Sphingosine Interaction with Acidic Leucine-rich Nuclear Phosphoprotein-32A (ANP32A) Regulates PP2A Activity and Cyclooxygenase (COX)-2 Expression in Human Endothelial Cells

Abstract

Sphingolipid metabolites regulate cell fate by acting on specific cellular targets. Although the influence of sphingolipids in cellular signaling has been well recognized, the exact molecular targets and how these targets influence cellular signaling mechanisms remain poorly understood. Toward this goal, we used affinity chromatography coupled with proteomics technology and identified acidic leucine-rich nuclear phosphoprotein-32A (ANP32A), an inhibitor of protein phosphatase 2A (PP2A) as a direct target of sphingosine, N,N-dimethyl sphingosine (DMS) and phytosphingosine but not dihydrosphingosine or sphingosine 1-phosphate. Treatment of human umbilical vein endothelial cells (HUVEC) with DMS, 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. Indeed, DMS relieved the ANP32A-mediated inhibition of PP2A enzyme complex in vitro. 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. These data identify ANP32A as a novel molecular target of sphingoid bases that regulates cellular signaling events and inflammatory gene expression.

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies

“…Accordingly, BCR-ABL1 uses Jak2 to induce SET-dependent PP2A inactivation. 24 To ensure that SET mediates PP2A inhibition in Jak2 V617F cells, we also targeted the related PP2A inhibitor I1PP2A (inhibitor 1 of PP2A) by administering DMS and sphingosine that, reportedly, 44 target I1PP2A and restore PP2A activity when it is repressed by I1PP2A. These compounds failed to induce PP2A activity in Jak2 V617F cells (not shown), indicating that I1PP2A unlikely plays a role in Jak2 V617F -induced PP2A inhibition.…”

Differential Splicing of ANP32A in Birds Alters Its Ability to Stimulate RNA Synthesis by Restricted Influenza Polymerase

“…HisGST-PB2 proteins were purified as before and dialyzed into 10 mM Tris-HCl pH 7.0, 200 mM NaCl, 10% glycerol (Tarendeau et al, 2008). In vitro protein interactions were performed by mixing 20 mg each of bait and prey proteins in 100 ml total interaction buffer (10 mM HEPES pH 7.5, 100 mM KCl, 10 mM NaCl, 5 mM MgCl 2 ) followed by rotating for 1 h at room temperature (Habrukowich et al, 2010). Bait proteins were then captured with 10 ml MagneGST glutathione particles (Promega).…”

Overexpression of the pp32r1 (ANP32C) oncogene or its functional mutant pp32r1Y140H confers enhanced resistance to FTY720 (Finguimod)

“…16 In this work pp32 was observed to interact with sphingosine or dimethyl sphingosine but not dihydrosphingosine or ceramide indicating that the presence of a double bond (present in sphingosine but absent in dihydrosphingosine) is essential for this interaction. (Fig.…”

“…16 We searched for similar compounds in the PubChem database (http://pubchem. ncbi.nlm.nih.gov/) and identified FTY720, a synthetic structural analog of sphingosine that is derived from the metabolite myriocin produced by the fungus Isaria sinclairii.…”