Protein Kinase D-mediated Phosphorylation and Nuclear Export of Sphingosine Kinase 2

Abstract

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, downregulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetateinduced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD overexpression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling. Sphingosine kinases (SPHKs)3 catalyze the formation of sphingosine 1-phosphate, a bioactive lipid that regulates a diverse range of cellular processes, including cell growth, survival, differentiation, motility, and cytoskeletal organization (1, 2). Some of these cellular processes are mediated by five sphingosine 1-phosphatespecific G protein-coupled receptors, whereas others appear to be controlled by intracellular sphingosine 1-phosphate through as yet unidentified intracellular targets (2, 3).Two distinct SPHK isoforms, SPHK1 and SPHK2, have been cloned and characterized (4, 5). Diverse external stimuli, particularly growth and survival factors, stimulate SPHK1, and intracellularly generated sphingosine 1-phosphate has been implicated in their mitogenic and anti-apoptotic effects (6 -15). Expression of SPHK1 enhanced proliferation, promoted the G 1 /S transition, protected cells from apoptosis (6,8,16), and induced tumor formation in mice (8, 9).In contrast to SPHK1 much less is known about SPHK2. Although highly similar in amino acid sequence and possessing five evolutionarily conserved domains found in all SPHKs (17), SPHK2 diverges in its N terminus and central regions. These two isoforms have different kinetic properties and differ in developmental and tissue expression (5) implying that they may have distinct physiological functions. In fact, studies from our laboratory have demonstrated that, in contrast to cytosolic distribution of SPHK1, SPHK2 enters nuclei and inhibits DNA synthesis or induces apoptosis under stressful conditions such as serum deprivation (18,19)....

Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner

“…In addition, the generation of a 'chimeric' SK-1 containing the NLS of SK-2 at its N-terminus was able to mimic the inhibitory effect on DNA synthesis . Moreover, it was shown that nuclear SK-2 can be phosphorylated by the protein kinase D, which is also known as protein kinase C-m, resulting in export of SK-2 to the cytoplasm (Ding et al, 2007). All these data suggest that there are subcellular pools of sphingolipids and that the knockout of either SK-1 or SK-2 may lead to subcellular and local changes in sphingolipid levels that are not detectable using our total cell lipid extraction method.…”

Hypoxia Enhances Sphingosine Kinase 2 Activity and Provokes Sphingosine-1-Phosphate-Mediated Chemoresistance in A549 Lung Cancer Cells

“…Tumor necrosis factor-a triggered phosphorylation of SphK1 by p42/44 MAPK (41), whereas epidermal growth factor induced a protein kinase Cydependent phosphorylation and the subsequent translocation of SphK1 to the plasma membrane (42). Phorbol 12-myristate 13-acetate triggered p42/44 MAPK and protein kinase D to phosphorylate SphK2, thus enhancing its catalytic activity and mediating its nuclear export (25,26). Activation of signaling pathways such as PI3K, extracellular signal-regulated kinase 1/2, or p38 MAPK, which are active under hypoxia (9,40), might therefore add to SphK2 regulation.…”

“…Activation of SphK2 in response to growth factors or phorbol 12-myristate 13-acetate via activation of extracellular signal-regulated kinase 1 and phosphorylation at Ser 351 and Thr 578 has also been shown (25). Ding et al identified two more serine residues within SphK2 (Ser 419 and Ser 421 ) that became phosphorylated by protein kinase D, which fostered the nuclear export of SphK2 as an alternative regulatory circuit (26).…”

Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes

“…Human SphK2-GFP and murine S1P 1 -CFP were constructed as described previously 41,50 . Murine S1P 3 was amplified and cloned into pECFP-N1 (Clontech) and pEGFP-N1 (Clontech) for making CFP-or GFP-tagged S1P 3 , respectively.…”