p38alpha antagonizes p38gamma activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating Ras transformation and stress response

p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.     

The stress kinase MRK contributes to regulation of DNA damage checkpoints through a p38gamma-independent pathway

DNA damage induced by ionizing radiation (IR) activates a complex cellular response that includes checkpoints leading to cell cycle arrest. The stress-activated mitogen-activated protein kinase (MAPK) p38gamma has been implicated in the G(2) phase checkpoint induced by IR. We recently discovered MRK as a member of the MAPK kinase kinase family that activates p38gamma. Here we investigated the role of MRK in the checkpoint response to IR. We identified autophosphorylation sites on MRK that are important for its kinase activity. A phosphospecific antibody that recognizes these sites showed that MRK is activated upon IR in a rapid and sustained manner. MRK depletion by RNA interference resulted in defective S and G(2) checkpoints induced by IR that were accompanied by reduced Chk2 phosphorylation and delayed Cdc25A degradation. We also showed that Chk2 is a substrate for MRK in vitro and is phosphorylated at Thr(68) by active MRK in cells. MRK depletion also increased sensitivity to the killing effects of IR. In addition, MRK depletion reduced IR-induced activation of p38gamma but had no effect on p38alpha activation, indicating that MRK is a specific activator of p38gamma after IR. Inhibition of p38gamma by RNA interference, however, did not impair IR-induced checkpoints. Thus, in response to IR MRK controls two independent pathways: the Chk2-Cdc25A pathway leading to cell cycle arrest and the p38gamma MAPK pathway.     

Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength

We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38alpha but not p38gamma. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38alpha by MKK6 correlated with more efficient binding of MKK6 to p38alpha than to p38gamma. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38alpha alone (low MKK6 activity) or both p38alpha and p38gamma (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.     

Novel regulation of p38gamma by dopamine D2 receptors during hypoxia

The p38 signalling pathway is part of the MAPK superfamily and is activated by various stressors. Our previous results have shown that two p38 isoforms, p38alpha and p38gamma, are activated by hypoxia in the neural-like PC12 cell line. PC12 cells also synthesize and secrete catecholamines, including dopamine, in response to hypoxia. We have now used this system to study the interaction between D2-dopamine receptor signalling and the p38 stress-activated protein kinases. Our results show that two D2 receptor antagonists, butaclamol and sulpiride, enhance hypoxia-induced phosphorylation of p38gamma, but not p38. This effect persists in protein kinase A (PKA)-deficient PC12 cells, demonstrating that p38gamma modulation by the D2 receptor is independent of the cAMP/PKA signalling system. We further show that removal of extracellular calcium blocks the hypoxia-induced increase in p38gamma activity. These results are the first to demonstrate that p38gamma can be regulated by the D2 receptor and calcium following hypoxic exposure.     

BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo

The compound BIRB796 inhibits the stress-activated protein kinases p38alpha and p38beta and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38gamma. This occurs at higher concentrations of BIRB796 than those that inhibit p38alpha and p38beta and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38gamma. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38alpha and p38beta, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38gamma as well as those of p38alpha and p38beta.     

Intrinsically active variants of all human p38 isoforms

The p38 mitogen-activated protein kinases are activated in response to various extracellular signals in eukaryotic cells and play a critical role in the cellular responses to these signals. The four mammalian isoforms (p38alpha, p38beta, p38gamma, and p38delta) are coexpressed and coactivated in the same cells. The exact role of each p38 isoform has not been entirely identified, in part due to the inability to activate each member individually. This could be resolved by the use of intrinsically active mutants. Based on previous studies on yeast p38/Hog1 [Bell M, Capone R, Pashtan I, Levitzki A & Engelberg D (2001) J Biol Chem276, 25351-2538] and human p38alpha[Diskin R, Askari N, Capone R, Engelberg D & Livnah O (2004) J Biol Chem279, 47040-47049] we have generated intrinsically active p38beta, p38gamma and p38delta mutants. In addition, we have identified a new activating mutation site in p38alpha. Most of the activating mutations are located in the L16 loop, in which conformational changes were shown to induce activation. We show that these changes impose substantial autophosphorylation activity, providing a mechanistic explanation for the intrinsic activity of the mutants. The new active variants maintain specificity towards substrates and inhibitors similar to that of the parental wild-type proteins, and are phosphorylated by mitogen-activated protein kinase kinase 6, their upstream activator. Thus, we have completed the development of a series of intrinsically active mutants of all p38 isoforms. These active variants could now become powerful tools for the elucidating the activation mechanism and specific biological roles of each p38 isoform.     

