PAK is regulated by PI3K, PIX, CDC42, and PP2Calpha and mediates focal adhesion turnover in the hyperosmotic stress-induced p38 pathway.
Fractionation of brain extracts and functional biochemical assays identified PP2Calpha, a serine/threonine phosphatase, as the major biochemical activity inhibiting PAK1. PP2Calpha dephosphorylated PAK1 and p38, both of which were activated upon hyperosmotic shock with the same kinetics. In comparison to growth factors, hyperosmolality was a more potent activator of PAK1. Therefore we characterize the PAK signaling pathway in the hyperosmotic shock response. Endogenous PAKs were recruited to the p38 kinase complex in a phosphorylation-dependent manner. Overexpression of a PAK inhibitory peptide or dominant negative Cdc42 revealed that p38 activation was dependent on PAK and Cdc42 activities. PAK mutants deficient in binding to Cdc42 or PAK-interacting exchange factor were not activated. Using a panel of kinase inhibitors, we identified PI3K acting upstream of PAK, which correlated with PAK repression by pTEN overexpression. RNA interference knockdown of PAK expression reduced stress-induced p38 activation and conversely, PP2Calpha knockdown increased its activation. Hyperosmotic stress-induced PAK translocation away from focal adhesions to the perinuclear compartment and resulted in disassembly of focal adhesions, which are hallmarks of PAK activation. Inhibition of PAK by overexpression of PP2Calpha or the kinase inhibitory domain prevented sorbitol-induced focal adhesion dissolution. Inhibition of MAPK pathways showed that MEK-ERK signaling but not p38 is required for full PAK activation and focal adhesion turnover. We conclude that 1) PAK plays a required role in hyperosmotic signaling through the PI3K/pTEN/Cdc42/PP2Calpha/p38 pathway, and 2) PAK and PP2Calpha modulate the effects of this pathway on focal adhesion dynamics.
Chan
Perry M
PM
GSK-IMCB Group, Institute of Molecular and Cell Biology, Proteos Building, 61 Biopolis Drive, Singapore 138673. perryc@imcb.a-star.edu.sg
Lim
Louis
L
Manser
Edward
E
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000502:physiology; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D009997:Osmotic Pressure; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D054648:Protein Phosphatase 2; D064067:Rho Guanine Nucleotide Exchange Factors; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C515903:PAK1 protein, human; C515906:Pak1 protein, rat; C517189:PPP2CA protein, human; C514014:Ppp2ca protein, rat; C494929:PTEN protein, human; C487216:Pten protein, rat