Ceramide Directly Activates Protein Kinase C Zeta 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 (PKCzeta) mediates SAPK signal complex formation and subsequent growth suppression. Ceramide directly activates both immunoprecipitated and recombinant human PKCzeta in vitro. Additionally, ceramide activates SAPK activity, which is blocked with a dominant-negative mutant of PKCzeta. Co-immunoprecipitation studies reveal that ceramide induces the association of SAPK with PKCzeta, but not with PKCepsilon. In addition, ceramide treatment induces PKCzeta 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 PKCzeta. Together, these studies demonstrate that ceramide induces cell cycle arrest by enhancing the ability of PKCzeta to form a signaling complex with MEKK1, SEK, and SAPK.