PMA stimulates PKCalpha‐dependent phosphorylation of ASAH1 in breast cancer cell lines.

Abstract

Acid ceramidase (ASAH1) catalyzes the hydrolysis of ceramide to form sphingosine. Studies have implicated deregulation of ASAH1 in cancer, where overexpression is linked to tumorigenesis. The aim of this study was to define the role of phosphorylation in regulating ASAH1 function in breast cancer cells. We show that phorbol 12‐myristate 13‐acetate (PMA) activated a time‐dependent increase in the phosphorylation of ASAH1 at threonine‐287 (T287) in both MCF‐7 and MDA‐MB‐231 breast cancer cells. Moreover, inhibiting calcium‐dependent protein kinase C (PKC) activity attenuated the ability of PMA to increase phosphorylation of ASAH1. Silencing PKCα protein attenuated ASAH1 PMA‐stimulated phosphorylation at T287. Interestingly, the level of expression of PKCα, which has been reported as a marker for breast cancer aggressiveness, is 6‐fold greater when compared to benign MCF10A mammary epithelial cells. In addition, PMA was unable to promote the phosphorylation of ASAH1 in the MCF10A cell line. Furthermore, alanine substitution at T287 reduced the stability of the protein and led to a 57% decrease in the half‐life of ASAH1. Collectively, these data link post‐translational modification of ASAH1 at T287 to the progression of a tumorigenic phenotype in breast cancer cells. This work is supported by NIH DK084178