PMA induces acid ceramidase degradation: a role for phosphorylation in controlling protein function

Acid ceramidase (ASAH1) hydrolyzes ceramide to form sphingosine and a free fatty acid. Because both ceramide and sphingosine are important signaling molecules, ASAH1 plays a critical regulatory role in lipid‐mediated cell signaling. ASAH1 enzymatic activity is regulated by many factors, including phorbol 12‐myristate 13‐acetate (PMA). However, aside from basal glycosylation of the β subunit, the role of post‐translational modification (PTM) in regulating ASAH1 function is largely unexplored. Therefore, we sought to identify signal‐dependent modifications that may regulate enzyme function. Mass spectrometric analysis revealed multiple PTMs on ASAH1, including phosphorylation at threonine‐289 (Thr289), a putative PKC target site. Given that PKC is a well‐established regulator of sphingolipid metabolism, we generated a phospho‐specific antibody against Thr289 to examine how cell signaling alters the phosphorylation status of the endogenous protein. We show that PMA acutely induces ASAH1 phosphorylation and stimulates protein degradation. Mutation of Thr289 alters the ability of ASAH1 to interact with protein partners, consistent with a key role for phosphorylation in controlling the function of ASAH1. Collectively, our findings reveal a novel role for PKC signaling in regulating ASAH1 function and sphingolipid metabolism. Research support from NIH DK084178 to MBS and AHA 10PRE3230019 to NCL.