An overview of mammalian p38 mitogen-activated protein kinases, central regulators of cell stress and receptor signaling

The p38 family is a highly evolutionarily conserved group of mitogen-activated protein kinases (MAPKs) that is involved in and helps co-ordinate cellular responses to nearly all stressful stimuli. This review provides a succinct summary of multiple aspects of the biology, role, and substrates of the mammalian family of p38 kinases. Since p38 activity is implicated in inflammatory and other diseases, we also discuss the clinical implications and pharmaceutical approaches to inhibit p38.

p38 mitogen-activated protein kinases p38α (originally named p38) was identified and cloned as a 38 kDa protein that was tyrosine-phosphorylated in response to LPS stimulation in mammalian cells 1,2 . Sequence comparison, on the day p38α was cloned, revealed that it belonged to the mitogen-activated protein kinase (MAPK) family and that a Saccharomyces cerevisiae osmotic response protein kinase HOG1 was a p38α homologue [3][4][5] . p38α was also named cytokine suppressive drug binding protein (CSBP) because it was identified as the target of a series of anti-inflammatory pyridinyl-imidazole compounds and as reactivating kinase (RK) because it phosphorylated and activated MK2 3-5 . There are four members of the p38 group of MAPKs encoded by four different genes in mammals: p38α (MAPK14, chromosome 6p21.31 in humans), p38β (MAPK11, SAPK2b, Chr22q13.33) 6 , p38γ (MAPK12, ERK6, SAPK3, Chr22q13.33) 7,8 , and p38δ (MAPK13, SAPK4, Serk4, Chr6p21.31) 9,10 . As can be surmised from their chromosomal locations, MAPK14/p38α and MAPK13/ p38δ are physically close and separated by just over 15 kb, as are MAPK12/p38β and MAPK11/p38γ, which are separated by less than 2 kb. All the p38s contain a conserved Thr-Gly-Tyr (TGY) dual phosphorylation motif within the kinase activation loop, and both Thr and Tyr phosphorylation are necessary to fully activate the kinase 11 . However, monophosphorylated p38α Thr 180 has some kinase activity in vitro, but a different substrate specificity, when compared with dual-site phosphorylated p38α 12 . p38 group members are expressed ubiquitously, but p38γ and p38δ are enriched in certain cell types and tissues, such as p38γ in skeletal muscle and p38δ in the salivary, pituitary, and adrenal glands 13 . p38β shares more amino acid sequence identity with p38α (~70%), while p38γ and p38δ share ~60% identity with p38α. p38γ and p38δ also share high sequence homology with cyclin-dependent kinases (CDKs) and are sensitive to some CDK inhibitors 14 .

Activation and inactivation of p38
p38α is involved in the response to almost all stressful stimuli, including LPS, UV light, heat shock, osmotic shock, inflammatory cytokines, T cell receptor ligation, glucose starvation, and oncogene activation 2,4,5,[15][16][17][18][19][20] . Under certain circumstances, it is also activated upon growth factor stimulation. It should be noted that the activation of p38 in some cases is cell type specific, since an activating stimulus in one cell type may inhibit p38 in other cell types 21 . The study of p38 group members other than p38α has been less intensive; however, where it has been examined, the other p38s are frequently co-activated with p38α 22 .
Like other MAPK signaling pathways, the activation of all p38s is mediated by a kinase cascade: MAPKKK (MAP3K), which activates MAPKK (MAP2K), which in turn activates MAPK. The MAP2K kinases MKK3 and MKK6 are the major upstream kinases for p38 activation [23][24][25] . Although MKK3 and MKK6 phosphorylate most p38 isoforms in vitro, selective activation and substrate specificity have been observed in vivo 26 . MKK4 has also been reported to phosphorylate p38α and p38δ in specific cell types 9 . A number of MAP3Ks have been reported to participate in p38 activation including TAK1 27 , ASK1 28 ,DLK 29 ,and MEKK4 29,30 . Low-molecular-weight GTP-binding proteins in the Rho family, such as Rac1 and Cdc42, can activate p38 through binding to MEK1 or MLK1, which function as upstream activators of MAP3K 31,32 .
