhaya, Sarah Kabbani & Ghassan Dbaibo

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    1. Rouba Hage-Sleiman
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    Rouba Hage-Sleiman and Asmaa B. Hamze have contributed equally to this work.

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    Hage-Sleiman, R., Hamze, A.B., El-Hed, A.F. et al. Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells. Immunol Res 64, 869–886 (2016). https://doi.org/10.1007/s12026-016-8787-9

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