Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1

3-Phosphoinositide-dependent kinase 1 (PDK1) has preciously been shown to p hosphorylate the activation loop of several AGC kinase family members. In t his study we show that p21-activated kinase I, the activity of which is reg ulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosph orylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurre d only in the presence of sphingosine, which increased PDK1 autophosphoryla tion 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentra tion-dependent manner and significantly increased phosphate incorporation i nto known PDK1 substrates. Studies on the lipid requirement for PDK1 activa tion found that both sphingosine isoforms and stearlyamine also increased P DK1 autophosphorylation. However, C-10-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that bot h a positive charge and a lipid tail containing at least a C-10-carbon back bone were required for PDK1 activation. Three PDK1 autophosphorylation site s were identified after stimulation with sphingosine in a serine-rich regio n located between the kinase domain and the pleckstrin homology domain usin g two-dimensional phosphopeptide maps and matrix assisted laser desorption/ ionization mass spectroscopy. Increased phosphorylation of endogenous Akt a t threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elev ated by treatment with sphingomyelinase. Sphingosine thus appears to be a t rue PDK1 activator.