Activation EAGLE 2005 Key
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Reduced TAO activation in cells lacking ATM. (A) Positions of SQ/TQ sites in TAOs. CC: coiled coils, φ: hydrophobic domain. (B) Human skin fibroblasts derived from a normal (1BR3) and an AT individual were treated with 10 Gy IR. p38 activity was assayed. (C) 1BR3 and ATV cells were treated with 10 Gy IR and TAO2 activity was determined.
G2/M arrest is caused by inhibition of mitotic cyclin/Cdc2. Cyclin/Cdc2 is tightly regulated by multiple phosphorylations. Cdc2 is inhibited by Myt1/Wee1 phosphorylation on Tyr15 (Lundgren et al, 1991; Parker and Piwnica-Worms, 1992). The key step in its activation is dephosphorylation of Tyr15 by Cdc25C. G2/M arrest by DNA damage causes the accumulation of inactive pTyr15-Cdc2. In cells transfected with control oligonucleotide, HU or UV treatment caused accumulation of pTyr15-Cdc2 (Figure 5C). However, knockdown of either TAO1 or TAO3 decreased the amount of pY15-Cdc2 substantially, indicating that Cdc2 was activated despite the presence of DNA damage. These data further support the idea that TAO-mediated regulation of p38 in response to DNA damage is critical for regulating entry into mitosis. Decreasing the expression of TAOs was sufficient to bypass inhibition of mitotic cyclin/Cdc2 in response to damaged DNA.
Multiple lines of evidence indicate that TAOs are important regulators of the response to genotoxic stress. The activation of these kinases is a critical determinant of the DNA damage response. Damage-induced p38 activation is blocked by catalytically deficient TAOs or TAO RNAi. RNAi of TAOs diminishes not only p38 activation but also impairs the DNA damage-activated G2/M cell cycle checkpoint. The inability to engage the damage-induced G2/M checkpoint in cells with reduced TAO expression parallels the persistent activation of mitotic cyclin/Cdc2. Importantly, reducing TAO expression enhanced sensitivity to γ-radiation in colony survival assays. In aggregate, these findings provide strong evidence that the TAOs are required for activation of p38 and the G2/M checkpoint upon DNA damage.
Further support comes from experiments that implicate TAO kinases as key intermediates in the activation of p38 by ATM/ATR. Significantly, TAO activation by DNA damage is diminished in AT cells, as is activation of p38. TAOs appear to be direct ATM substrates. They contain canonical ATM/ATR phosphorylation sites, are in vitro substrates of ATM, and at least one of the TAOs is phosphorylated on an SQ site in response to DNA damage. Mutation of this site on TAO3 interferes with the IR- and UV-induced checkpoint. Furthermore, the IR-induced phosphorylation of TAO3 is dependent on ATM. Similar experiments with candidate phosphorylation sites on TAO1 reduced p38 activation by IR and UV (data not shown) and interfered with engagement of the checkpoint, suggesting that these sites are required for activation of TAO1 by ATM/ATR and its function in regulating p38 in response to DNA damage.
Why is p38 required for checkpoint activation when protein kinases such as Chk1 and Chk2 engage these checkpoints in response to DNA damage? DNA is damaged not only by radiation but also as a consequence of cell stress by other agents. For example, osmotic stress also induces double-strand DNA breaks (Kultz and Chakravarty, 2001; Dmitrieva et al, 2003) but does not induce Chk1 phosphorylation. Hence, pathways alternative to Chk1/2 must exist that sense DNA damaged by other events. p38 is ideal for this purpose because of its stress sensitivity. An array of MAP3Ks are linked to p38, providing the wiring to engage p38 as a consequence of many cellular insults. Specific MAP3Ks are responsive to overlapping stresses. TAOs are activated more strongly by DNA-damaging agents than by other cellular stresses. This suggests that among their primary functions is p38 activation in response to DNA damage, either due to activation of ATM/ATR or through other mechanisms.
Loss of integrin engagement can influence ERK activation at several points within the signaling cascade. Growth factor receptors have been shown to associate in complexes with integrins in an extracellular matrix-dependent fashion (47), which may concentrate receptors at sites on the plasma membrane to enhance receptor activation (42). Cell detachment from the substratum leads to activation of phosphatases (38) that inhibit ERK activation directly. The organization of the cytoskeleton can also influence kinases, such as protein kinase C (PKC), Src, or p21-activated kinase (PAK) (34), which facilitate Raf-1 or MEK activation. Finally, even with forced activation of ERK in suspended cells, loss of cytoskeletal integrity inhibits ERK translocation to the nucleus (3).
