V-Raf murine sarcoma 3611 viral oncogene homolog aRaf²¹ interacts with small GTPase rRas, MAP2K2, MAP2K-like protein PDZ-binding kinase (PBK), casein kinase-2β, phosphoinositide 3-kinase regulatory subunit PI3K_r1, as well as 14-3-3γ, 14-3-3η, and 14-3-3σ, among others [251]. Hence, whereas bRaf and cRaf bind to all the 14-3-3 isoforms, aRaf links to some isoforms (14-3-3ε, -σ, and -τ [547]) in vitro and to a lesser degree. Enzyme aRaf transduces signals from mitogens and growth factors and activates MAP2K1.

Like other RAF family members, aRaf binds lipids to enable membrane connection, thereby regulating the kinase activity. Like cRaf, aRaf tethers to monophosphorylated phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) and phosphatidylinositol (3,5)-bisphosphate. In addition, aRaf also links specifically to PI(4,5)P₂ and PI(3,4)P₂ and to phosphatidic acid [547].

Like cRaf, 2 ARAF genes exist in the human genome, one of which is functional (ARAF1). The ARAF2 gene is a pseudogene [547]. Two alternative splice aRaf variants have been detected without kinase domain (but with the Ras-binding domain): daRaf1 and daRaf2. They bind to activated Ras, but act as antagonists of the Ras–ERK pathway.

Activity of aRaf is regulated by phosphorylation as well as lipidic and proteic interactions. Protein aRaf is able to form homo- and heterodimers as well as trimers with cRaf and bRaf. Heterodimer Raf has properties distinct from those of Raf monomer and homodimer, such as elevated kinase activity toward MAP2K.

Enzyme aRaf exhibits different effects according to the type of receptor Tyr kinase. It is recruited to EGFR upon EGF stimulation. However, it is constitutively associated with PDGFR.

Enzyme aRaf is weakly activated by small GTPase hRas and Src kinase with respect to bRaf and cRaf.²² Full aRaf activation needs Ras GTPase and other tyrosine kinases. Kinase aRaf may have the lowest kinase activity among Raf kinases for both MAP2K1 and MAP2K2 [547].

Casein kinase CK2β activates aRaf. Interleukin-3 and phosphoinositide 3-kinase contribute to aRaf activity. Kinase aRaf interacts with P85 regulatory subunit of PI3K (P85_PI3K) in the presence or not of growth factor [547]. It also regulates the transition from the inactive dimeric form to the active tetrameric form of pyruvate kinase-M2. Enzyme aRaf interacts with TOM and TIM proteins involved in the mitochondrial transport system. It also targets trihydrophobin-1, a component of the negative elongation factor complex that hinders transcriptional elongation by RNA polymerase-2. In addition, aRaf acts as a dynamic interactor of scaffold Kinase suppressor of Ras KSR2 [547]. Protein aRaf binds constitutively to pro-apoptotic kinase mammalian Sterile 20-like kinase STK3 (MST2) [547].

Other interactors include [251, 547]: argininosuccinate synthase ASS1,²³ Rab geranylgeranyltransferase β subunit, negative elongation factor proteins NElFc²⁴ that precludes transcriptional elongation by RNA polymerase-2, translocase of inner mitochondrial membrane homolog TIMM44, PRP6 pre-mRNA processing factor PRPF6 (or U5 small nuclear ribonucleoprotein particle-binding protein) that is involved in pre-mRNA splicing, nucleoside uridine diphosphate-linked moiety X-type motif NUDT14, lymphokine-activated killer T-cell-originated protein kinase (TOPK or PBK), COP9 signalosome complex subunit-3, mitochondrial carbamoyl-phosphate synthase CPS1, and EGF-containing fibulin-like extracellular matrix protein EFEMP1 (or fibulin-3).

V-Raf murine sarcoma viral oncogene homolog-B1 bRaf is also known as BRaf1, and RafB1. The Ras–Raf–ERK signal transduction pathway controls multiple processes, such as proliferation, differentiation, senescence, and apoptosis, depending on the duration and strength of the external stimulus and the cell type. The Ras–Raf–ERK pathway is activated in most human tumors, especially tumor cells with bRaf²⁵ or Ras gene mutations. When the MAPK cascade is inhibited, tumor growth is completely hindered in bRaf mutant and only partly in Ras-mutant tumors [554]. In addition, bRaf, but neither aRaf nor cRaf, contributes to vascular patterning in the placenta, although Raf kinases play redundant roles during initial stages of embryogenesis [556].

Kinase bRaf interacts with kinases PKB, ERK1, and cRaf, small GTases hRas and mRas, Rap1 GTPase-activating protein, Ras-related protein RaP1A, and 14-3-3β, -γ, -ε, -ζ, -σ, and -τ [251]. It mobilizes ERK1 and ERK2 via MAP2K1 and MAP2K2 and potentially ERK3.

Protein Ser/Thr kinase (MAP3K) cRaf, or Raf1 (v-Raf1 murine leukemia viral oncogene homolog), binds and operates downstream from membrane-associated Ras GTPase. Activated cRaf phosphorylates (activates) dual-specificity protein kinases MAP2K1 and MAP2K2 that, in turn, phosphorylate (activate) extracellular signal-regulated kinases ERK1 (MAPK3) and ERK2 (MAPK1 or MAPK2). The kinetics (sustained or transient activation) and intensity of Raf–ERK pathway stimulation by growth factors specify the signaling type. Kinase cRaf is a direct effector of Ras GTPase. Its activation involves plasmalemmal recruitment and binding to RasGTP. It is inactivated by protein phosphatase-5 (Sect. 8.3.5) [555].

Pleiotropic cRaf interacts with receptor PDGFRB, dihydrotestosterone receptor, nuclear receptor NR3c1, transcription factor Myc, G-protein subunits Gβ2 and Gγ4, monomeric GTPases Rap1a, eRas, hRas, kRas, mRas, nRas, rRas, RasD2, and RHEB, guanine nucleotide-exchange factor Vav1, arrestin-β2, kinases MAP3K5 (ASK1), MAP2K1, MAPK1 (ERK2), MAPK3 (ERK1), MAPK7 (ERK4), Mst4 (or MASK), bRaf, Src, Fyn, LCK, LTK, JaK1, JaK2, PAK1, PKG1, and PKCε, phosphatase CDC25A, phosphorylase kinase-α2, lipoprotein receptor-related protein associated protein LRPAP1, TNFR2–TRAF-signaling complex proteins Inhibitor of apoptosis proteins IAP2 and IAP3,²⁶ adaptor GRB10, interactor MAPK8IP3 (or JIP3), connectors enhancers of kinase suppressor of Ras CnKSR1 and CnKSR1, phosphatidylethanolamine-binding protein PEBP4, sprouty-2, transcriptional modulator prohibitin, transcriptional regulator TSC22-related protein TSC22D3, Soc2 suppressor of clear homolog SHOC2, chaperonin-containing TCP1 subunit CCT3, heat shock protein Hsp90α, heat shock protein-A-binding protein-2, voltage-dependent anion channel VDAC1 (porin), ubiquitin-conjugating enzyme UbE2d2, ubiquitin ligase CHIP (or STUB1), and 14-3-3β, -γ, -ζ, -η, and -τ [251].

