Title

Identification of novel sphingosine binding proteins and their roles in cellular signal transduction

Date of Completion

January 2009

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Sphingolipid metabolites regulate cell fate by acting on specific cellular targets. We used affinity chromatography coupled with proteomics technology and identified the scaffolding protein, 14-3-3 epsilon, as a direct target of sphingoid bases. In addition, we also found that acidic leucine-rich nuclear phosphoprotein-32A (ANP32A), an inhibitor of protein phosphatase 2A (PP2A), is a direct target of sphingosine, N,N'-dimethyl sphingosine (DMS) and phytosphingosine but not dihydrosphingosine. ^ We have determined that sphingosine specifically binds to all isoforms of 14-3-3. R18, the peptide identified by a phage display library binding within the amphipathic ligand binding groove of 14-3-3 and inhibiting 14-3-3 from interacting with its binding partners, competitively blocks the 14-3-3:sphingosine interaction in a dose-dependent manner. This suggests the phospho-serine recognition site of the 14-3-3 ligand binding groove may be interacting with sphingosine. We suggest that sphingosine can inhibit the interaction of 14-3-3 with its binding partners to regulate cell signaling. ^ Treatment of human umbilical vein endothelial cells (HUVEC) with DMS, an analog of sphingosine which is not phosphorylated by sphingosine kinases, led to the activation of PP2A activity. Suppression of ANP32A with siRNA enhanced basal and DMS activated PP2A activity suggesting that the sphingoid base binds to and relieves the inhibitory action of ANP32A on the PP2A complex. Interestingly, DMS treatment induced the p38 stress-activated protein kinase (SAPK) and expression of cyclooxygenase (COX)-2 transcript and protein. Knockdown of ANP32A expression further induced p38 SAPK and COX-2. We suggest that sphingoid bases bind to ANP32A and regulate cellular signaling resulting in changes in gene expression. ^ These studies further our understanding of how sphingosine modulates cellular behavior. ^