Mimicking Protein Kinase C Phosphorylation Inhibits Arc/Arg3.1 Palmitoylation and Its Interaction with Nucleic Acids.

Activity-regulated cytoskeleton-associated protein (Arc) plays essential roles in diverse forms of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity. In addition, it assembles into virus-like particles that may deliver mRNAs and/or other cargo between neurons and neighboring cells. Considering this broad range of activities, it is not surprising that Arc is subject to regulation by multiple types of post-translational modification, including phosphorylation, palmitoylation, SUMOylation, ubiquitylation, and acetylation. Here we explore the potential regulatory role of Arc phosphorylation by protein kinase C (PKC), which occurs on serines 84 and 90 within an α-helical segment in the N-terminal domain. To mimic the effect of PKC phosphorylation, we mutated the two serines to negatively charged glutamic acid. A consequence of introducing these phosphomimetic mutations is the almost complete inhibition of Arc palmitoylation, which occurs on nearby cysteines and contributes to synaptic weakening. The mutations also inhibit the binding of nucleic acids and destabilize high-order Arc oligomers. Thus, PKC phosphorylation of Arc may limit the full expression of LTD and may suppress the interneuronal transport of mRNAs.

ASCL1-ERK1/2 Axis: ASCL1 restrains ERK1/2 via the dual specificity phosphatase DUSP6 to promote survival of a subset of neuroendocrine lung cancers.

The transcription factor achaete-scute complex homolog 1 (ASCL1) is a lineage oncogene that is central for the growth and survival of small cell lung cancers (SCLC) and neuroendocrine non-small cell lung cancers (NSCLC-NE) that express it. Targeting ASCL1, or its downstream pathways, remains a challenge. However, a potential clue to overcoming this challenage has been information that SCLC and NSCLC-NE that express ASCL1 exhibit extremely low ERK1/2 activity, and efforts to increase ERK1/2 activity lead to inhibition of SCLC growth and surival. Of course, this is in dramatic contrast to the majority of NSCLCs where high activity of the ERK pathway plays a major role in cancer pathogenesis. A major knowledge gap is defining the mechanism(s) underlying the low ERK1/2 activity in SCLC, determining if ERK1/2 activity and ASCL1 function are inter-related, and if manipulating ERK1/2 activity provides a new therapeutic strategy for SCLC. We first found that expression of ERK signaling and ASCL1 have an inverse relationship in NE lung cancers: knocking down ASCL1 in SCLCs and NE-NSCLCs increased active ERK1/2, while inhibition of residual SCLC/NSCLC-NE ERK1/2 activity with a MEK inhibitor increased ASCL1 expression. To determine the effects of ERK activity on expression of other genes, we obtained RNA-seq from ASCL1-expressing lung tumor cells treated with an ERK pathway MEK inhibitor and identified down-regulated genes (such as SPRY4, ETV5, DUSP6, SPRED1) that potentially could influence SCLC/NSCLC-NE tumor cell survival. This led us to discover that genes regulated by MEK inhibition suppress ERK activation and CHIP-seq demonstrated these are bound by ASCL1. In addition, SPRY4, DUSP6, SPRED1 are known suppressors of the ERK1/2 pathway, while ETV5 regulates DUSP6. Survival of NE lung tumors was inhibited by activation of ERK1/2 and a subset of ASCL1-high NE lung tumors expressed DUSP6. Because the dual specificity phosphatase 6 (DUSP6) is an ERK1/2-selective phosphatase that inactivates these kinases and has a pharmacologic inhibitor, we focused mechanistic studies on DUSP6. These studies showed: Inhibition of DUSP6 increased active ERK1/2, which accumulated in the nucleus; pharmacologic and genetic inhibition of DUSP6 affected proliferation and survival of ASCL1-high NE lung cancers; and that knockout of DUSP6 "cured" some SCLCs while in others resistance rapidly developed indicating a bypass mechanism was activated. Thus, our findings fill this knowledge gap and indicate that combined expression of ASCL1, DUSP6 and low phospho-ERK1/2 identify some neuroendocrine lung cancers for which DUSP6 may be a therapeutic target.

Modulating p38 MAPK signaling by proteostasis mechanisms supports tissue integrity during growth and aging.