p38α can also be activated by MAP2K-independent mechanisms. TAB1 (TAK1-binding protein 1) directly interacts with p38α and can promote trans autophosphorylation on Thr 180 and Tyr 182 and thus full activation of p38α 33 . A subsequent study revealed that autophosphorylation of Thr 180 and Tyr 182 requires a conserved Thr 185 residue 34 . TAB1-dependent p38α activation has been implicated in ischemic myocardial injury and T cell anergy 35,36 . TAB1 is also claimed to play a role in Sestrin-mediated p38α activation 12 . Another MAP2K-independent activation is mediated by ZAP70 after T cell receptor ligation. ZAP70 can directly phosphorylate p38α/β on Tyr 32318 , leading to autophosphorylation on Thr 180 , one of the dual phosphorylation sites. As discussed, mono-Thr 180 phosphorylated p38 still has some kinase activity 37 , and loss of ZAP70-mediated p38 activation in p38αβ Y323F double knock-in mice reduces autoimmunity and inflammation in several autoimmune disease models [38][39][40] . Interestingly, p38α also phosphorylates ZAP70, resulting in a decrease in the size and persistence of the T cell receptor signaling complex, and therefore acts as a feedback regulator of ZAP70 41 .
Conversely, de-phosphorylation of both threonine and tyrosine residues in the activation loop inactivates MAPKs, and this is mainly carried out by dual-specificity phosphatases of the MAPK phosphatase (MKP)/dual specificity phosphatase (DUSP) family 42 . Although several MKPs have been reported to dephosphorylate p38α, MKP1/DUSP1, MKP5/DUSP10, MKP8/DUSP26, and DUSP8 are more potent inhibitors of p38α and JNK than ERK 43 . A recent report showed that DUSP12 is also a p38α phosphatase 44 . While there are a number of p38α DUSPs, no DUSP for p38γ or p38δ has been reported, and these two p38s are resistant to several known p38α MKPs such as MKP1, 3, 5, and 7 45 . p38α-dependent upregulation of MKP1 was reported and is believed to be part of a negative feedback loop of p38α activation 46 . Other types of phosphatases have also been reported to target p38 MAPKs, such as CacyBP/SIP 47 , Wip1 48 , and PP2C 49,50 . The substrate specificity between p38 and phosphatases and the related physiological functions in vivo still need further investigation. p38γ has also been reported to be degraded by a p38/JNK/ubiquitin-proteasome-dependent pathway, which represents an additional mechanism by which p38 kinases may cross regulate each other 51 . Yet other ways of regulating p38 are suggested from studies in Caenorhabditis elegans, where a genetic screen for resistance against bacterial infection identified RIOK-1, an atypical serine kinase and human RIO kinase homolog, as a suppressor of the p38 pathway 52 . As RIOK-1 is a transcriptional target of the p38 pathway in C. elegans, this suggests that RIOK-1 is part of a negative feedback loop. A brief summary of the p38 pathway is shown in Figure 1.

Downstream substrates of p38
Protein kinases The p38 MAPK cascade does not end at p38. Members of the MAPK-activated protein kinase (MAPKAPK) family such as MK2, MK3, and MK5 (PRAK) are all p38 substrates 3,4,53-55 . The MKs have a broad range of substrates that extend the range of functions regulated by p38 kinases. Mitogen-and stressactivated protein kinase-1/2 (MSK1/2), which are important for CREB activation and chromosome remodeling, have also been identified as substrates of p38α 56 . MNK1/2, kinases that phosphorylate the eukaryotic initiation factor-4e (eIF-4E), are phosphorylated by p38α 57,58 . p38α has also been reported to inactivate murine GSK3β by phosphorylating Ser 389 , and since GSK3β is required for the continuous degradation of β-catenin in the Wnt signaling pathway, this can lead to an accumulation of β-catenin 59,60 . It was also reported that p38δ negatively regulates insulin secretion by catalyzing an inhibitory phosphorylation of PKD1 61 . A number of p38 protein kinase substrates are summarized in Table 1.
