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Upon activation, p38 MAPK alpha activates MK2 by phosphorylating Thr-222, Ser-272, and Thr-334 (Ben-Levy et al. 1995).
The phosphorylation of MK2 at Thr-334 attenuates the affinity of the binary complex MK2:p38 alpha by an order of magnitude and leads to a large conformational change of an autoinhibitory domain in MK2. This conformational change unmasks not only the MK2 substrate-binding site but also the MK2 nuclear export signal (NES) thus leading to the MK2:p38 alpha translocation from the nucleus to the cytoplasm. Cytoplasmic active MK2 then phosphorylates downstream targets such as the heat-shock protein HSP27 and tristetraprolin (TTP) (Meng et al. 2002, Lukas et al. 2004, White et al. 2007).
MAPKAPK (MAPK-activated protein) kinase 3 (MK3, also known as 3pK) has been identified as the second p38 MAPK-activated kinase that is stimulated by different stresses (McLaughlin et al. 1996; Sithanandam et al. 1996; reviewed in Gaestel 2006). MK3 shows 75% sequence identity to MK2 and, like MK2, is activated by p38 MAPK alpha and p38 MAPK beta. MK3 phosphorylates peptide substrates with kinetic constants similar to MK2 and phosphorylates the same serine residues in HSP27 at the same relative rates as MK2 (Clifton et al. 1996) indicating an identical phosphorylation-site consensus sequence. Hence, it is assumed that its substrate spectrum is either identical to or at least overlapping with MK2.
p38 MAPK alpha phosphorylates MK2 at Thr222, Ser272, and Thr334. The phosphorylation of Thr334 but not the kinase activity of MK2 has been demonstrated to be critical for the nuclear export of the p38 alpha - MK2 complex. Phosphorylation of Thr334 is believed to induce a conformational change in the complex exposing NES prior to interaction with the leptomycin B-sensitive nuclear export receptor.
The major cytosolic target of activated ERKs are RSKs (90 kDa Ribosomal protein S6 Kinase). Active RSKs translocates to the nucleus and phosphorylates such factors as c-Fos(on Ser362), SRF (Serum Response Factor) at Ser103, and CREB (Cyclic AMP Response Element-Binding protein) at Ser133. In the nucleus activated ERKs phosphorylate many other targets such as MSKs (Mitogen- and Stress-activated protein kinases), MNK (MAP interacting kinase) and Elk1 (on Serine383 and Serine389). ERK can directly phosphorylate CREB and also AP-1 components c-Jun and c-Fos. Another important target of ERK is NF-KappaB. Recent studies reveals that nuclear pore proteins are direct substrates for ERK (Kosako H et al, 2009). Other ERK nuclear targets include c-Myc, HSF1 (Heat-Shock Factor-1), STAT1/3 (Signal Transducer and Activator of Transcription-1/3), and many more transcription factors.
Activated p38 MAPK is able to phosphorylate a variety of substrates, including transcription factors STAT1, p53, ATF2 (Activating transcription factor 2), MEF2 (Myocyte enhancer factor-2), protein kinases MSK1, MNK, MAPKAPK2/3, death/survival molecules (Bcl2, caspases), and cell cycle control factors (cyclin D1).
JNK, once activated, phosphorylates a range of nuclear substrates, including transcription factors Jun, ATF, Elk1, p53, STAT1/3 and many other factors. JNK has also been shown to directly phosphorylate many nuclear hormone receptors. For example, peroxisome proliferator-activated receptor 1 (PPAR-1) and retinoic acid receptors RXR and RAR are substrates for JNK. Other JNK targets are heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Other adaptor and scaffold proteins have also been characterized as nonnuclear substrates of JNK.
MAP3K5 phosphorylates and activates MAP2K3 (MKK3) and MAP2K6 (MKK6) (Ichijo et al. 1997, Takekawa et al. 2005), which act as p38 MAPK kinases, as well as MAP2K4 (SEK1) (Ichijo et al. 1997, Matsuura et al. 2002), which, together with MAP2K7 (MKK7), acts as a JNK kinase.
