Abstract
Akt activation requires phosphorylation of Thr(308) and Ser(473) by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser(473) phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.
Highlights
Akt activation requires phosphorylation of Thr308 and Ser473 by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively
Inhibition of p38 kinase did not alter fMLPstimulated ERK activity by either of the two methods. These data suggest that ERK does not participate in the Akt signaling pathway, despite the requirement of phosphatidylinositol 3-kinase (PI-3K) for ERK activation. Both Akt and p38 kinase are rapidly activated in neutrophils by a number of inflammatory mediators, and one or both kinases participate in respiratory burst activity, chemotaxis, priming, and apoptosis [17, 18, 28, 29, 31, 32]
Our study provides evidence for the first time that p38 kinase participates in the signal transduction pathway leading to Akt activation
Summary
Vol 276, No 5, Issue of February 2, pp. 3517–3523, 2001 Printed in U.S.A. p38 Kinase-dependent MAPKAPK-2 Activation Functions as 3-Phosphoinositide-dependent Kinase-2 for Akt in Human Neutrophils*. Akt activation requires phosphorylation of Thr308 and Ser473 by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp and that p38-dependent MK2 activation functions as PDK2 in human neutrophils. Phosphorylation of Ser473 is dependent on products of PI-3K; the identity of this kinase, termed PDK2, is unknown [11, 12] Cellular stresses, such as heat shock and hyperosmolarity, stimulate both p38 kinase and Akt activity [13]. MAP kinase-activated protein kinase-2 (MK2), a direct target of p38, has been reported to phosphorylate Ser473 of Akt in vitro [19], and activated Akt associates with a substrate of MK2, heat shock protein 27 (Hsp27) [20]. We report that Akt forms a stable complex with p38 kinase, MK2, and Hsp and that, upon stimulation with fMLP, Hsp dissociates from this complex
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