Transcription factors p38 targets a large number of transcription factors, including myocyte-specific enhancer factor 2 (MEF2) family members, cyclic AMP-dependent transcription factor 1, 2, and 6 (ATF-1/2/6), CHOP (growth arrest and DNA damage inducible gene 153, or GADD153), p53, C/EBPβ, MITF1, DDIT3, ELK1/4, NFAT, and STAT1/4. p38 phosphorylation of transcription factors predominantly leads to enhanced transcriptional activity. However, in some cases, it represses transcription, and this is summarized in Table 2. Transcription factor phosphorylation by p38 is often stimulus and cell type dependent and plays a role in the cellular response to inflammation, DNA damage, metabolic stress, and many other stresses 62-76 . The effects of p38 on transcription seem to constitute the major part of p38's responses to stress stimuli.
in Table 3. The SWI-SNF complex subunit BAF60 is phosphorylated and inactivated by p38 during skeletal myogenesis 109,110 , and EZH2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2), was also found to be phosphorylated by p38, particularly in ER-negative breast cancer samples 111 . Besides its transcriptional function, dATF-2 is also involved in heterochromatin formation, and stress-induced phosphorylation of dATF-2 by p38 disrupts heterochromatin in Drosophila 112 .

Other substrates
Given the wide range of responses that p38 is involved in, it is not surprising that many p38 substrates cannot be so easily categorized into groups, and these miscellaneous substrates are summarized in Table 4. Some of them are involved in metabolism such as Raptor phosphorylation by p38β, which enhances mTORC1 activity in response to arsenite-stress 113 , and DEPTOR (mTOR-inhibitory protein) phosphorylation by p38γ and p38δ, leading to its degradation and mTOR hyperactivation 114 . p38α phosphorylation of Tip60 at Thr 158 promotes senescence and DNA-damage-induced apoptosis 115,116 . Some p38 substrates are cell death regulators. In the ER stress response, p38α locates to the lysosome and phosphorylates the chaperone-mediated autophagy (CMA) receptor LAMP2A, leading to activation of CMA and thus protecting cells from ER stress-induced death 117 .

Biological functions of the p38 pathway
Embryo development p38α is required for embryo development, since the mouse Mapk14 -/embryo dies between embryonic days (E) 10.5 and 12.5 118-121 . Mutant mice with a single Thr 180 to Ala mutation or with the double T180A Y182F mutation are also embryonic lethal 122,123 . Surprisingly, given the importance of the dual phosphorylation for complete p38 activation, substitution of Tyr 182 with Phe results in mice that have reduced p38 signaling but are nevertheless viable 123 , although this is consistent with previous studies showing that the p38 phosphorylated on Thr 180 alone retains some activity in vitro 37 . Histological analysis demonstrates that p38α is required for placental angiogenesis, but not embryonic cardiovascular development, and tetraploid rescue of the placental defect in Mapk14 -/embryos confirmed that p38α is  CDK, cyclin-dependent kinase; EGFR, epidermal growth factor receptor; FBP1, far upstream binding protein; FGF1, fibroblast growth factor 1; FGFR1, fibroblast growth factor receptor 1; FRS2, fibroblast growth factor receptor substrate 2; GDI, GDP dissociation inhibitor; KSRP, hnRNPK-homology type splicing regulatory protein; MAPK, mitogen-activated protein kinase; mTORC1, mammalian target of rapamycin complex 1; NADPH, nicotinamide adenine dinucleotide phosphate; NGF, nerve growth factor; NHE1, Na + /H + exchanger isoform 1; PHD3, prolyl hydroxylase 3; PLA2, phospholipase A2; SAP97, synapse-associated protein 