MKK3 and MKK6 phosphorylate and activate p38 MAPK alpha (MAPK14) and beta (MAPK11) (Raingeaud et al. 1996), enabling p38 MAPKs to phosphorylate and activate MAPKAPK2 (MK2) and MAPKAPK3 (MK3) (Ben-Levy et al. 1995, Clifton et al. 1996, McLaughlin et al. 1996, Sithanandam et al. 1996, Meng et al. 2002, Lukas et al. 2004, White et al. 2007), as well as MAPKAPK5 (PRAK) (New et al. 1998 and 2003, Sun et al. 2007).
Phosphorylation of JNKs (MAPK8, MAPK9 and MAPK10) by MAP3K5-activated MAP2K4 (Deacon and Blank 1997, Fleming et al. 2000) allows JNKs to migrate to the nucleus (Mizukami et al. 1997) where they phosphorylate JUN. Phosphorylated JUN binds FOS phosphorylated by ERK1 or ERK2, downstream of activated RAS (Okazaki and Sagata 1995, Murphy et al. 2002), forming the activated protein 1 (AP-1) complex (FOS:JUN heterodimer) (Glover and Harrison 1995, Ainbinder et al. 1997).
Activation of p38 MAPKs and JNKs downstream of MAP3K5 (ASK1) ultimately converges on transcriptional regulation of CDKN2A locus. In dividing cells, nucleosomes bound to the CDKN2A locus are trimethylated on lysine residue 28 of histone H3 (HIST1H3A) by the Polycomb repressor complex 2 (PRC2), creating the H3K27Me3 (Me3K-28-HIST1H3A) mark (Bracken et al. 2007, Kotake et al. 2007). The expression of Polycomb constituents of PRC2 (Kuzmichev et al. 2002) - EZH2, EED and SUZ12 - and thereby formation of the PRC2, is positively regulated in growing cells by E2F1, E2F2 and E2F3 (Weinmann et al. 2001, Bracken et al. 2003). H3K27Me3 mark serves as a docking site for the Polycomb repressor complex 1 (PRC1) that contains BMI1 (PCGF4) and is therefore named PRC1.4, leading to the repression of transcription of p16INK4A and p14ARF from the CDKN2A locus, where PCR1.4 mediated repression of p14ARF transcription in humans may be context dependent (Voncken et al. 2005, Dietrich et al. 2007, Agherbi et al. 2009, Gao et al. 2012). MAPKAPK2 and MAPKAPK3, activated downstream of the MAP3K5-p38 MAPK cascade, phosphorylate BMI1 of the PRC1.4 complex, leading to dissociation of PRC1.4 complex from the CDKN2A locus and upregulation of p14ARF transcription (Voncken et al. 2005). AP-1 transcription factor, formed as a result of MAP3K5-JNK signaling, as well as RAS signaling, binds the promoter of KDM6B (JMJD3) gene and stimulates KDM6B expression. KDM6B is a histone demethylase that removes H3K27Me3 mark i.e. demethylates lysine K28 of HIST1H3A, thereby preventing PRC1.4 binding to the CDKN2A locus and allowing transcription of p16INK4A (Agger et al. 2009, Barradas et al. 2009, Lin et al. 2012).
p16INK4A inhibits phosphorylation-mediated inactivation of RB family members by CDK4 and CDK6, leading to cell cycle arrest (Serrano et al. 1993). p14ARF inhibits MDM2-mediated degradation of TP53 (p53) (Zhang et al. 1998), which also contributes to cell cycle arrest in cells undergoing oxidative stress. In addition, phosphorylation of TP53 by MAPKAPK5 (PRAK) activated downstream of MAP3K5-p38 MAPK signaling, activates TP53 and contributes to cellular senescence (Sun et al. 2007).