The role of sphingosine kinase in plant cell signaling.

PANAGOPULOS M; WORRAL D; LIANG YK; ALVAREZ SE; HOLROYD G; SPIEGEL S; GRAY J; HETHERINGTON A

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR AND INTEGRATIVE PHYSIOLOGY

ELSEVIER SCIENCE INC

Lugar: Amsterdam; Año: 2008

1095-6433

Sphingolipids are ubiquitous components of all eukaryotic cellular membranes. In yeastand mammals, sphingolipid metabolites have been attracting considerable attention dueto their involvement in the control of a wide range of biological processes such asgrowth, motility, stress responses and cell survival. Recent reports reveal that not onlydo plants contain components of eukaryotic sphingolipid signaling pathways, they alsoposses novel plant-specific sphingolipid regulatory systems. The sphingolipid metabolite,sphingosine-1-phosphate (S1P), has been shown to regulate calcium mobilization activein Arabidopsis guard cell drought/abscisic acid (ABA) responses. Previously, we haveshown that the enzyme responsible for S1P production, sphingosine kinase (SphK), isstimulated by the phytohormone abscisic acid in guard cells of Arabidopsis thaliana andthat S1P regulates guard cell turgor via a heterotrimeric G protein. Arabidopsis containsfour gene loci that are phylogenetically distinct and code for proteins that contain the fiveconserved domains previously identified in SphKs. NP_566064 and NP_193885 are notyet characterized, whereas At-LCBK1 and ACD5 are bona fide dihydrosphingosinekinase and ceramide kinase, respectively. In the current study, we have clonedNP_566064 and NP_193885 and characterized their enzymatic activities. Lysates ofHEK 293 cells overexpressing NP_193885 had high kinase activity with sphingosine, aminor Arabidopsis free sphingoid base, and dihydrosphingosine and slightly loweractivity with phytosphingosine, when substrates were presented as BSA complexesrather than in Triton X-100 mixed micelles, a property similar to that of mammalian SphKtype 1. The phosphorylating activity was also markedly enhanced by addition of 1M KCl,which has been shown to selectively stimulate SphK2 and inhibit SphK1. Neitherceramide nor N,N-dimethylsphingosine, a potent inhibitor of both mammalian SphKs,were phosphorylated by these plant SphKs regardless of the method of substratepresentation. Interestingly, lysates of HEK 293 cells overexpressing NP_566064 did nothave any significant kinase activity compared to untransfected cells. Taken together,these data indicate that the NP_193885 is a bona fide sphingosine kinase that is distinctfrom At-LCBK1, and each fall into separate plant clusters that are distinguishable fromboth ceramide kinase cluster enzymes. Supported by R 37 GM043880 (SS)