97; TAB, transforming growth factor-β-activated protein kinase-1-binding protein; TACE, tumor necrosis factor-alpha-converting enzyme; TAK1, transforming growth factor β-activated kinase 1; TGF, transforming growth factor. essential for extraembryonic development 120,121 . Given the important role that p38 and MK2 plays in regulating TNF-induced cell death [179][180][181][182] , it is intriguing that the Mapk14 -/embryonic lethal phenotype is very similar to that observed in other mice with defects in the TNF death pathway. Caspase-8, FADD, and cFLIP knock-out mice also die at E10.5, and this is due to TNFdependent endothelial cell death and disruption of the vasculature in the yolk sac 183,184 . Other p38 isoforms are not necessary for embryo development, but p38α and p38β have overlapping functions, as Mapk14 loxp/loxp Mapk11 -/-Sox2-Cre embryos die before E16.5 with spina bifida that correlates with neural hyperproliferation and increased apoptosis in the liver, which was not observed in Mapk14 ∆/∆ Sox2-Cre embryos 185 . Remarkably, p38α appears to have a very specific function during embryogenesis because when p38α was replaced by p38β in the Mapk14 chromosomal locus, which thereby placed p38β under the control of the endogenous p38α promoter, it was unable to rescue the embryonic lethality induced by loss of p38α 185 .
Immune responses p38 is activated by many inflammatory stimuli, and its activity is important for inflammatory responses. Macrophage-specific deletion of Mapk14 inhibits inflammatory cytokine production and protects mice from CLP-induced sepsis 186 . p38α controls the production of inflammatory cytokines, such as TNF and IL-6, at many levels. It directly phosphorylates transcription factors, such as MEF2C 62,186 , and regulators of mRNA stability, such as hnRNPK-homology (KH) type splicing regulatory protein (KSRP) 187 . MEF2C appears to play an anti-inflammatory role in endothelial cells in vivo 188 . Via MK2/MK3, p38 also upregulates cytokine mRNA transcription by the serum response transcription factor (SRF) 189 , and similarly, via MK2/MK3, p38 regulates mRNA stability by phosphorylating and inactivating TTP/Zfp36, a protein that promotes rapid turnover of AU-rich mRNAs, many of which are cytokine mRNAs 187,190 . p38 activation also induces the expression of inflammatory mediators such as COX-2, MMP9, iNOS, and VCAM-1, which are involved in tissue remodeling and oxidation regulation [191][192][193][194] . The p38 pathway also regulates adaptive immunity. p38α participates in antigen processing in CD8 + cDCs 195 , and ZAP70-mediated p38α/β activation is important for T cell homeostasis and function 18 . In B cells, p38α is important for CD40-induced gene expression and proliferation of B cells 196 , and the p38α-MEF2c axis is believed to be necessary for germinal center B (GCB) cell proliferation and survival 197,198 . Excessive activation of p38α has been observed in many inflammatory diseases, such as inflammatory bowel disease (IBD), asthma, rheumatoid arthritis, and steatohepatitis [199][200][201] . The other members of the p38 family also play roles in immune responses. For example, p38γ and p38δ are required for neutrophil migration to damaged liver in non-alcoholic fatty liver disease 202 and inhibition of eukaryotic elongation factor 2 in LPS-induced liver damage 203 . p38δ is required for neutrophil accumulation in acute lung injury 204 . These observations, and the role that p38s play in TNF production, led to enormous pharmaceutical efforts to develop p38 inhibitors to treat chronic inflammatory diseases. However, unfortunately, these drugs were not efficacious in these diseases 205 .