The conserved p38 MAPK family is activated by phosphorylation during stress responses and inactivated by phosphatases. C. elegans PMK-1 p38 MAPK initiates innate immune responses and blocks development when hyperactivated. Here we show that PMK-1 signaling is enhanced during early aging by modulating the stoichiometry of non-phospho-PMK-1 to promote tissue integrity and longevity. Loss of pmk-1 function accelerates progressive declines in neuronal integrity and lysosome function compromising longevity which has both cell autonomous and cell non-autonomous contributions. CED-3 caspase cleavage limits phosphorylated PMK-1. Enhancing p38 signaling with caspase cleavage-resistant PMK-1 protects lysosomal and neuronal integrity extending a youthful phase. PMK-1 works through a complex transcriptional program to regulate lysosome formation. During early aging, the absolute phospho-p38 amount is maintained but the reservoir of non-phospho-p38 diminishes to enhance signaling without hyperactivation. Our findings show that modulating the stoichiometry of non-phospho-p38 dynamically supports tissue-homeostasis during aging without hyper-activation of stress response.

ERK2 Mutations Affect Interactions, Localization, and Dimerization.

The most frequent ERK2 (MAPK1) mutation in cancers, E322K, lies in the common docking (CD) site, which binds short motifs made up of basic and hydrophobic residues present in the activators MEK1 (MAP2K1) and MEK2 (MAP2K2), in dual specificity phosphatases (DUSPs) that inactivate the kinases, and in many of their substrates. Also, part of the CD site, but mutated less often in cancers, is the preceding aspartate (D321N). These mutants were categorized as gain of function in a sensitized melanoma system. In Drosophila developmental assays, we found that the aspartate but not the glutamate mutant caused gain-of-function phenotypes. Here, we catalogued additional properties of these mutants to accrue greater insight into their functions. A modest increase in nuclear retention of E322K was noted. Binding of ERK2 E322K and D321N to a small group of substrates and regulatory proteins was similar, in spite of differences in CD site integrity. Interactions with a second docking site, the F site, which should be more accessible in E322K, were modestly reduced rather than increased. The crystal structure of ERK2 E322K also indicated a disturbed dimer interface, and reduced dimerization was detected by a two-hybrid test; yet, it was detected in dimers in EGF-treated cells, although to a lesser extent than D321N or wt ERK2. These findings indicate a range of small differences in behaviors that may contribute to increased function of E322K in certain cancers.

WNK1 collaborates with TGF-ß in endothelial cell junction turnover and angiogenesis.

Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-ß signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-ß-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-ß interactome. However, molecular events jointly regulated by TGF-ß and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-ß-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-ß-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-ß. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-ß-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-ß-regulated functions.

UBR5 is a novel regulator of WNK1 stability.

The with no lysine (K) 1 (WNK1) protein kinase maintains cellular ion homeostasis in many tissues through actions on ion cotransporters and channels. Increased accumulation of WNK1 protein leads to pseudohypoaldosteronism type II (PHAII), a form of familial hypertension. WNK1 can be degraded via its adaptor-dependent recruitment to the Cullin3-RBX1 E3 ligase complex by the ubiquitin-proteasome system. Disruption of this process also leads to disease. To determine if this is the primary mechanism of WNK1 turnover, we examined WNK1 protein stability and degradation by measuring its rate of decay after blockade of translation. Here, we show that WNK1 protein degradation exhibits atypical kinetics in HeLa cells. Consistent with this apparent complexity, we found that multiple degradative pathways can modulate cellular WNK1 protein amount. WNK1 protein is degraded by not only the proteasome but also the lysosome. Non-lysosomal cysteine proteases calpain and caspases also influence WNK1 degradation, as inhibitors of these proteases modestly increased WNK1 protein expression. Importantly, we discovered that the E3 ubiquitin ligase UBR5 interacts with WNK1 and its deficiency results in increased WNK1 protein. Our results further demonstrate that increased WNK1 in UBR5-depleted cells is attributable to reduced lysosomal degradation of WNK1 protein. Taken together, our findings provide insights into the multiplicity of degradative pathways involved in WNK1 turnover and uncover UBR5 as a previously unknown regulator of WNK1 protein stability that leads to lysosomal degradation of WNK1 protein.

WNK1 Enhances Migration and Invasion in Breast Cancer Models.

Metastasis is the major cause of mortality in patients with breast cancer. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase with no lysine (K) 1 (WNK1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here, we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype, and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.

Functional divergence caused by mutations in an energetic hotspot in ERK2.

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila, only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.