Kinase cRaf is a regulator of apoptosis, as it can phosphorylate (inactivate) pro-apoptotic protein BCL2 antagonist of cell death (BAD) in addition to potentiating BCL2-mediated resistance to apoptosis. It connects to retinoblastoma proteins Rb1 and Rb2, B-cell lymphoma protein BCL2, BCL2-associated athanogene BAg1 that enhances BCL2 anti-apoptotic effect, BCL2-like protein BCL2l1, and caspase-like apoptosis regulatory protein ClARP (Caspase homolog [CasH], Casp8 and FADD-like apoptosis regulator [CFLAR], or usurpin-β) [251].

Mitogen-activated protein kinase kinase kinase-9 (MAP3K9)²⁷ contains an N-terminal glycine-rich region, an SH3 domain, a kinase domain similar to both Tyr and Ser/Thr kinases, 2 Leu/Iso (or Ile) zipper motifs, and a polybasic sequence similar to nuclear localization signals near the C-terminus [557, 558]. Kinase MLK1 requires autophosphorylation of its activation loop (Thr304, Thr305, Ser308, and Thr312) for full activity. It is predominantly produced in the central nervous system, kidney, spleen, stomach, and testis [559].

Kinase MLK1 phosphorylates dual-specificity kinase MAP2K4 to activate Jun N-terminal kinases in coordination with pro-apoptotic scaffold proteins such as Plenty of SH3 domains POSH1, especially for JNK-mediated neuronal apoptosis [559]. Scaffold POSH interacts with activated Rac to stimulate a subset of MLK family members, notably CDC42–Rac-interactive binding domain-containing MLK1 to MLK3. It forms a complex with MAP3K kinase (MLK1–MLK3 or MAP3K12), MAP2K (MAP2K4 or MAP2K7), and JNK1 or JNK2. It is inhibited by PKB. It operates as a pro-apoptotic protein in fibroblasts and mature neurons.

Enzyme MAP3K10²⁸ possesses a structure similar to that of MAP3K9, i.e., an SH3 sequence, a kinase motif that is associated with both Tyr and Ser/Thr specificity, a double Leu/Iso zipper region, and a basic domain similar to nuclear localization signals, as well as a large C-terminus. The production of MAP3K10 is relatively restricted, as it is predominantly detected in the central nervous system, kidney, skeletal muscle, and testis [560]. Highest expression level is observed in brain and skeletal muscles.

In neurons, huntingtin interacts with MAP3K9 and huntingtin-associated protein HAP1 that connect to neuron-specific basic helix–loop–helix transcription factor neurogenic differentiation protein NeuroD1 (or β-cell E-box transactivator β2).²⁹ Enzyme MAP3K9 phosphorylates (activates) NeuroD [561]. This effect is facilitated by huntingtin and huntingtin-associated HAP1 protein.

Other MAP3K10 interactors include MAP2K4, MAPK8IP1 and MAPK8IP2, CDC42, dynamin-1, kinesin-3A, kinesin-associated protein KiFAP3, clathrin heavy chain-1, Disc large homolog DLg4, keratin-associated protein-3 pseudogene product KrtAP3P1, human growth factor-regulated tyrosine kinase substrate (HGS or HRS), hippocalcin, and 14-3-3ε [251].

Active small GTPases CDC42 and Rac1 tether MAP3K10 and might then relieve its auto-inhibition. P21 (CKI1a)-activated kinase PAK1 binds MAP3K10 to partly control its capacity to activate JNK [560]. The Rho target and scaffold Connector enhancer of kinase suppressor of Ras CnKSR1 connects to MAP3K10 possibly for Rho activation of JNK kinase. Ubiquitin conjugase UbE2d3.2 expressed in a tissue-restricted fashion during development interacts with MAP3K10 kinase [560].

Although it can phosphorylate MAP2K3 and MAP2K6, MAP3K10 kinase is a weak activator of the P38MAPK and ERK pathways [560]. Its major substrates include MAP2K4 and MAP2K7. It is rather specific to the JNK pathway, operating as a JN3K kinase. Members of scaffold POSH and JIP families that can dimerize bind directly to MAP3K10 to facilitate the activation of the JNK pathway. Conversely, JNK2 phosphorylates MAP3K10 kinase.

MAP3K10 participates in the regulation of vesicular transfer. It localizes to clathrin-coated vesicles [560]. It binds to dynamin-1. In addition, like MAP3K9, MAP3K10 is involved in neuronal apoptosis.

Enzyme MAP3K11³⁰ is widespread. It has a structure identical to MLK1 and MLK2 enzymes. Enzyme MAP3K11 colocalizes with prenylated, activated monomeric GTPase CDC42 at the plasma membrane [562]. Its activation requires phosphorylation of multiple sites in the activation loop.

Enzyme MAP3K11 activates the JNK pathway in response to gonadotropin-releasing hormone, epidermal and platelet-derived growth factors, tumor-necrosis factor-α, transforming growth factor-β, T-cell receptor costimulation, and interferon-γ [562]. It contributes to cell proliferation, especially to microtubule organization during the cell division cycle.

Enzyme MAP3K11 can be phosphorylated (activated; Ser281) by MAP4K1 (or hematopoietic progenitor kinase HPK1) [560]. In addition to MAP3K1, MAP3K11 is, indeed, another effector for MAP4K1 that also interacts with adaptors GRB2, CRK, and CRKL for signaling, as they recruit MAP4K1 to autophosphorylated receptor Tyr kinases at the plasma membrane. Enzyme MAP3K11 can also tether directly to CDC42 [267]. Enzyme MAP4K2 (GCK) interacts directly with and activates MAP3K11 kinase [560].

Substrates of MAP3K11 include MAP2K1, MAP2K3, as well as MAP2K4 and MAP2K7 that activate ERK, P38MAPK, and JNK, respectively [562]. Type of scaffold proteins directs MAP3K11 signaling mode. MAPK-interacting proteins, such as JNK-interacting proteins JIP1 and JIP3 as well as SH3 domain-containing ring finger protein SH3RF1,³¹ promote MAP3K11-mediated JNK activation, whereas JIP2 enhances MAP3K11-induced P38MAPK stimulation [562]. In addition, MAP3K11 phosphorylates IκB kinases IKKα and -β.

Substrates of PKB comprise glycogen synthase kinase-α and -β, cRaf, bRaf, MAP3K5, MAP3K11, and MAP2K4, as well as Fox transcription factors. In addition, PKB1 isoform (but not PKB2) interacts with scaffold JNK-interacting protein JIP1 and prevents formation of JNK signaling complex. On the other hand, PKB2 targets SH3RF1 scaffold for the JNK signaling axis. This scaffold binds to activated Rac and is able to activate the JNK pathway and promote nuclear translocation of NFκB factor. Protein kinase-B2 binds to the SH3RF1–MAP3K11–MAP2K–JNK complex and phosphorylates MLK3, thereby causing complex disassembly and impeding the JNK pathway [564].³²

Small Rho GTPases, such as CDC42 and Rac, as well as MAP4Ks, such as MAP4K1 and MAP4K2, provoke MAP3K11 activity. Binding of a GTPase to the CDC42–Rac-interactive binding site (CRIB) disrupts auto-inhibition generated by the SH3 domain of MAP3K11 on its kinase motif. Ceramide also activates MAP3K11. Dimerization or oligomerization enhances MAP3K11 function. Conversely, MAP3K11 phosphorylation (Ser674) by PKB lowers its catalytic activity. Kinase-specific cochaperone Hsp90 regulates MAP3K11 stability.