Cell cycle p38 has been implicated in G1 and G2/M phases of the cell cycle in several studies. The addition of activated recombinant p38α caused mitotic arrest in vitro, and an inhibitor of p38α/β suppressed activation of the checkpoint by nocodazole in NIH3T3 cells 206 . G1 arrest caused by Cdc42 overexpression is also dependent on p38α in NIH3T3 cells 207 . Besides, p38γ is specially required for gamma-irradiation-induced G2 arrest 208 . The link between p38 and cell cycle control has been proposed through the regulation of several p38 substrates. Both p38α and p38γ regulate cell cycle progression via Rb but in opposite directions 14,209 . HBP1 represses the expression of cell cycle regulatory genes during cell cycle arrest in a p38-dependent manner 210 ; p53 and p21 activation by p38α prevented G1 progression through blockade of CDK activity 211,212 . The p38 pathway is also involved in cell cycle progress, as it is essential for self-renewal of mouse male germline stem cells 213 and its regulation of G1-length plays a role in cell size uniformity 214 .

Cell differentiation
Participation of p38 in cell differentiation has been reported in certain cell types. p38α activity is essential for neuronal differentiation in PC-12 cells and EPO-induced differentiation in SKT6 cells 20,215 . Treatment of 3T3-L1 fibroblasts with specific p38α/β inhibitors prevents their differentiation into adipocytes by reducing C/EBPβ phosphorylation 83 , and p38α-dependent phosphorylation of MEF2C and BAF60 is critical for myogenic differentiation 110,216 . Intestinal epithelial cell-specific deletion of p38α also influences goblet cell differentiation in a Notchdependent manner 200 .
Cell metabolism p38 group members participate in many cellular events related to metabolism. The p38β-PRAK axis specifically phosphorylates Rheb and suppresses mTORC1 activity under energy depletion conditions 22 . DEPTOR, an inhibitor of mTORC, can be phosphorylated by p38γ and p38δ, leading to its degradation 123 . Meanwhile, p38δ directly phosphorylated p62 to enhance mTORC1 activity in response to amino acids 175 . In brown adipocytes, p38α functions as a central mediator in β-adrenergic-induced UCP1 expression 217,218 , while in white adipocytes, p38α inactivation leads to elevated white-to-beige adipocyte reprogramming and resistance to diet-induced obesity 219,220 . In hepatocytes, p38α controls lipolysis and protects against nutritional steatohepatitis. Thus, mice with hepatocyte-specific loss of p38α developed more severe steatohepatitis than wild type mice when fed high-fat or -cholesterol diets. Intriguingly, macrophage specific deletion of p38 had the opposite effect in the same high-fat diets and resulted in less steatohepatitis than in wild type mice, which probably reflects the inflammatory role of p38 in macrophages 199 . p38α also directly phosphorylates Xbp1s to enhance its nuclear migration for maintaining glucose homeostasis in obesity 75 . However, p38α also functions as a negative regulator of AMPK signaling in maintaining gluconeogenesis, and hepatic p38α could be a drug target for hyperglycemia 221 . It was also reported that p38γ directly phosphorylated p62 under imidazole propionate stimulation to promote mTORC1 activity in hepatocytes 176 . Interestingly, AMPK also triggers the recruitment of p38α to scaffold protein TAB1 for p38α autoactivation in human T cells 222 .
Cell senescence p38α appears to play a pivotal role in senescence. Constitutive activation of the p38 pathway by active MKK3 or MKK6 induces senescence in several cell types 223,224 , and p38α activity is responsible for senescence induced by multiple stimuli, such as telomere shortening 225,226 , H 2 O 2 exposure 227,228 , and chronic oncogene activation 19,223,229 . p38α/β-specific inhibitors have been successfully used to prevent cellular senescence in cultivated human corneal endothelial cells 230 . Since cellular senescence is considered a defense strategy against oncogene activation, the p38 pathway plays important roles in tumorigenesis 231 . Meanwhile, p38α activity is important for senescence-associated secretory phenotype (SASP), and its inhibition markedly reduces the secretion of most SASP factors, suggesting multiple roles for the p38 pathway in senescence 232-235 .

