Abstract 5578: Cell Polarity Factor Par3 Binds SPTLC1 Modulating Monocyte/Macrophage Serine Palmitoyltransferase Activity, De Novo Ceramide Synthesis and Chemotaxis

Abstract

Elevated sphingolipids associate with increased cardiovascular disease. Conversely, atherosclerosis is reduced in mice by blocking de novo synthesis of sphingolipids catalyzed by serine palmitoyltransferase (SPT). The SPT enzyme is composed of the SPTLC1 and 2 subunits, and we have identified a novel protein-protein interaction between SPTLC1 and the PDZ protein Partitioning Defective Protein 3 (Par3). Mammalian SPTLC1 orthologs have a highly conserved C-terminus that conforms to a type II PDZ protein interaction motif, and by screening PDZ domain protein arrays with a SPTLC1 C-terminal peptide we found it bound the 3 rd PDZ domain of Par3. Overlay and immuno-precipitation assays confirmed this interaction and indicate Par3 associates with the SPTLC1/2 holoenzyme by binding the C-terminal PDZ motif of SPTLC1, and this binding can re-localize SPTLC1 to the periphery of the cell at sites of cell-cell contact and in cell protrusions. The SPTLC1/2-Par3 complex was detected at endogenous expression levels in mouse liver and primary macrophages demonstrating its physiologic existence, and siRNA inhibition of Par3 in human THP-1 monocytes significantly reduced SPT activity and the de novo synthesis of ceramide and sphingomyelin by nearly 40%. Given monocyte recruitment into inflamed vessels promotes atherosclerosis, and since Par3 and sphingolipids have been associated with polarized cell migration, we tested and found that Par3 and SPTLC1 are required for the ability of THP-1 monocytes to migrate towards monocyte chemoattractant protein 1 (MCP-1). Knockdown of Par3 significantly reduced MCP-1 induced chemotaxis of THP-1 monocytes, as did knockdown of SPTLC1, and this Par3 effect depended upon SPT activity. Migration in response to other inflammatory G-protein coupled chemokines including fractalkine and fMLP was also inhibited in these cells and in primary mouse macrophages treated with the anti-atherosclerotic SPT inhibitor myriocin. In conclusion, we have identified a novel SPTLC1-Par3 interaction that promotes monocyte serine palmitoyltransferase activity and chemotaxis towards inflammatory chemokine signals.