Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue.

Yong-Seok Oh,Sung Ho Ryu,Sun Sik Bae,Sang Hoon Ha,Pann-Ghill Suh,Jong Bae Park,Qiming Jane Wang

doi:10.1371/journal.pone.0143695

Abstract

Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.

Highlights

Sphingolipids such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P) are ubiquitous constituents of eukaryotic membranes that regulate cell growth, survival, apoptosis, differentiation, migration, and immune responses [1,2,3,4]
We observed that S373 of mSK1a is directly phosphorylated by conventional PKCs (cPKCs) in response to Phorbol 12-myristate 13-acetate (PMA) treatment
Our results demonstrate that phosphorylation of mSK1a is strongly elicited by PMA, a potent activator of PKC, but not by forskolin, an activator of PKA, indicating that this phosphorylation of mSK1a is kinase-specific

Summary

Introduction

Sphingolipids such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P) are ubiquitous constituents of eukaryotic membranes that regulate cell growth, survival, apoptosis, differentiation, migration, and immune responses [1,2,3,4]. S1P is important for direct modulation of the activity of histone deacetylase [7], the ubiquitin ligase activity of TRAF2 [8], activation of MAP kinase [9], and Ca2+ mobilization [10, 11] In another context, S1P functions as an extracellular ligand for a family of S1P-specific cell-surface G protein-coupled receptors (GPCRs) [5, 12]. MSK1 harbors multiple Ca2+/calmodulin-binding sequences [32] and both isoforms have a proline-rich motif that might be involved in the interaction with SH3 domain-containing protein(s) [33], suggesting that the two isoforms might be differentially regulated through both specific and common signaling pathways. We analyzed the phosphorylation pattern of mSK isoforms in response to extracellular stimuli. Based on the initial observation that mSK1a is robustly phosphorylated in response to phorbol ester, we characterized the molecular pathway leading to mSK1a phosphorylation and elucidated its influence on SK activity

Material and Methods

Results

Discussion