Abstract
3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C(10)-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C(10)-carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator.
Highlights
Three Phosphoinositide-dependent kinase 1 (PDK1) autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy
Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase
A significant increase in [32P]phosphate incorporation into protein kinase A (PKA) K72H and into the activation loops of protein kinase CII (PKCII), p21 activated kinase 1 (PAK1), and Akt was observed in the presence of PDK1 and sphingosine over samples incubated with PDK1 alone, indicating that sphingosine serves as a true PDK1 activator
Summary
Materials—All lipids used in these studies were from Sigma except sn-1,2, dipalmitoyl PtdIns bis-4,5 phosphate, sn-1,2, dipalmitoyl PtdIns bis-3,4 phosphate, and sn-1,2, dipalmitoyl PtdIns bis-3,4,5 phosphate, which were purchased from Matreya. The phospho-Akt threonine 308 phate; MALDI, matrix assisted laser desorption/ionization; MS, mass spectroscopy; PKA, protein kinase A; IGF-1, insulin-like growth factor-1; GST, glutathione S-transferase; Pipes, piperazine-N,NЈ-bis-(2ethanesulfonic acid). Kinase Assays—COS-7 lysates containing various PDK1 or PAK1 constructs were incubated with the anti-Myc (9E10) monoclonal antibody in binding buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 10 mM Pipes, pH 7.3, 1.0 mM dithiothreitol, and 1.0 mM phenylmethylsulfonyl fluoride) overnight at 4 °C, followed by a 1-h incubation with a 1:1 slurry of bovine serum albumin (Sigma)-coated protein G-Sepharose beads (Amersham Pharmacia Biotech).
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