Enzyme MAP3K12³³ activates the JNK (SAPK) pathway via MAP2K7 enzyme. It is involved in the regulation of apoptosis, cAMP response element-binding protein transcriptional activity, and terminal differentiation of keratinocytes [565]. It can form homodimers. Oligomerization-dependent autophosphorylation is required for its activation.

Unlike most of the MLKs that are widely expressed, MAP3K12 exhibits a more restricted pattern of expression. It activates JNK for neuronal migration during cerebral cortex development [556]. Scaffolds MAPK-binding inhibitory proteins MAPK8IP1 (or JNK-interacting protein JIP1) and MAP3K12IP1 (or MUK-binding inhibitory protein [MBIP]) regulate MAP3K12 activity by preventing its oligomerization and activation. Protein phosphatases PP1 and PP2A dephosphorylate MAP3K12.

Leucine zipper-bearing kinase (LZK), or MAP3K13, activates the Jun N-terminal kinase pathway, but not the ERK axis. It is detected in the human brain.

Enzyme MAP3K13 interacts with itself and MAP2K7, as well as mitogen-activated protein kinase-8 interacting protein MAPK8IP1 (or JNK-interacting protein JIP1) and peroxiredoxin-3 [251]. Enzyme MAP3K13 stimulates inhibitor of nuclear factor-κB kinase IKKβ, hence transcription factor NFκB. Anti-oxidant protein AOP1 that localizes to mitochondria binds to MAP3K13 to modulate its activity. Protein AOP1 enhances MAP3K13-induced NFκB activation [566].

Mixed lineage kinase-4 (MLK4) has several splice variants. Isoform MLK4α contains a different, shorter C-terminus than MLK4β subtype. Its subcellular compartment comprise the plasma membrane and cytoplasm.

Mixed lineage kinase-7 (MLK7)³⁴ is ubiquitous. In cells, it is located in the cytosol and nucleus [567]. Two splice variants exist: MLK7α and MLK7β.

In response to stress, both isoforms phosphorylate MAP2K4 and MAP2K7 to activate the JNK pathway as well as MAP2K3 and MAP2K6 to excite the P38MAPK axis [567]. Overexpression of MLK7α causes cell proliferation and transformation as well as degradation of stress fibers. In the heart, MLK7β induces cardiac hypertrophy and fibrosis. Enzyme MLK7α, but not MLK7β, phosphorylates histone-3 in response to epidermal growth factor cue. Enzyme MLK7β contributes to cell cycle arrest after DNA damage.

Enzyme MLK7α is phosphorylated by PAK1. Enzyme MLK7β phosphorylates checkpoint kinase ChK2 [567]. Osmotic shock and ionizing radiation cause MLK7β autophosphorylation (activation; Thr161 and Ser165).

Mitogen-activated protein kinase kinase kinase MAP3K1 is a ubiquitous Ser/Thr kinase that transduces many mitogenic and metabolic stimuli, such as insulin and growth factors, as well as pro-inflammatory cues. Enzyme MAP3K1 regulates diverse functions in a tissue- and cell type-specific manner. In most cases, MAP3K1 preferentially activates MAP2K4 and MAP2K7 that, in turn, stimulate Jun N-terminal kinases and/or P38MAPKs [568].³⁵ It can also activate extracellular signal-regulated kinases ERK1 and ERK2 via MAP2K1 and MA2PK2 enzymes. Besides, it can phosphorylate IκB kinases.

Protein MAP3K1 not only phosphorylates (activates) its effector kinases, but also has a ubiquitin ligase activity for ERK1 and ERK2 kinases. It also acts as a scaffold protein that binds to components (e.g., cRaf, MAP2K1, and ERK2) of the 3-tier ERK module as well as to the module activator Ras. It also uses additional scaffold proteins such as MAPK8IP3 to regulate the signaling kinetics and threshold.

Numerous input signals trigger MAP3K1 activation, such as osmotic stress, growth factors, and TNFSF proteins. Context-specific signaling of MAP3K1-based modules requires specific scaffold proteins to yield signaling routes with milestones made of proteic complexes.

WD repeat domain-containing protein WDR68³⁶ couples MAP3K1 to 2 protein Ser/Thr kinases: dual-specificity Tyr phosphorylation-regulated kinase DYRK1a and DYRK1b and homeodomain-interacting protein kinase HIPK2 [569].³⁷ Proteins MAP3K1 and DYRK1a suppress NFAT signaling, as NFAT is sequestered into the cytoplasm, thereby counteracting T-cell activation. Both MAP2K1 and HIPK2 target P300 acetyltransferase.³⁸ In addition, both MAP3K1 and HIPK2 can bind to axin adaptor.³⁹

It is a key MAP3K in the regulation of cell migration, especially that involved in vascular remodeling as well as epithelial cell movement primed by developmental factors FGF2, TGFβ, and activin B.

Enzyme MAP3K1 contributes to heart hypertrophy triggered by Gq subunit that is activated by angiotensin-2, endothelin, and adrenergic signals. Furthermore, it protects cardiomyocytes from oxidative stress [556].

Enzyme MAP3K1 participates in the modulation of immune responses. It favors B-cell proliferation via TNFRSF5-dependent stimulation of JNK and P38MAPK.⁴⁰ In T lymphocytes, MAP3K1 precludes T-cell-mediated autoimmune diseases.

Enzyme MAP3K1 is a prosurvival and anti-apoptotic factor in a cell-type- and stimulus-dependent manner, but when overexpressed, it can be pro-apoptotic [568]. In addition, MAP3K1 can phosphorylate transcription factors such as STAT3 and cofactors, such as transducer of regulated CREB activity TORC1 (but not TORC2) and P300. Enzyme MAP3K1 stimulates P300-dependent transcription; P300 mediates apoptosis.

Enzyme MAP3K1 interacts with MAP4K1 (HPK1), MAP4K2 (GCK), MAP4K4 (HGK or NIK), cRaf, MAP2K1, MAP2K4 (JNKK1), MAPK1 (ERK2), and MAPK8 (JNK1), as well as MAPK8IP3 [251]. Enzyme MAP3K1 is an effector for MAP4K1 that also interacts with adaptors GRB2, CRK, and CRKL for signaling, as they recruit MAP4K1 to autophosphorylated receptor Tyr kinases at the plasma membrane. Active GTPase CDC42^GTP binds and promotes MAP3K1 homodimerization and activation [267]. Other interactors include nuclear factor-κB inhibitor kinase IKKβ, morphogen disheveled-2, Wnt inhibitor axin-1, adaptor GRB2, RhoGAP4, NCK-interacting kinase (NIK), focal adhesion kinase, actin-crosslinking protein α-actinin, TNFR-associated factors TRAF2 and TRAF6, FK506-binding protein FKBP5, ribosomal proteins RPL4, RPL30, and RPS13, cell division cycle protein CDC37, a chaperone that complexes with HSP90 and many kinases (CDK4, CDK6, Src, cRaf, MOK,⁴¹ and eIF2αK), heat shock protein HSP90α, ubiquitin conjugase UbE2l, and 14-3-3ε [251]. Various intracellular molecules function as activators of MAP3K1, such as RhoA GTPase, filamin-B, which organizes interferon signaling, TRAF2, and glycogen synthase kinase GSK3β, whereas protein Ser/Thr kinase STK38 and multifunctional Parkinson disease protein Park7 of the peptidase C56 family sequester MAP3K1 and hinder MAP3K1 activity [568]. Receptor-interacting protein Ser/Thr kinase RIPK1 phosphorylates MAP3K1 enzyme.