OSR1 regulates a subset of inward rectifier potassium channels via a binding motif variant.

The with-no-lysine (K) (WNK) signaling pathway to STE20/SPS1-related proline- and alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinase is an important mediator of cell volume and ion transport. SPAK and OSR1 associate with upstream kinases WNK 1-4, substrates, and other proteins through their C-terminal domains which interact with linear R-F-x-V/I sequence motifs. In this study we find that SPAK and OSR1 also interact with similar affinity with a motif variant, R-x-F-x-V/I. Eight of 16 human inward rectifier K+ channels have an R-x-F-x-V motif. We demonstrate that two of these channels, Kir2.1 and Kir2.3, are activated by OSR1, while Kir4.1, which does not contain the motif, is not sensitive to changes in OSR1 or WNK activity. Mutation of the motif prevents activation of Kir2.3 by OSR1. Both siRNA knockdown of OSR1 and chemical inhibition of WNK activity disrupt NaCl-induced plasma membrane localization of Kir2.3. Our results suggest a mechanism by which WNK-OSR1 enhance Kir2.1 and Kir2.3 channel activity by increasing their plasma membrane localization. Regulation of members of the inward rectifier K+ channel family adds functional and mechanistic insight into the physiological impact of the WNK pathway.

Amino Acids Regulate mTORC1 by an Obligate Two-step Mechanism.

The mechanistic target of rapamycin complex 1 (mTORC1) coordinates cell growth with its nutritional, hormonal, energy, and stress status. Amino acids are critical regulators of mTORC1 that permit other inputs to mTORC1 activity. However, the roles of individual amino acids and their interactions in mTORC1 activation are not well understood. Here we demonstrate that activation of mTORC1 by amino acids includes two discrete and separable steps: priming and activation. Sensitizing mTORC1 activation by priming amino acids is a prerequisite for subsequent stimulation of mTORC1 by activating amino acids. Priming is achieved by a group of amino acids that includes l-asparagine, l-glutamine, l-threonine, l-arginine, l-glycine, l-proline, l-serine, l-alanine, and l-glutamic acid. The group of activating amino acids is dominated by l-leucine but also includes l-methionine, l-isoleucine, and l-valine. l-Cysteine predominantly inhibits priming but not the activating step. Priming and activating steps differ in their requirements for amino acid concentration and duration of treatment. Priming and activating amino acids use mechanisms that are distinct both from each other and from growth factor signaling. Neither step requires intact tuberous sclerosis complex of proteins to activate mTORC1. Concerted action of priming and activating amino acids is required to localize mTORC1 to lysosomes and achieve its activation.

Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding.

The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183. ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligonucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site.

Isoxazole Alters Metabolites and Gene Expression, Decreasing Proliferation and Promoting a Neuroendocrine Phenotype in ß-Cells.

Novel strategies are needed to modulate ß-cell differentiation and function as potential ß-cell replacement or restorative therapies for diabetes. We previously demonstrated that small molecules based on the isoxazole scaffold drive neuroendocrine phenotypes. The nature of the effects of isoxazole compounds on ß-cells was incompletely defined. We find that isoxazole induces genes that support neuroendocrine and ß-cell phenotypes and suppresses genes important for proliferation. Isoxazole alters ß-cell metabolites and protects glucose-responsive signaling pathways under lipotoxic conditions. Finally, we show that isoxazole improves glycemia in a mouse model of ß-cell regeneration. Isoxazole is a prime candidate to alter cell fate in different contexts.

Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK.

The related protein kinases SPAK and OSR1 regulate ion homeostasis in part by phosphorylating cation cotransporter family members. The structure of the kinase domain of OSR1 was determined in the unphosphorylated inactive form and, like some other Ste20 kinases, exhibited a domain-swapped activation loop. To further probe the role of domain swapping in SPAK and OSR1, we have determined the crystal structures of SPAK 63-403 at 3.1 Å and SPAK 63-390 T243D at 2.5 Å resolution. These structures encompass the kinase domain and different portions of the C-terminal tail, the longer without and the shorter with an activating T243D point mutation. The structure of the T243D protein reveals significant conformational differences relative to unphosphorylated SPAK and OSR1 but also has some features of an inactive kinase. Both structures are domain-swapped dimers. Sequences involved in domain swapping were identified and mutated to create a SPAK monomeric mutant with kinase activity, indicating that monomeric forms are active. The monomeric mutant is activated by WNK1 but has reduced activity toward its substrate NKCC2, suggesting regulatory roles for domain swapping. The structure of partially active SPAK T243D is consistent with a multistage activation process in which phosphorylation induces a SPAK conformation that requires further remodeling to build the active structure.

Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals.

The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 and STE20/SPS1-related proline-, alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2 phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection of the PI3K pathway through mTORC2 to a Ste20 protein kinase and ion homeostasis.

Interactions with WNK (with no lysine) family members regulate oxidative stress response 1 and ion co-transporter activity.

Two of the four WNK (with no lysine (K)) protein kinases are associated with a heritable form of ion imbalance culminating in hypertension. WNK1 affects ion transport in part through activation of the closely related Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-, alanine-rich kinase (SPAK). Once activated by WNK1, OSR1 and SPAK phosphorylate and stimulate the sodium, potassium, two chloride co-transporters, NKCC1 and NKCC2, and also affect other related ion co-transporters. We find that WNK1 and OSR1 co-localize on cytoplasmic puncta in HeLa and other cell types. We show that the C-terminal region of WNK1 including a coiled coil is sufficient to localize the fragment in a manner similar to the full-length protein, but some other fragments lacking this region are mislocalized. Photobleaching experiments indicate that both hypertonic and hypotonic conditions reduce the mobility of GFP-WNK1 in cells. The four WNK family members can phosphorylate the activation loop of OSR1 to increase its activity with similar kinetic constants. C-terminal fragments of WNK1 that contain three RFXV interaction motifs can bind OSR1, block activation of OSR1 by sorbitol, and prevent the OSR1-induced enhancement of ion co-transporter activity in cells, further supporting the conclusion that association with WNK1 is required for OSR1 activation and function at least in some contexts. C-terminal WNK1 fragments can be phosphorylated by OSR1, suggesting that OSR1 catalyzes feedback phosphorylation of WNK1.

Serum and glucocorticoid-induced kinase (SGK) 1 and the epithelial sodium channel are regulated by multiple with no lysine (WNK) family members.

The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.

Radial spoke protein 3 is a mammalian protein kinase A-anchoring protein that binds ERK1/2.

Initially identified in Chlamydomonas, RSP3 (radial spoke protein 3) is 1 of more than 20 identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be characterized. We found mammalian RSP3 to bind to the MAPK ERK2 through a yeast two-hybrid screen designed to identify interacting proteins that have a higher affinity for the phosphorylated, active form of the protein kinase. Consistent with the screening result, the human homolog, RSPH3, interacts with and is a substrate for ERK1/2. Moreover, RSPH3 is a protein kinase A-anchoring protein (AKAP) that scaffolds the cAMP-dependent protein kinase holoenzyme. The binding of RSPH3 to the regulatory subunits of cAMP-dependent protein kinase, RIIalpha and RIIbeta, is regulated by ERK1/2 activity and phosphorylation. Here we describe an ERK1/2-interacting AKAP and suggest a mechanism by which cAMP-dependent protein kinase-AKAP binding can be modulated by the activity of other enzymes.

The roles of MAPKs in disease.

MAP kinases transduce signals that are involved in a multitude of cellular pathways and functions in response to a variety of ligands and cell stimuli. Aberrant or inappropriate functions of MAPKs have now been identified in diseases ranging from cancer to inflammatory disease to obesity and diabetes. In many cell types, the MAPKs ERK1/2 are linked to cell proliferation. ERK1/2 are thought to play a role in some cancers, because mutations in Ras and B-Raf, which can activate the ERK1/2 cascade, are found in many human tumors. Abnormal ERK1/2 signaling has also been found in polycystic kidney disease, and serious developmental disorders such as cardio-facio-cutaneous syndrome arise from mutations in components of the ERK1/2 cascade. ERK1/2 are essential in well-differentiated cells and have been linked to long-term potentiation in neurons and in maintenance of epithelial polarity. Additionally, ERK1/2 are important for insulin gene transcription in pancreatic beta cells, which produce insulin in response to increases in circulating glucose to permit efficient glucose utilization and storage in the organism. Nutrients and hormones that induce or repress insulin secretion activate and/or inhibit ERK1/2 in a manner that reflects the secretory demand on beta cells. Disturbances in this and other regulatory pathways may result in the contribution of ERK1/2 to the etiology of certain human disorders.

TAO kinases mediate activation of p38 in response to DNA damage.

Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Full-length and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM.