SUBSTRATE SPECIFICITY OF NEW ARABIDOPSIS SPHINGOSINE KINASES

ALVAREZ SE; WORRAL D; LYNCH DV; HETHERINGTON A; MILSTIEN S; SPIEGEL S

Cashiers, NC USA

Congreso; XXXX Southeastern Regional Lipid Conference; 2005

Sphingolipids are ubiquitous components of all eukaryotic cellular membranes. In yeast and mammals, sphingolipid metabolites have been attracting considerable attention due to their involvement in the control of a wide range of biological processes such as growth, motility, stress responses and cell survival. Recent reports reveal that not only do plants contain components of eukaryotic sphingolipid signaling pathways, they also posses novel plant-specific sphingolipid regulatory systems. The sphingolipid metabolite, sphingosine-1-phosphate (S1P), has been shown to regulate calcium mobilization active in Arabidopsis guard cell drought/abscisic acid (ABA) responses. Previously, we have shown that the enzyme responsible for S1P production, sphingosine kinase (SphK), is stimulated by the phytohormone abscisic acid in guard cells of Arabidopsis thaliana and that S1P regulates guard cell turgor via a heterotrimeric G protein. Arabidopsis contains four gene loci that are phylogenetically distinct and code for proteins that contain the five conserved domains previously identified in SphKs. NP_566064 and NP_193885 are not yet characterized, whereas At-LCBK1 and ACD5 are bona fide dihydrosphingosine kinase and ceramide kinase, respectively.  In the current study, we have cloned NP_566064 and NP_193885 and characterized their enzymatic activities. Lysates of HEK 293 cells overexpressing NP_193885 had high kinase activity with sphingosine, a minor Arabidopsis free sphingoid base, and dihydrosphingosine and slightly lower activity with phytosphingosine, when substrates were presented as BSA complexes rather than in Triton X-100 mixed micelles, a property similar to that of mammalian SphK type 1. The phosphorylating activity was also markedly enhanced by addition of 1M KCl, which has been shown to selectively stimulate SphK2 and inhibit SphK1. Neither ceramide nor N,N-dimethylsphingosine, a potent inhibitor of both mammalian SphKs, were phosphorylated by these plant SphKs regardless of the method of substrate presentation. Interestingly, lysates of HEK 293 cells overexpressing NP_566064 did not have 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 distinct from At-LCBK1, and each fall into separate plant clusters that are distinguishable from both ceramide kinase cluster enzymes. Supported by R 37 GM043880 (SS).