p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils.
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.
Rane
M J
MJ
Department of Medicine, University of Louisville Health Sciences Center and the Veterans Affairs Medical Center, Louisville, Kentucky 40202, USA. mrane@louisville.edu
Coxon
P Y
PY
Powell
D W
DW
Webster
R
R
Klein
J B
JB
Pierce
W
W
Ping
P
P
McLeish
K R
KR
D064413:3-Phosphoinositide-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011725:Pyridines / Q000494:pharmacology; D012694:Serine / Q000235:genetics; D013912:Threonine / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2
D064413:3-Phosphoinositide-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010716:Phosphatidylinositols; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011725:Pyridines; D012694:Serine; D013912:Threonine; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C494918:AKT1 protein, human; C578562:PDPK1 protein, human; C482530:MAP2K3 protein, human; C482566:MAP2K6 protein, human; C093642:SB 203580