Enzyme MAP3K1 can also interact with small GTPases CDC42, Rac1, and Ras. Activation of MAP3K1 by protein Tyr kinases requires the coordinated recruitment of MAP3K1 and its adaptors and activators to the plasma membrane. Activated EGFR bound to phosphorylated adaptor SHC can recruit: (1) the GRB2–MAP3K1 complex; (2) adaptors CRK or CRKL and MAP4K1; or (3) the Ras-activating GRB2–SoS complex [267]. On the other hand, EPH receptor recruits and activates MAP4K4 and NCK.

Enzyme MAP3K2 selectively activates JNK and ERK5 kinases. It also phosphorylates (activates) IκB kinases, thus initiating NFκB signaling. Enzyme MAP3K2 interacts with MAP2K4 (JNKK1), MAP2K5, MAP2K7 (JNKK2), and MAPK8 (JNK1), as well as T-lymphocyte specific adaptor SH2 domain-containing protein SH2D2a, apoptosis inhibitor IAP3,⁴² and 14-3-3ε and 14-3-3σ [251].

Enzyme MAP3K2 contributes to the maturation and function of cellular immunity, as it modulates T-cell receptor stimulation of the JNK pathway and favors cytokine production in response to IgE [556].

Ubiquitous MAP3K3 regulates ERK1 and ERK2, ERK5, JNK, and P38MAPK signaling as well as NFκB transcription factor. It intervenes in cardiovascular development via MAP2K5 and ERK5, especially in the maturation of primary vasculature and endocardium. In addition, activation of ERK1 and ERK2, JNK, P38MAPK, and NFκB signaling pathways is necessary for epithelial-to-mesenchymal transition, particularly during the formation of the cardiovascular and nervous system [556].

Enzyme MAP3K3 interacts with MAP2K5 as well as microtubule affinity-regulating kinase-2 (MARK2). Other interactors include GRB2-associated binding protein GAB1, transcription factor zinc finger and BTB domain-containing ZBTB16, TNFR-associated factor TRAF7, P53- and DNA-damage-regulated protein PDRG1, cell division cycle homolog CDC37, DNA-damage repair mediator breast cancer susceptibility protein BrCa1, JNK-associated scaffold protein sperm-associated antigen SpAg9, prefoldin-2, heat shock proteins HSP90α (HSPCal3) and HSPa4, and 14-3-3γ and 14-3-3ε [251].

Hypertonicity (e.g., high NaCl concentration)⁴³ activates the osmolarity-responsive NFAT5 transcription factor.⁴⁴ Small Rac1 GTPase indeed complexes with cerebral cavernous malformation-2 homolog (CCM2)⁴⁵ MAP3K3, and MAP2K3 that activates P38MAPK in response to hypertonicity. However, NFAT5 activation results from signaling via phospholipase-Cγ1 and the Rac1-CCM2 axis, but not the Rac1-CCM2-MAP3K3-MAP2K3-P38MAPK pathway [571].

Enzyme MAP3K4⁴⁶ activates JNKs and P38MAPKs, but not MAP2K1, in response to environmental stresses, such as osmotic shock, UV irradiation, wound stress, and inflammatory factors. It is a major activator of the P38MAPK pathway, but a minor effector of the JNK pathway. The JNK signalosome is a complex constituted by Jun N-terminal kinase and dual-specificity kinases MAP2K4 and MAP2K7 that can activate Jun transcription factor, a AP1 component.

Enzyme MAP3K4 contributes to the regulation of cell proliferation, apoptosis, and migration. It participates in neural, cardiac (valvulogenesis, chamber partitioning, and formation of the coronary vasculature), and skeletal development [556]. Wnt signaling can operate via MAP3K4. Angiotensin-2 provokes calcium-dependent phosphorylation by FAK2 of MAP3K4 that, in turn, activates MAP2K6 in smooth muscle cells. In addition, both MAP2K6 and calcium-regulated phosphatase calcineurin (PP2B or PP3) are involved in T-cell signaling and activation.

Enzyme MAP3K4 interacts with MAP2K1, MAP2K3, MAP2K4 (JNKK1), and MAP2K6, small GTPase CDC42 and Rac1, and growth arrest and DNA-damage-inducible GADD45α, -β, and -γ [251]. These 3 GADD45 polypeptides may contribute to activation of both P38MAPKs and JNKs via MAP3K4 enzyme. However, GADD45 homologs remains at low level when JNKs are fully activated during DNA damage [267].

Enzyme MAP3K5, or apoptosis signal-regulating kinase ASK1, functions in both the MAP2K4–JNK (or JN2K1–JNK) and MAP2K7–JNK (or JN2K2–JNK), as well as MAP2K3–P38MAPK (or SKK2–P38MAPK) and MAP2K6–P38MAPK (or SKK3–P38MAPK) cascades. It intervenes in apoptosis under various stress types, such as oxidative and endoplasmic reticulum stresses. These stresses causes MAP3K5 homo-oligomerization and autophosphorylation (Thr838). Apoptosis that depends on MAP3K5 is mainly mediated by mitochondria-dependent caspase activation (caspase-9 and -3) [572].

Enzyme MAP3K5 also mediates signaling for cell differentiation and survival, in particular, in neurons and keratinocytes [572]. It is also involved in inflammation and innate immunity. Production of inflammatory cytokines (e.g., TNFα, IL6, and IL1β) relies on MAP3K5 in splenocytes and bone marrow-derived dendritic cells.

Oligomerization of MAP3K5 is required for its activation by trans-autophosphorylation. Enzyme MAP3K5 is phosphorylated (activated; Thr838) [572]. In addition, MAP3K6 hetero-oligomerizes with MAP3K5 (ASK1–ASK2 complex) and directly phosphorylates MAP3K5 (Thr838). The MAP3K5–MAP3K6 heteromer stabilizes MAP3K6 and improves MAP3K6 efficiency for its subtrates in the JNK and P38MAPK modules. The MAP3K5–MAP3K6 complex causes apoptosis.

