Mechanism of Ceramide-Induced Activation of Protein Phosphatase 2A
Krishna K Narra, Scott A Summers. University of Utah, Salt Lake City, UT; Duke-NUS Graduate Medical School, Singapore, Singapore
Background: Ceramide regulates cell metabolism, growth, and death. Inhibiting this sphingolipid delays or prevents disease onset in animal models of diabetes, cardiomyopathy, insulin resistance, atherosclerosis, and hepatic steatosis. Therapeutic strategies for treating these metabolic diseases will benefit from understanding the mechanism of action of ceramide. Among other molecular mechanisms, ceramide activates protein phosphatase 2A (PP2A), which dephosphorylates and inactivates Akt2 kinase, an insulin signaling intermediate. PP2A comprises of A (scaffolding), B (regulatory), and C (catalytic) subunits. The B subunit isoforms impart substrate and regulator specificity. In vitro, ceramide binds to inhibitor 2 of protein phosphatase 2A (I2PP2A), which then dissociates from PP2A thus activating the phosphatase. Here we show novel in vivo subcellular and molecular mechanisms by which ceramide regulates I2PP2A and PP2A.
Design: PP2A activity was measured in immunoprecipitates from H4iie (rat hepatoma) and U-87MG (human glioblastoma) cell cultures with in vitro and in vivo incubation with ceramide and also from dihydroceramide desaturase (DES1) knockout fibroblasts which completely lack ceramide. Akt phosphorylation was evaluated with knockdown of I2PP2A. PP2A activity and Akt phosphorylation were evaluated with overexpression and knockdown of select PP2A B subunit isoforms and with ceramide exposure. Using immunofluorescence, the effect of ceramide on subcellular localization of I2PP2A was studied. High fat diet (HFD) increases ceramide levels. Liver from HFD-fed C57b/6 mice treated with myriocin, a ceramide synthesis inhibitor, was evaluated for I2PP2A protein levels.
Results: In our experimental system designed to study allosteric mechanisms, ceramide did not activate PP2A by either in vitro or in vivo incubation; however, PP2A activity was reduced in DES1 knockout fibroblasts. Manipulation of expression of select B subunit isoforms did not affect ceramide-induced PP2A activity or Akt phosphorylation. Ceramide caused membrane localization of I2PP2A. Knocking down I2PP2A inhibited phosphorylation of Thr 308 but not Ser 473 residue of Akt. I2PP2A protein levels were decreased with HFD and were restored by reducing ceramide levels.
Conclusions: Ceramide-induced PP2A activation is not a direct allosteric mechanism. Ceramide causes subcellular translocation of I2PP2A thus dissociating the inhibitor from PP2A and activating the phosphatase. Ceramide causes long term regulation by decreasing I2PP2A protein expression.
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 280, Monday Morning