Cell survival and death
The role of the p38 pathway in cell fate is cell type and stimulus dependent. For example, p38α becomes activated upon NGF withdrawal in PC-12 cells, and p38α activated by overexpression of MKK3 induced apoptosis in NGF differentiated PC-12 cells 211 . Similarly, inhibition of p38 with PD169316 blocked NGF withdrawal-induced apoptosis in PC-12 cells 236,237 . The interplay between the p38 pathway and caspases, the central regulators/executors of apoptosis, is complicated because p38α activity can be elevated in a caspase-dependent manner in death stimulus treated cells 238,239 , and caspase activity can also be elevated in MKK6E (dominant active form) overexpressed cells 239,240 . In contrast, inhibition of caspase-8 and caspase-3 by p38α-mediated phosphorylation in neutrophils was also reported 140 . Recent studies show that p38-activated MK2 directly phosphorylates RIPK1 in TNF-treated cells or pathogen-infected cells, limiting TNF-induced cell death 180-182 . This represents an interesting link between cytokine production induced by TNF and cell death because TNF-induced MK2/MK3 phosphorylation of tristetraprolin/Zfp36 inactivates it and leads to increased stability of cytokine mRNAs 190 . Aberrant p38α activity is observed in many tumor cells, and inhibition of p38α/β enhances cell death in these cells 241,242 .
Perspectives p38 is one of the most researched of all proteins, let alone kinases, and a search in PubMed for p38 MAPK or p38 kinase returns more than 36,000 publications, which is a higher number than some proteins listed in a review of the "top 10" most studied genes 243 . By contrast, searches for the kinases Raf and Src return about 17,000 and 25,000 hits, respectively. In 2018, there were more than 2,000 publications that mention p38, and it is clearly impractical to summarize such a vast amount of literature. As might be surmised from the preceding commentary, the studies are on a wide range of topics; however, the publications are more concentrated in some areas than others. The role of the p38 pathway in cancers (>10,000) 244-246 , inflammation (>8,000) 247-249 , and infections (>3,600) 250,251 was intensively studied. About 1,600 publications include the specific term "p38 inhibitor". This reflects the previously mentioned enormous interest of the pharmaceutical industry in developing p38 inhibitors to treat chronic inflammatory diseases, such as rheumatoid arthritis. Yet other publications report natural products that can activate or inhibit p38, with the ultimate aim of using them clinically 252-258 . In 2011, the European Commission approved Esbriet (pirfenidone), which was described as a p38γ inhibitor, for the treatment of idiopathic pulmonary fibrosis 259 . However, when this drug was approved by the FDA in 2014 for treating the same disease, it was described as a compound that acts on multiple pathways. In 2008, there were 27 clinical trials listed testing the use of p38 inhibitors in inflammatory disease settings 205 , while a search today for p38 inhibitors in clinicaltrials.gov returns 44 studies for conditions as diverse as pain, asthma, cognitive impairment, rheumatoid arthritis, cancer, myelodysplastic syndrome, and depression ( and that there is therefore a need for more specific inhibitors of each of the p38 group members and more basic research to fully understand how the pathway, especially how each member of the p38 family, is utilized and regulated.
One consequence of the massive pharmaceutical effort over the last 20 years is a large number of very specific, well-tolerated, and readily bioavailable drugs that can enable such basic research. For example, one study using a boutique panel of kinase inhibitors was able to demonstrate that 11 potent and specific p38 inhibitors synergized with Smac-mimetic drugs to kill a subset of AML leukemias, providing the strongest evidence implicating p38 in Smac-mimetic-induced killing 179 . Since several of these p38 inhibitors had already been clinically trialed, this presents an opportunity to fast-track such combinations into the clinic. In our opinion, it is likely that this is where the future of p38 research and p38 inhibitors lies, in revealing the intricate web of inter-connections and inter-dependencies of this core and central regulator of cell stress. We also believe that greater efforts to genetically assess the role of p38 and p38 isoforms in the pathophysiology of inflammatory and other diseases need to be made in order to push forward the clinical application of our burgeoning knowledge.