Enzyme MAP3K5 interacts not only with itself and MAP3K6, but also with MAP3K7 (TAK1). It then disrupts TRAF6–MAP3K7 interaction and inhibits IL1-induced NFκB activity [572]. It connects to MAP2K6 and MAP2K7 [251]. It is also linked to MAPK8IP3 (or JNK-interacting protein JIP3), thioredoxin, glutaredoxin, TNFR-associated factors TRAF1 to TRAF3, TRAF5 and TRAF6, arrestin-β2, glutaminyl-tRNA synthase, glutathione S-transferase-M1, dual-specificity phosphatase DuSP19, cell division cycle CDC25a phosphatase, cRaf, growth arrest and DNA-damage-inducible protein GADD45β, cyclin-dependent kinase inhibitor CKI1a, programmed cell death PCD6,⁴⁷ endoplasmic reticulum-to-nucleus signaling ERN1, and 14-3-3ζ protein, among others (Table 6.11).

Various factors regulate MAP3K5 oligomerization. Both TRAF2 and TRAF6 as well as adaptor death-associated protein DAP6,⁴⁸ facilitate MAP3K5 oligomerization and activation. On the other hand, Vacuolar protein sorting-associated protein-28 homolog (VPS28)⁴⁹ is an anti-apoptotic protein that suppresses MAP3K5 oligomerization [572].

Conversely, MAP3K5 is inactivated by phosphorylation at Ser83, Ser966, and Ser1033 in humans [572]. Protein kinase-B phosphorylates (inactivates) MAP3K5 (Ser83). Phosphoinositide-dependent protein kinase PDK1 also phosphorylates (inhibits) MAP3K5 (Ser966). The receptor Tyr kinase insulin-like growth factor receptor also phosphorylates (inactivates) MAP3K5. In addition, 14-3-3 protein prevents MAP3K5 activity, as it binds to a phosphorylated Ser residue (Ser966) in the 14-3-3-binding motif of MAP3K5 agent [572].

On the other hand, protein kinase-D and Disabled homolog 2-interacting protein (Dab2IP)⁵⁰ foster the release of 14-3-3 inhibitor from MAP3K5 enzyme. Homeodomain-interacting protein kinase HIPK1 is an MAP3K5–Dab2IP complex-associated protein that activates MAP3K5, as it dissociates the inhibitors 14-3-3 and thioredoxin from MAP3K5. Kinase HIPK1 assists activation of the MAP3K5–JNK axis by TNFα and ROS agents [459].

In addition, Ca–calmodulin-dependent protein kinase CamK2 phosphorylates (activates) MAP3K5 in neurons [573]. Ste20-like kinase (SLK) interacts with and activates MAP3K5 and provokes apoptosis [572].

Phosphatase PP2 supports MAP3K5 activity, as it dephosphorylates an inactivating phosphorylation site (Ser966) and dissociates 14-3-3 from MAP3K5 enzyme [572]. Calcium–calmodulin-activated protein phosphatase PP3 also activates MAP3K5, as it promotes its release from 14-3-3 proteins in cardiomyocytes. Moreover, PP3 dephosphorylates the inactivating phosphorylation site of MAP3K5, thereby exciting its activity. On the other hand, protein Ser/Thr phosphatase PP5 inhibits MAP3K5, as it dephosphorylates an activating phosphorylation site (negative feedback).

Phosphatase CDC25a inhibits MAP3K5 independently of its phosphatase activity, as it impedes MAP3K5 oligomerization [572]. Dual-specificity phosphatase DuSP19⁵¹ enhances MAP3K5 signaling, as it acts as a scaffold for this kinase.

Activation of the MAP3K5–P38MAPK pathway upon stimulation by reactive oxygen species is required for extracellular ATP-induced apoptosis in macrophages, which is initiated by the ATP-ligated P2X₇ receptor, an ATP-gated ionotropic channel [572].

Adaptors TRAF2 and TRAF6, which link plasmalemmal receptors and kinases, are recruited to the ROS-stimulated MAP3K5 signalosome and activate MAP3K5 (full MAP3K5 activation), after dissociation of thioredoxin from inactivated MAP3K5, the MAP3K5 signalosome enlarging with respect to its resting state [572].

Redox sensing oxidoreductase thioredoxin, only in its reduced state, inhibits MAP3K5, as it sequesters MAP3K5 away from TRAF2 until TRAF2-dependent production of reactive oxygen species provokes the conversion of thioredoxin to its oxidative form, hence the dissociation of the MAP3K5–thioredoxin complex [267]. Interaction between MAP3K5 and TRAF6 is also prevented by thioredoxin.

Calcium- and integrin-binding protein-1 (CIB1)⁵² that interacts with α_2B integrin in particular, binds to MAP3K5, interferes with the recruitment of TRAF2 to MAP3K5, and inhibits MAP3K5 autophosphorylation, thereby repressing MAP3K5 activation [573]. Inhibitor CIB1 thus competes with TRAF2 to connect to MAP3K5 kinase. Calcium influx relieves inhibition of CIB1 on MAP3K5 kinase.

Retinoblastoma RB1-inducible coiled-coil protein RB1CC1,⁵³ a widespread inhibitor of FAK1 and FAK2 and regulator of the retinoblastoma gene⁵⁴ contributes to the regulation of cell proliferation and migration as well as regulates TRAF2–MAP3K5 interaction [575].⁵⁵ Eukaryotic translation initiation factor-2α kinase-2,⁵⁶ an element of the antiviral response, links to MAP3K5 to activate the JNK and P38MAPK pathways [576].

Enzyme MAP3K5 is also regulated by ubiquitination. Suppressor of cytokine signaling SOCS1 constitutively binds to MAP3K5 and causes its ubiquitination and degradation in resting cells [576]. Cellular inhibitor of apoptosis protein IAP1⁵⁷ is a ubiquitin–protein ligase for MAP3K5 that can terminate TNFα-induced MAPK activation (negative feedback).

Balance between cell survival and death relies on activation of the PI3K–PKB and MAP3K5–JNK pathways. Activated MAP3K5 triggers cell death in response to diverse apoptotic stimuli, such as TNFα or reactive oxygen species. Scaffold protein Disabled homolog Dab2-interacting protein (Dab2IP), a Ras-GTPase activating protein (RasGAP)⁵⁸ causes cell cycle arrest and promotes apoptosis in response to stress. It actually suppresses the PI3K–PKB pathway and enhances MAP3K5 activation to impede cell survival and provoke cell death [577].⁵⁹

Enzyme MAP3K5 interacts with cell cycle regulators. Cyclin-dependent kinase inhibitor CKI1a links to MAP3K5 and inhibits the MAP3K5 signaling [572].

Several scaffold proteins, such as MAPK8IP3 and β-arrestin, interact with MAP3K5 to facilitate its signaling [572]. The scaffold Gem (nuclear Gemini of Cajal bodies)-associated protein Gemin-5, a component of the survival of motor neurons (SMN) complex,⁶⁰ interacts with ASK1 and potentiates activation of the MAP3K5–MAP2K4–JNK1 pathway. Parkinson disease protein Park7 tethers to and inhibits MAP3K5 enzyme.

Enzyme MAP3K5 phosphorylates Programmed cell death PDCD6,⁶¹ a member of the pentaEF hand Ca-binding protein family; this phosphorylation is counteracted by cRaf [572].

