p38 MAPK signaling review

Mitogen-activated protein kinases (MAPK) such as p38 MAPK, JNK1/2 & ERK1/2 are a family of Ser/Thr kinases that phosphorylate a dynamic protein network which regulates cellular programs including proliferation, apoptosis, transcription, motility and metabolism [Cuevas et al., 2007]. MAPKs become activated following a range of diverse stimuli including growth factors, cytokines, drugs, cell adherence, UV light, oxygen free radicals, and in response to change in light and temperature [Chen et al., 2001].

In mammals, there are over a dozen members of the MAPK family. The MAPK cascade consists of a core of three protein kinases, and despite this seemingly minimalist cascade, they respond to and activate a wide range of signalling pathways. MAPKs are the terminal kinase in a phosphorylation signalling cascade. MAPKs are phosphorylated and activated by mitogen activated protein kinase kinases (MAPKKs), which in turn become phosphorylated and activated by mitogen activated protein kinase kinase kinases (MAPKKKs) [Cuevas et al., 2007].

Members of the MAPK signalling module have been intensively researched and defined over the past twenty years. In humans the MAPKKKs contain over 20 genes, followed by the MAPKs with eleven genes and finally the MAPKKs with seven genes [Uhlik et al., 2004]. The best known MAPK family members are the c-Jun N-terminal kinase (JNK 1-3), the extracellular signal related kinases 1 and 2 (ERK1/2) and p38 which regulate a diverse range of cellular functions [Pearson et al., 2001].

The first member of the p38 MAPK family was originally independently identified by four groups and was found to be a homologue of the S. cerevisiae protein,Hog1 [Cuadrado and Nebreda, 2010]. The p38 MAPK family contains four genes which include MAPK14 which encodes p38 , MAPK11 which encodes p38 , MAPK12 which encodes p38 and MAPK13 which encodes p38 Wagner and Nebreda, 2009.

p38 is activated following phosphorylation within the activation loop sequence Thr-Gly-Tyr. The dual-specificity MAPKKs phosphorylate p38 MAPKs following a range of stimuli. MAPKK6 can phosphorylate all four p38 MAPK family members,whereas MAPKK3 can phosphorylate p38 , with MAPKK4 only phosphorylating p38 [Cuadrado and Nebreda, 2010].

Activated p38 phosphorylates transcription factors such as p53, activating transcription factor 2 (ATF-2), myocyte specific enhancer 2 (MEF2) and also protein kinases including mitogen and stress activated protein kinase 1 (MSK1), MAP kinase-interacting Ser/Thr kinase 1 and 2 (MNK1/2) and MAPK activated kinase 2 (MAPKK/MK2) [Wagner and Nebreda, 2009]. The p38 MAPK promotes G1 arrest through the phosphorylation of cyclin D, resulting in its degradation and the induction of Cdk2/cyclin E inhibitor p21CIP1 [Densham et al., 2008; Todd et al., 2004]. Furthermore, Taxol treatment has been shown to activate JNK and p38 MAPK, resulting in downregulation of the reactive oxygen species (ROS) inhibitor, UCP2 (mitochondrial uncoupling protein 2) and activation of the AP-1 transcription factor complex proteins, ATF-2 and ELK1 [Selimovic et al., 2008].

Treatment of cells with the chemotherapeutic agent, sodium arsenite, results in the p38-dependent activation of the forkhead transcription factor, FOXO3a,resulting in the upregulation of BimEL [Cai and Xia, 2008]. Furthermore, p38 MAPK has also been shown to phosphorylate BimEL in response to arsenite treatment resulting in cell death [Cai et al., 2006].

The Ras-Raf-MEK-ERK/MAPK pathway is highly conserved in metazoans and controls many of the principal pathways involved in cell motility, metabolism, cell proliferation, differentiation and apoptosis [Kolch, 2005]. Initiation of the Ras-Raf-MEK/MAPK pathway can occur through receptor tyrosine kinase (RTK) activation. RTKs act as entry points for many extracellular cues as they contain an N-terminal extracellular ligand binding domain and C-terminal intracellular tyrosine kinase domain [McKay and Morrison, 2007; Pawson, 2002]. Upon binding of their respective ligands, RTKs undergo dimerisation and autophosphorylation of tyrosine residues within their C-terminal intracellular domain.

Activation of Ras GTPase is a key step allowing for signal transduction from RTKs to ERK1/2 MAPK. Ras GTPases are a superfamily of small monomeric GTPases which include NRAS, KRAS and HRAS [Downward, 2003]. One of the most intensively studied activators of Ras is the SOS (Son of sevenless homologue 1) guanine nucleotide exchange factor (GEF) [Downward, 1996].

GRB2 mediates the translocation of SOS from the cytosol to the plasma membrane upon RTK activation, resulting in the activation of Ras through GTP exchange. Activated Ras then binds to and recruits the Raf kinase to the cell membrane.

The RAF gene produces three isoforms (A-Raf, B-Raf and Raf-1), and each isoform has specific functions. The Raf kinase is the apical kinase in a three-tier phosphorylation activation cascade, where Raf phosphorylates and activates MEK1 and MEK2, which in turn phosphorylates activates and ERK1/2 [Vigil et al., 2010].