In NIH 3T3 fibroblasts, Ras becomes equally GTP loaded in adherent or suspended cells treated with growth factors, indicating that the signaling pathway is intact to this point (41, 45). We have found that activation of Raf-1 is a key adhesion-dependent step downstream of Ras (41), although others have suggested that abrogation of growth factor signaling in nonadherent cells primarily involves MEK (45). Raf-1 is regulated by functional interactions with many proteins, including kinases (Src, PAK, PKA, PKC, and Akt), phosphatases (PP1 and PP2A), and scaffolding proteins (14-3-3, Hsp90, KSR, and RKIP) (22). Unlike many kinases in which simple phosphorylation of a catalytic loop leads to activation, Raf-1 contains many phospho-regulatory sites, including serines (43, 233, 259, 338, 339, 491, and 621), threonine (494), and tyrosines (340 and 341). The PAKs (10, 49, 51) as well as a rho-dependent kinase (39) seem to play an important role in anchorage-dependent regulation in the ERK cascade. PAK3 phosphorylates Raf-1 on serine 338 (S338), a step that is required for efficient Raf-1 and ERK activation (10). Mutation of Raf-1 S338 to alanine results in a nonactivatable kinase (10, 13). Phosphorylations at this site are not activating for Raf-1 but are thought to relieve an autoinhibitory state to permit activation (17, 51). Importantly, the PAK family kinases are poorly activated in suspended cells due to lack of interaction between PAK and GTP-loaded Rac or cdc42 (19, 20) and because of inhibition by PKA (34).
PKA is a promiscuous kinase also involved in adhesion-dependent signaling to ERK. PKA is transiently activated upon detachment from the substratum (34) and can phosphorylate and inhibit both PAK (34) and Raf-1 (25, 30). PKA activation can have many effects throughout the cell that may impinge on Raf-1 signaling, including cytoskeletal disruption and phosphatase activation. PKA can also directly phosphorylate Raf-1 on at least two critical sites. Phosphorylation of Raf-1 S43 inhibits the Ras-Raf-1 interaction, which is crucial for Raf-1 translocation and activation (4). Phosphorylation of Raf-1 S233 or S259 is thought to inhibit Raf-1 by enhancing 14-3-3 binding to these sites and restricting Raf-1 intra- or intermolecular interactions (26).
The goal of this study was to investigate how cells translate physical adhesion to ECM into critical biochemical events that allow for efficient signaling within the cell. We examined the complex interactions between Raf-1, PKA, and PAK to determine their roles in the adhesion dependence of signaling from EGF to ERK in NIH 3T3 fibroblasts and HEK293 cells. We conclude that the adhesion-dependent regulation of ERK in EGF-treated cells occurs at the level of Raf-1. Unlike adherent cells, suspended cells do not phosphorylate Raf-1 S338 efficiently upon EGF treatment. Raf-1 from suspended cells is generally dephosphorylated on S43 and S259, and so it seems that it is loss of the permissive S338 phosphorylation, rather than excessive inhibitory phosphorylation, that is critical. Restoration of phosphorylation at S338, either by expression of a highly active version of PAK1 (PAK165) or by expression of a phospho-mimetic mutation (Raf-1 S338D) leads to a partial rescue of ERK activation in suspended cells.
Raf-1 S338 phosphorylation is a critical adhesion-dependent step in ERK activation. (A) Cell adhesion affects ERK and Raf 338 phosphorylation. Suspended and adherent NIH 3T3 fibroblasts were treated with EGF. Whole-cell lysates were probed for pERK and ERK, and Raf-1 immunoprecipitates were probed for Raf-1 and pS338 Raf-1. (B) Effect of activated PAK on Raf 338 phosphorylation. NIH 3T3 fibroblasts expressing Flag-Raf-1 and vector or constitutively active PAK1 (PAK165) were starved and treated as in panel A. Flag immunoprecipitates were probed with Raf-1 and pS338 antibodies. (C) Effect of activated PAK on ERK kinase activity. NIH 3T3 fibroblasts expressing HA-ERK and vector or PAK165 were assayed for adhesion-dependent HA-ERK activation. A representative blot of immunoprecipitated HA-ERK and an autoradiograph for 32P incorporation into MBP are shown. Bars represent means and standard errors of three experiments.
Loss of ERK activation parallels Raf-1 S338 phosphorylation in cells treated with forskolin and IBMX. (A) Effects of forskolin or cell suspension on phosphorylation of ERK, Raf-1, and CREB. Adherent NIH 3T3 fibroblasts were pretreated for 30 min with 100 μM IBMX and 0, 2, 10, or 50 μM forskolin and compared to cells placed in suspension for 15, 45, or 120 min. Cells were treated with 10 ng/ml EGF for 5 min as indicated. Whole-cell lysates were probed for ERK phosphorylation, and PKA activation was assessed with phospho-specific CREB antibodies. Raf-1 immunoprecipitates were assessed for pS338, pS259, and Raf-1. (B) Active PAK antagonizes the inhibitory effect of forskolin on ERK. NIH 3T3 fibroblasts expressing GFPERK, PAK165, and PKI as indicated were pretreated with 100 μM IBMX and 2, 10, or 50 μM forskolin for 30 min before EGF treatment. Whole-cell lysates were probed for pERK and ERK. (C) Effects of forskolin, active PAK, and cell suspension on phosphorylation of ERK and Raf-1. HEK293 cells were cotransfected with GFPERK, Flag-Raf-1 wt, and PAK165 as indicated. Adherent cells were pretreated for 30 min with DMSO (first two lanes) or 100 μM IBMX and 2, 10, or 50 μM forskolin. Adherent cells were compared to cells placed in suspension for 15 min or 2 h. Whole-cell lysates were probed for pERK and ERK. Flag (Raf-1) immunoprecipitates were probed for pS43, pS259, pS338, and Flag (Raf-1). 2b1af7f3a8