Enzyme MAP3K5 contributes to cardiogenesis, as cardiac chamber partitioning and valve formation require appropriate timing of apoptosis, as well as left ventricular remodeling by inducing apoptosis after myocardial infarct via the P38MAPK and JNK pathways [556].

Enzyme MAP3K6 interacts with MAP3K5 (ASK1). Alone, MAP3K6 weakly activates JNKs and does not stimulate ERKs and P38MAPKs [578]. Its kinase activity happens only in association with MAP3K5 enzyme. The latter may confer kinase activity to MAP3K6; it may allosterically activate MAP3K6, hence causing its autophosphorylation (Thr806). Activation of MAP3K6 does not depend on MAP3K5 kinase activity. In the presence of MAP2K5, MAP3K6 phosphorylates (activates) MAP2K4 and MAP2K6, thereby priming the MAP2K4–JNK and MAP2K6–P38MAPK pathways, to the same extent as MAP3K5 alone [578]. Reactive oxygen species leads to MAP3K6 autophosphorylation (Thr806) [578].

In addition to its heteromerization with MAP3K5, MAP3K6 can homodimerize. This homodimer may enable MAP3K6 trans-autophosphorylation. Protein 14-3-3 is a MAP3K6-interacting partner; this connection is reversibly regulated by phosphorylation.

Enzyme MAP3K6 regulates the expression of vascular endothelial growth factor under both normoxia and hypoxia in vitro [579]. Consequently, MAP3K6 favors tumor angiogenesis and tissue response to ischemia.

Extracellular signal-regulated kinases ERK1 and ERK2 are expressed in many tissues. They are activated by growth factors to regulate cell adhesion, growth, proliferation, differentiation, migration (during both chemotaxis and haptotaxis), and apoptosis. In particular, activated ERK1 and ERK2 regulates membrane protrusions and focal adhesion turnover by phosphorylating (activating) myosin light-chain kinase and calpain. They also regulate focal adhesion dynamics by influencing the interaction between paxillin and focal adhesion kinase. Unlike adhesion signaling, growth factors activate both MAP2K1 and -2.

The corresponding multipotent pathway achieves response specificity especially owing to interactions with scaffold proteins. Scaffolds include kinase repressor of Ras, β-arrestin, IL17Rd, and IQGAP1 proteins. Molecule KSR produces a docking platform at the plasma membrane. Agent IQGAP1 links the MAP2K–ERK pathway to the cytoskeleton.

In unstimulated cells, scaffold protein KSR1 prevents improper ERK cascade activation by sequestering MAP2K away from Raf kinase. In response to growth factors, KSR1 that localizes to the plasma membrane contributes to the spatiotemporal control of the Raf–MAP2K–ERK signaling. Both MAP2K1 and MAP2K2 kinases possess a KSR-binding motif that allows the formation of the bRaf–MAP2K–KSR ternary complex [592]. Docking of active ERK to KSR1 scaffold allows ERK to phosphorylate KSR1 and bRaf, hence ensuring a feedback control. Phosphorylation of KSR1 and bRaf actually promotes their dissociation and causes KSR1 release from the plasma membrane. Therefore, KSR1 first potentiates signal transmission, as it colocalizes cascade components to facilitate Raf–MAP2K interaction, thereby increasing efficiency and specificity of signaling, and then attenuates ERK activation, thereby controlling intensity and duration of ERK signaling.

The Ras–Raf–MAP2K–ERK signaling cascade triggered by growth factors and intercellular contacts controlled by IQGAP family proteins controls cell proliferation. Protein IQGAP3 is specifically expressed by proliferating cells to cooperate with Ras GTPase in the cell cycle [593].

Phosphoprotein-enriched in astrocytes PEA15 binds ERK and RSK2 for sequestration in the cytoplasm. Protein RSK2 is an ERK substrate kinase that can activate CREB-mediated transcription. Protein PEA15 independently binds ERK and RSK2 and enhances ERK binding to and phosphorylation (activation) of RSK2 in a concentration-dependent manner [594].

Endosome location of the MAP2K1–ERK pathway is required for full activation of ERK. Adaptor LAMTOR1⁹⁹ anchored to membrane rafts of late endosome connects late endosomes to the MAP2K1–ERK pathway via the LAMTOR2–MP1 complex that serves as a scaffold for MAP2K1 [595].¹⁰⁰ LAMTOR1 is required for vesicle dynamics that comprise Rab11-mediated endosome recycling and lysosome processing, thus participating in the regulation of vesicular interactions and material transport along the cytoskeleton.

Enzymes of the MAP3K set in the ERK1 and ERK2 cascades¹⁰¹ are usually members of the RAF family (aRaf–cRaf) that bind to Ras GTPase to be activated. Upstream activators bind to receptor Tyr kinases or G-protein-coupled receptors. Activation of the ERK pathway can result from stimulation of plasmalemmal proteins Ras via receptor Tyr kinases in association with GRB2 adaptor and Son of sevenless, a guanine nucleotide-exchange factor. Activation of Ras triggers Raf recruitment to the plasma membrane and stimulation, phosphorylation (activation) of MAP2K1 and/or MAP2K2, and then activation of ERK1 and ERK2 kinases. Enzymes ERK1 and ERK2 have many targets, such as transcription factors (NFκB, Jun, and ELk1), kinases (MAPKAPK1 and MAPKAPK2), plasmalemmal receptors (EGFR), upstream MAPK tier activators (cRaf), SOS affector, and paxillin.

Recruitment of scaffolds and adaptors, phosphorylation, and dephosphorylation yields temporospatial regulation of the ERK pathway. Upon activation by growth factors and other stimulators such as urokinase plasminogen activator of the Ras–Raf–MAP2K1/2–ERK1/2 cascade, Enzymes MAP2K1 and MAP2K2 are phosphorylated by kinase Raf (Ser218 and Ser222 of MAP2K1 and Ser222 and Ser226 of MAP2K2) and phosphorylate substrates ERK1 and ERK2 (on specific tyrosine and threonine residues). The Ras–Raf–MAP2K1/2–ERK1/2 cascade initiates not only a rapid signal amplification, but also a negative feedback loop via MAP2K1/2 heterodimer formation and ERK-mediated phosphorylation of MAP2K1 [597]. Agent MAP2K1 restricts Raf-induced MAP2K2 phosphorylation. Enzyme ERK can also exert negative feedback on several upstream components of the pathway, such as SOS, PAK, cRaf, and bRaf.

Homo- and heterodimerization are common events in ERK signaling. In particular, bRaf and cRaf can form heterodimers with stronger kinase activity than that of monomers or homodimers [598]. Heterodimerization is enhanced by proteins 14-3-3 and by mitogens independently of ERK. Phosphorylation by ERK of bRaf promotes Raf heterodimer disassembly.

Nearly half of ERK1 and ERK2 are bound to microtubules with a role in polymerization dynamics. Kinases ERK1 and ERK2 also localize to adherens junctions (intercellular contact) and focal adhesions (cell–matrix contact), thereby operating in cell motility. Enzymes ERK1 and ERK2 also reside in the nucleus. In the cytoplasm, they can phosphorylate certain transcription factors prior to nuclear entry. Proteins that alter the subcellular localization of ERK1 and ERK2 can generate diseases [599].

