PAK Is Regulated by PI3K, PIX, CDC42, and PP2Cα and Mediates Focal Adhesion Turnover in the Hyperosmotic Stress-induced p38 Pathway

Abstract

Fractionation of brain extracts and functional biochemical assays identified PP2C␣, a serine/threonine phosphatase, as the major biochemical activity inhibiting PAK1. PP2C␣ 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, PP2C␣ knockdown increased its activation. Hyperosmotic stressinduced 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 PP2C␣ 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/PP2C␣/p38 pathway, and 2) PAK and PP2C␣ modulate the effects of this pathway on focal adhesion dynamics. PAK,2 the p21-activated kinase, is an effector kinase for the small Rho GTPases Cdc42 and Rac (1). PAKs mediate cytoskeletal rearrangements promoted by the activated GTPases such as loss of focal adhesions and actin stress fibers and the generation of filopodia (2, 3). PAK has also been implicated in other cellular events, including protection from apoptosis through phosphorylation of BAD (4, 5), mitosis through phosphorylation of RAF-1 (6, 7), and hormone signaling through estrogen receptor phosphorylation (8). The mitogen-activated protein kinase (MAPK) pathway is linked to PAK through Cdc42-mediated activation of p38, JNK (9), and ERK (10). The signaling pathways of extracellular stimuli leading to PAK and MAPK activation are not well characterized.Changes in extracellular osmolality rapidly induce the activation of MAPKs (11); however, little is known of the regulators of the MAPK pathway. In Saccharomyces cerevisiae, stress response is mediated through specific osmosensing pathways, of which components include the MAPKs (12). The mammalian counterpart of these osmosensors has not been conclusively identified, although clustering of epidermal growth factor receptor has been proposed (13). PAK has been implicated in the stress response pathway through its activation upon hyperosmotic shock (14, 15).The mechanis...

The Apical Polarity Determinant Crumbs 2 Is a Novel Regulator of ESC-Derived Neural Progenitors

“…A well-described downstream target of activated Cdc42 is serine/threonine protein kinase 1 that stimulates the FGF signaling pathway [52][53][54]. Interestingly, Par6 overexpression is able to induce EGF/ FGF independence of epithelial cells, in a Cdc42-and aPKCdependent manner [55], providing further evidence for a possible link between apical polarity complex proteins and FGF signaling.…”

Akt1 promotes focal adhesion disassembly and cell motility through phosphorylation of FAK in growth factor-stimulated cells

“…We have previously shown that GF-activated PAK serves as a scaffold protein of the Akt1 pathway that regulates the target specificity of Akt1 (Higuchi et al, 2008). Since it has been reported that PAK is localized at FAs (Stoletov et al, 2001;Brown et al, 2002;Chan et al, 2008;Delorme-Walker et al, 2011) and that PAK forms a complex with FAK (Stoletov et al, 2001), it is possible that PAK may also serve as a scaffold protein that facilitate the association between Akt1 and FAK at FAs, resulting in Akt1-mediated phosphorylation and activation of FAK in GF-stimulated cells. Since it has been shown in a recent study that Ser695 is necessary for pressure-induced activation of FAK in Caco-2 cells (Wang and Basson, 2011), it would also be worth examining whether the PAK-Akt1 pathway is also involved in pressure-induced activation of FAK in these cells.…”

Group I Paks Promote Skeletal Myoblast Differentiation In Vivo and In Vitro

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