Balance between MKK6 and MKK3 mediates p38 MAPK associated resistance to cisplatin in NSCLC

The p38 MAPK signaling pathway has been proposed as a critical mediator of the therapeutic effect of several antitumor agents, including cisplatin. Here, we found that sensitivity to cisplatin, in a system of 7 non-small cell lung carcinoma derived cell lines, correlated with high levels of MKK6 and marked activation of p38 MAPK. However, knockdown of MKK6 modified neither the response to cisplatin nor the activation of p38 MAPK. Deeper studies showed that resistant cell lines also displayed higher basal levels of MKK3. Interestingly, MKK3 knockdown significantly decreased p38 phosphorylation upon cisplatin exposure and consequently reduced the response to the drug. Indeed, cisplatin poorly activated MKK3 in resistant cells, while in sensitive cell lines MKK3 showed the opposite pattern in response to the drug. Our data also demonstrate that the low levels of MKK6 expressed in resistant cell lines are the consequence of high basal activity of p38 MAPK mediated by the elevated levels of MKK3. This finding supports the existence of a regulatory mechanism between both MAPK kinases through their MAPK. Furthermore, our results were also mirrored in head and neck carcinoma derived cell lines, suggesting our observations boast a potential universal characteristic in cancer resistance of cisplatin. Altogether, our work provides evidence that MKK3 is the major determinant of p38 MAPK activation in response to cisplatin and, hence, the resistance associated with this MAPK. Therefore, these data suggest that the balance between both MKK3 and MKK6 could be a novel mechanism which explains the cellular response to cisplatin.     

Role for Lyn tyrosine kinase as a regulator of stress-activated protein kinase activity in response to DNA damage

The cellular response to DNA damage includes activation of the nuclear Lyn protein tyrosine kinase. Using cells deficient in Lyn expression, the present studies demonstrate that Lyn is required in part for induction of the stress-activated protein kinase (SAPK) in the response to 1-beta-D-arabinofuranosylcytosine (ara-C) and other genotoxic agents. By contrast, exposure of Lyn-deficient cells to ara-C, ionizing radiation, or cisplatin had no effect on activation of extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase. Similar findings were obtained in cells stably expressing a kinase-inactive, dominant-negative Lyn(K-R) mutant. Coexpression studies demonstrate that Lyn, but not Lyn(K-R), induces SAPK activity. In addition, the results demonstrate that Lyn activates SAPK by an MKK7-dependent, SEK1-independent mechanism. As MEKK1 functions upstream to MKK7 and SAPK, the finding that a dominant-negative MEKK1(K-M) mutant blocks Lyn-induced SAPK activity supports involvement of the MEKK1-->MKK7 pathway. The results also demonstrate that inhibition of Lyn-induced SAPK activity abrogates the apoptotic response of cells to genotoxic stress. These findings indicate that activation of SAPK by DNA damage is mediated in part by Lyn and that the Lyn-->MEKK1-->MKK7-->SAPK pathway is functional in the induction of apoptosis by genotoxic agents.     

The structure of phosphorylated p38gamma is monomeric and reveals a conserved activation-loop conformation.

BACKGROUND: Mitogen-activated protein (MAP) kinases mediate the cellular response to stimuli such as pro-inflammatory cytokines and environmental stress. P38gamma is a new member of the MAP kinase family, and is expressed at its highest levels in skeletal muscle. P38gamma is 63% identical in sequence to P38alpha. The structure of P38alpha MAP kinase has been determined in the apo, unphosphorylated, inactive form. The structures of apo unphosphorylated ERK2, a related MAP kinase, and apo phosphorylated ERK2 have also been determined. RESULTS: We have determined the structure of doubly phosphorylated P38gamma in complex with an ATP analog by X-ray crystallography. This is the first report of a structure of an activated kinase in the P38 subfamily, and the first bound to a nucleotide. P38gamma residue phosphoryl-Thr183 forms hydrogen bonds with five basic amino acids, and these interactions induce an interdomain rotation. The conformation of the activation loop of P38gamma is almost identical to that observed in the structure of activated ERK2. However, unlike ERK2, the crystal structure and solution studies indicate that activated P38gamma exists as a monomer. CONCLUSIONS: Interactions mediated by phosphoryl-Thr183 induce structural changes that direct the domains and active-site residues of P38gamma into a conformation consistent with catalytic activity. The conformation of the phosphorylation loop is likely to be similar in all activated MAP kinases, but not all activated MAP kinases form dimers.     

RACK1 and Stratifin Target ΔNp63α for a Proteasome Degradation in Head and Neck Squamous Cell Carcinoma Cells upon DNA Damage

P53 family members with a transactivation domain induce cell cycle arrest and promoteapoptosis. However, ΔNp63 isotypes lacking the transactivation (TA)- domain promote cellproliferation and tumorigenesis in vitro and in vivo. Although p53, TAp63 or TAp73 are stabilizedupon DNA damage, we found that the genotoxic stress agents induced a dramatic decrease andphosphorylation of ΔNp63α in squamous cell carcinoma cells. Further work revealed that RACK1physically associated with the p63α C-terminal domain through its WD40 domain. However,stratifin binds with phosphorylated ΔNp63α in response to cisplatin. Upon DNA damage inducedby cisplatin, stratifin mediated a nuclear export of ΔNp63α into cytoplasm and then RACK1targeted latter into a proteasome degradation pathway possibly serving as an E3 ubiquitin ligase.Moreover, siRNA knockdown of both stratifin and RACK1 inhibited a nuclear export and proteindegradation of ΔNp63α, respectively. Our data suggest that modification and down regulation ofΔNp63α is one of the major determinants of the cellular response to DNA damage in human headand neck cancers.