Protein kinase-C can activate cRaf, and hence plays a role at the MAP4K stage. In endothelial cells, activation of ERK1 and ERK2 that depends on shear stress¹⁰² relies on PKCε [600].¹⁰³ Affector cRaf, an ERK1/2 activator, is recruited to the plasma membrane, and activated by Ras, possibly phosphorylated by CSK on the one hand and by diacylglycerol-regulated PKCα and PKCε on the other hand [601].

Protein kinases contains a site aimed at selectively binding nucleotides and inhibitors. Inhibitors prevent autophosphorylation. A gatekeeper residue in ERK2 impedes auto-activation [603].¹⁰⁴

Urocortin (Ucn), a member of the corticotropin-releasing factor family and its homologs urocortin-3, or stresscopin (Scp), and urocortin-2, or stresscopin-related peptide (SRP), are potent cardioprotectors in cells exposed to hypoxia–reoxygenation events. Whereas urocortin binds to both types of CRF G-protein-coupled receptors, CRFR1 and CRFR2, urocortin-2 and -3 tether exclusively and with high affinity to CRFR2. However, all 3 peptides activate the ERK1/2 and PKB pathways [604].

The signaling adaptor sequestosome-1 regulates osteoclastogenesis via ubiquitin ligase TRAF6 and activation of nuclear factor-κB. It also promotes Ras-induced cell transformation. Moreover, sequestosome-1 impedes adipogenesis and insulin resistance, without alterations in feeding or locomotion, but favors energy combustion [605]. Sequestosome-1 sequesters extracellular signal-regulated kinase ERK1 into cytoplasm in adipose tissue. Its synthesis is induced during adipocyte differentiation (negative feedback) to prevent excessive adipogenesis. Ribosomal S6 kinase is an ERK substrate in adipogenesis.

Extracellular signal-regulated kinase-3 (ERK3)¹⁰⁵ and -4 (ERK4)¹⁰⁶ define a distinct subfamily of MAPKs, the ERK3 subfamily, that exists exclusively in vertebrates [606].

Jun N-terminal kinase JNK1¹¹⁴ has various splice variants [267]: JNK1α1,¹¹⁵ JNK1α2,¹¹⁶ JNK1β1¹¹⁷ and JNK1β2.¹¹⁸

This major Jun N-terminal kinase is activated by most known JNK inducers and is responsible for most known JNK effects. It contributes to cell differentiation and proliferation. It also intervenes in TNFα-regulated activities, such as cell death and inflammation.¹¹⁹

Enzyme JNK1 activates Ub-ligase Itch of the HECT (NEDD4) family¹²⁰ that specifically ubiquitinates the caspase-8 inhibitor Casp8 and FADD-like apoptosis regulator (CFLAR)¹²¹ to trigger its proteasomal degradation [608].

The pro-inflammatory cytokine tumor-necrosis factor-α exerts its pleiotropic activities via multiple effectors such as JNK1 kinase. Transcription factor Myc-interacting zinc finger protein-1 (MIZ1)¹²² that operates in transcriptional activation in the nucleus in response to cytoskeletal changes and impedes cell proliferation (transcriptional activator and repressor) also acts in the cytoplasm as a pathway-specific regulator, as it selectively suppresses TNFα-induced JNK1 activation and cell death [609]. Protein MIZ1 specifically regulates TNFα-induced TRAF2 Lys63-linked polyubiquitination. It, indeed, affects neither JNK1 activation by IL1β, nor TNFα-mediated activation of P38MAPK, ERK, and IκB kinase. Upon TNFα stimulation, MIZ1 is degraded rapidly by the proteasome to relieve its inhibition on JNK1 kinase.

Antigen-presenting cells, such as macrophages and dendritic cells, present antigen to naive T lymphocytes via T-cell receptors, CD3 subunit of which, in conjunction with CD28, initiates the TCR–CD28 costimulatory pathway. The latter promotes: (1) proliferation and maturation of CD4 + CD8 + cells to CD4 + T_H lymphocytes that then become interferon-synthesizing T_H1 and cytokine-producing T_H2 effectors due to IL12 and IL4 as well as (2) production of some cytokines such as IL2. Lymphocytes T_H1 produce interferon-γ and promote Ig switching in B lymphocytes, hence stimulating phagocytosis. On the other hand, T_H2 cells produce IL4 and IL5 that induce switching to IgE and IgG4 in B lymphocytes that both bind to Fc receptor, either on mastocytes and basophils (IgE) or phagocytic cells (IgG4), and activate eosinophils, respectively. The TCR–CD28 costimulation generates a strong JNK1–AP1 activation [267]. Transcription factor AP1 is required for T-cell maturation and development of acquired immunity. However, JNK1 suppresses the differentiation of T_H lymphocytes into T_H2 cells, as it impedes NFAT1 activation and TCR-stimulated IL4 production, hence favoring T_H1 cell differentiation.

The Jun N-terminal kinase JNK2¹²³ has diverse splice variants [267]: JNK2α1,¹²⁴ JNK2α2,¹²⁵ JNK2β1,¹²⁶ and JNK2β2.¹²⁷ An additional isoform JNK2γ has been detected in human B lymphocytes.

Dimerization of JNK2 can support JNK2 activation. Nitrosylation and ubiquitination modify JNK2 activity. Extensive S-nitrosylation inhibits JNK2 phosphorylation [610].

Like all JNKs, JNK2 promotes cell death in various cell types. However, under some circumstances, JNK2 acts as an anti-apoptotic agent. Unlike ERK1 and ERK2, JNK2 is not strongly activated by mitogens, but vigorously by: (1) environmental stresses (heat shock, ionizing radiation, oxidative stress, DNA-damaging chemicals, reperfusion injury, shear stress, and protein synthesis inhibitors); (2) inflammatory cytokines of the TNF superfamily, such as TNF, interleukin-1, TNFSF5, TNFSF6, TNFSF7, TNFSF11a, etc.; and (3) vasoactive peptides, such as endothelin and angiotensin-2 [267]. According to the cell type and context, JNK2 is either preferentially activated by MAP2K4 or MAP2K7 [610].

Among JNK2 kinases, JNK2α1 and JNK2α2 have a very high affinity for Jun, whereas JNK2β1 and JNK2β2 link preferentially to Activating transcription factor ATF2. In addition, JNK2 phosphorylates P53 factor. It also activates nuclear factor of activated T-cells NFAT3 [610].

Enzyme JNK2 is involved in T-cell activation, as it enhances TCR–CD28 costimulation of T-cell proliferation and TCR-stimulated production of IL2, IL4, and Ifnγ by activated T lymphocytes [267]. Enzyme JNK2 also contributes to B-cell activation. It is also involved in the control of the helper T_H1 and T_H2 cells [610].

The Jun N-terminal kinase JNK3¹²⁸ has several splice variants [267]: JNK3α1,¹²⁹ JNK3α2,¹³⁰ JNK3β1,¹³¹ and JNK3β2.¹³² It is predominantly expressed in the central nervous system. It is found, to a lesser extent, in the heart and testis. According to the context, JNK3 acts as a pro- or anti-apoptotic regulator; in particular, it preserves the pancreatic β-cell pool [611]. Unphosphorylated (inactive) JNK3 lodges mostly in the cytoplasm.

