UC San Diego

PUMA is a Bcl-2 pro-apoptotic protein known for its ability to mediate cell death by apoptosis and, more recently, autophagocytosis. Previously three serine phosphorylation sites have been identified which are involved in the post-translational regulation of PUMA, S- 10 -96 and -106. Thus far, serine-10 is best understood for promoting cell survival when phosphorylated by destabilizing PUMA and decreasing apoptosis, however the regulatory function of S-96 and S-106 phosphorylation is still unknown. To address this question we developed a PUMA S96,106A non-phosphorylation mutant and compared its function with wild-type PUMA. We first used flow cytometry to determine the regulatory function of S-96 and S-106 in apoptosis and found and insignificant difference between wild-type and mutant PUMA. Next, flow cytometry was used to quantify autophagy in cells overexpressing PUMA wt and S96,106A, revealing that simultaneous elimination of S-96 and S-106 phosphorylation significantly decreases cellular autophagy. This result was confirmed by quantifying Light Chain-3-II (LC3-II) by protein immunoblotting and fluorescence microscopy. Lastly, the protein stability of wild-type and mutant constructs was quantified by western blot showing that elimination of S-96 and S-106 phosphorylation slightly decreased PUMA stability, however these results were not significant enough to draw any definite conclusions. We conclude that serine-96 and -106 phosphorylation sites are involved in regulating PUMA's mediated autophagy but not apoptosis or protein stability. The aim of this study is to better understand how PUMA is post-translationally regulated important for the application of PUMA in pathologies including but not limited to cancer, alzheimer's and colitis