Ceramide directly activates protein kinase C ζ to regulate a stress-activated protein kinase signaling complex

We have previously shown that interleukin 1 (IL-1)receptor-generated ceramide induces growth arrest in smooth muscle pericytes by activating an upstream kinase in the stress-activated protein kinase (SAPK) cascade. We now report the mechanism by which ceramide activates the SAPK signaling pathway in human embryonic kidney cells (HEK-293). We demonstrate that ceramide activation of protein kinase C zeta (PKC zeta) mediates SAPK signal complex formation and subsequent growth suppression. Ceramide directly activates both immunoprecipitated and recombinant human PKC zeta in vitro. Additionally, ceramide activates SAPK activity, which is blocked with a dominant-negative mutant of PKC zeta Coimmunoprecipitation studies reveal that ceramide induces the association of SAPK with PKC zeta, but not with PKC epsilon. In addition, ceramide treatment induces PKC zeta association with phosphorylated SEK and MEKK1, elements of the SAPK signaling complex. The biological role of ceramide to induce cell cycle arrest is mimicked by overexpression of a constitutively active PKC zeta. Together, these studies demonstrate that ceramide induces cell cycle arrest by enhancing the ability of PKC zeta to form a signaling complex with MEKK1, SEK, and SAPK.