Once activated ERK1/2 phosphorylates a spectrum of substrates in both the cytosol and nucleus which include phosphatases, kinases,cytoskeletal proteins and transcription factors which play an integral role in cell death and cell proliferation [Yoon and Seger, 2006].Deregulation of the ERK pathway occurs in up to one-third of all cancers. Due to its anti-apoptotic and proliferative affects, ERK1/2 is a central player in the propagation of many forms of cancer. ERK becomes constitutively active following aberrant expression of tyrosine kinases and following sustained paracrine or autocrine signalling, and B-Raf or Ras mutations [Dhillon et al.,2007]. For example, malignant melanoma is a highly aggressive cancer, that is resistant to chemotherapy [Houghton and Polsky, 2002]. Approximately 60% of melanomas contain somatic B-Raf missense mutations which are found within the kinase domain of B-Raf. The V600E missense mutation accounts for 80% of all mutations [Davies et al., 2002]. The B-RafV600E mutant results in a dramatic elevation in its kinase activity leading to increased ERK1/2 kinase activity in vivo [Wan et al., 2004].

Constitutive activation of B-Raf V600E allows for chemo-resistance through the ERK1/2-dependent phosphorylation of the pro-apoptotic protein, Bim, resulting in its ubiquitin mediated degradation. Furthermore, ERK1/2 phosphorylates Bad preventing its release from 14-3-3 proteins, thus increasing the threshold required for cell death activation [Sheridan et al., 2008a].

The JNK MAPK proteins are encoded by three genes, MAPK8 encodes JNK1 protein, MAPK9 encodes JNK2 and MAPK10 encodes JNK3, which are also alternatively spliced and give rise to at least 10 different isoforms [Gupta et al., 1996]. Activation of JNK1/2 MAPK occurs following osmotic stress, UV irradiation, cytokine withdrawal, mitogenic signalling and MTA treatment [Manning and Davis, 2003; Shi et al., 2008].

Activation of JNK1/2 MAPKs occurs through the mixed-lineage kinases, which phosphorylate and activate the upstream MAPKKs such as MAPKK4 and MAPKK7, which in turn co-operatively phosphorylate and activate the JNK1/2 MAPKs. Activation of JNK1/2 MAPK can also occur through the MAPKKKs including, the MEK kinases (MEKKs), transforming growth factor (TGF) activated kinase 1 and apoptosis inducing kinase 1 (ASK1) [Gallo and Johnson, 2002].

Each JNK MAPK isoform can phosphorylate c-Jun, JunB, JunD and ATF2 which forms part of the AP-1 (activator protein-1) transcription factor complex [Davis,2000]. The AP-1 transcription factor complex targets a range of genes involved in cell cycle progression, cell survival and apoptosis [Wagner and Nebreda, 2009].

JNK was originally identified as having a role in apoptosis following the withdrawal of growth factors from PC12 pheochromocytoma cells [Xia et al.,1995]. Overexpression of the upstream JNK MAPK activator, MEKK1, results in sustained JNK activation and upregulation of the death receptor ligand FasL [Faris et al., 1998]. Gene disruption studies in mice have shown that JNK contributes to cell death initiation. In vivo JNK3 is required for cell death in hippocampal neurons following excitotoxic stress, whereas, JNK1 and JNK2 are required for thymocyte apoptosis following engagement of the T-cell receptor [Sabapathy et al., 1999; Yang et al., 1997]. JNK1/2 MAPK activation alone can stimulate cytochrome C release and apoptosis through the intrinsic cell death pathway; however, Bax and Bak are essential to JNK1/2 MAPK mediated apoptosis [Lei and Davis, 2003].

JNK and the p38 MAPK kinase are found to phosphorylate Bax following a rangeof cellular stresses resulting in the activation and translocation of Bax from the cytosol to the mitochondria and subsequent apoptosis [Kim et al., 2006].The activation of JNK1/2 MAPK varies with MTA and cell type [Shi et al., 2008]. JNK1/2 MAPK becomes activated within minutes following treatment of K562 cells with the MTA, pyrrolo-1,5-benzoxazepine (PBOX-6) [Mc Gee et al., 2002].However, activation of JNK1/2 MAPK occurs in a cell type specific manner upon Taxol treatment [Shi et al., 2008].

Overexpression of JNK has been shown to result in the phosphorylation of Bcl-2[Maundrell et al., 1997]. However, the requirement of JNK activity for the phosphorylation of Bcl-2 and Bcl-XL following MTA treatment has been a matter of debate. Small molecule inhibitors of JNK1/2 MAPK block the phosphorylation of Bcl-2 and Bcl-XL following Taxol treatment in ovarian and prostate cancer cells [Basu and Haldar, 2003; Brichese et al., 2004]. In contrast to this study Chambers and colleagues, illustrated that following vinblastine treatment the phosphorylation of Bcl-2 and Bcl-XL occurs independent of JNK1/2 MAPK activity, and that Cdk1 was the kinase responsible for the phosphorylation of Bcl-2 and Bcl-XL [Du et al., 2004; Terrano et al., 2010].

JNK1/2 MAPK has also been shown to phosphorylate the BH3-only proteins, Bim and Bmf [Lei and Davis, 2003]. JNK phosphorylates the BH3-only proteins, Bim and Bmf following UV irradiation. JNK1/2-dependent phosphorylation of BimL within its DLC1-binding motif results in the release of BimL from DLC-1 and initiation of cell death in a Bax/Bak dependent manner. Following UV irradiation JNK has also been shown to phosphorylate Bmf resulting in its release from the DLC2, a component of the myosin V motor complex [Lei and Davis, 2003].