Protein JNK3 is phosphorylated (activated) by 2 dual-specificity kinases — MAP2K4 and MAP2K7 — (Thr221 and Tyr223). These activating kinases are themselves activated by MAP3K1 to MAP3K4 (MEKK family), MAP3K5 and MAP3K6 (ASK group), MAP3K7, MAP3K8, and MAP3K9 to MAP3K13 (MLK family). It is dephosphorylated (inactivated) by dual-specificity phosphatase MKP7 (or DuSP16) [611].

Scaffold proteins can facilitate the phosphorylation and dephosphorylation of JNK3 by linking to JNK3 and appropriate kinase(s) or phosphatase(s). These scaffolds include MAPK8-interacting proteins (MAPK8IP1–MAPK8IP3) [611]. Protein MAPK8IP1 aggregates MAP3K11, MAP2K7, and JNK3 enzymes to form a complete MAPK module. Scaffold β-arrestin-2 can selectively bind to JNK3 and assemble MAP3K5, MAP2K4, and JNK3 proteins.¹³³ Both MAPK8IP1 and β-arrestin-2 can bind MKP7 (DuSP16) phosphatase. Phosphorylated MAPK8IP3 may interact with MAP2K4, MAP2K7, and JNK3 enzymes.¹³⁴ Moreover, MAPK-binding protein MAPKBP1 (or JNKBP1) bind to JNK3, in addition to JNK1 and JNK2.

In addition, cyclin-dependent kinase CDK5 may phosphorylate (inactivate) JNK3 protein [611]. Cyclin-dependent kinase inhibitor CKI2a can also bind to JNK3, thereby suppressing its activity without affecting JNK3 phosphorylation. On the other hand, S-nitrosylation of JNK3 by NO can improve the phosphorylation level and activity of JNK3 kinase.

Activated JNK3 can phosphorylate transcriptional factors, such as Jun, ELk1, and ATF2 to increase the transcriptional activity [611]. Other substrates of JNK3 include P53, kinesin-1,¹³⁵ microtubule-associated protein Tau (MAPT), stathmin-like 2,¹³⁶ mitochondrial protein Second mitochondria-derived activator of caspases (SMAC),¹³⁷ and Rab3 GDP–GTP-exchange factor (Rab3GEP).¹³⁸

Enzyme P38MAPKα¹⁴¹ resides in both the cytoplasm and nucleus. In humans, 3 splice variants have been detected in addition to full-length P38MAPKα [614]: (1) cytokine suppressive anti-inflammatory drug-binding protein CSBP1; (2) exon skip Exip (as it skips exon 10); and (3) MAX-interacting protein MxI2, a shorter splice variant and a kinase that may interact with heterodimeric partner MyC-associated factor-X (MAX) of MyC transcription factor.

Enzyme P38MAPKα is required in erythropoietin synthesis and angiogenesis. It transmits chemotactic signals in neurons, endothelial and epithelial cells, neutrophils, mastocytes, monocytes, and vascular smooth muscle cells [614].

Its activation results from dual phosphorylation of the Thr180–Gly181–Tyr182 motif on the activation loop by MAP2K3, MAP2K4, and MAP2K6 enzymes (in vivo, mainly by MAP2K3 and MAP2K6, and under certain circumstances, by MAP2K4, an activator of JNKs). In addition to the these phosphorylation sites (Thr180 and Tyr182), P38MAPKα can be phosphorylated on Ser2, Thr16, and Thr241 [614].

Enzyme P38MAPKα is dephosphorylated (Thr180) by magnesium-depen-dent PPM1d protein phosphatase. Phosphatase PPM1a dephosphorylates MAP2K6 activator and P38MAPKα [614]. MAPK phosphatases, such as MKP1 (or DuSP1), MKP2 (or DuSP4), MKP5 (or DuSP10), and MKP7 (or DuSP16), and other dual-specificity phosphatases, such as DuSP2 and DuSP8, also dephosphorylate (inactivate) P38MAPKα.

Scaffold MAPK8IP2 binds to MAP3K11, MAP2K3, P38MAPKα, and TIAM1 (RacGEF) and RasGRF1 [614]. * In brown adipocytes, β-adrenergic receptor and protein kinase-A stimulate P38MAPKα via JIP2 that links to MAP2K3 activator. In addition, JIP4 (MAPK8IP4), which binds to, but does not activate JNK, supports MAP2K3- and MAP2K6-dependent activation of P38MAPKα, but does not bind MAP2K3 and MAP2K6, whereas it connects to MAP2K4 activator [614].

Cerebral cavernous malformation CCM2 homolog, or osmosensing scaffold for MAP3K3 (Osm), tethers to Rac, MAP3K3, and MAP2K3, and indirectly enables P38MAPKα activation in response to osmotic stress. In addition, MAP3K7IP1 (or TAB1) serves as a P38MAPKα binding partner. Last, but not least, P38MAPKα interacts with P38MAPK-interacting protein (P38IP) that regulates its activity.

Once it is activated by stress, it re-activates MAPKAPK2 kinase that phosphorylates heat shock protein HSP27 in response to interleukin-1β and LIMK1 kinase in response to VEGF in endothelial cells [614]. In addition, P38MAPKα targets caldesmon, an actin- and myosin-binding protein involved in the assembly of actin filaments, in smooth muscle cells, as well as paxillin, a phosphoprotein of focal adhesions [614].

Enzyme P38MAPKα participates in cell adaptation to stress, cell cycle regulation, inducing cell division arrest at various checkpoints. and control of cellular death (promotion or protection against cell death according to circumstances) [614].

Enzyme P38MAPKα is implicated in inflammation and its associated increase in vasculature permeability to enable influx of molecular and cellular mediators. It controls the production of COx2 cyclooxygenase and promotes prostaglandin synthesis, in particular in fibroblasts and endothelial cells [614]. It also regulates the synthesis of pro-inflammatory cytokines IL1 and TNFα in monocytes and neutrophils. It mediates the activation by distinct pattern-recognition receptors on airway epithelial cells.

Enzyme P38MAPKα is involved in viral and bacterial infections [614]. It provokes degranulation of neutrophils; in these cells, it also phosphorylates NADPH oxidase, thereby priming oxidative burst. In macrophages, P38MAPKα upregulates scavenger receptors for phagocytosis of bacteria in response to Toll-like receptor activation.

Enzyme P38MAPKα is involved in the activation of the transcription regulators of the superfamily-2B3A of nucleus-resident nuclear factors Activator protein AP1 and transcription factors of the ATF–CREB family,¹⁴² in conjunction with other MAPKs [614].¹⁴³ In particular, G-protein-coupled receptor activates the Jun promoter via P38MAPKα and AP1 activation in monocytes depends on JNK and P38MAPKα. Moreover, in some circumstances, P38MAPKα enables activation of nuclear factor-κB.

Signaling by P38MAPKβ¹⁴⁴ depends on HDAC3, a class-1 histone deacetylase.¹⁴⁵ During inflammation, the induction of immediate-early genes such as that of TNFα is regulated by signaling cascades, such as the JaK–STAT, NFκB, and P38MAPK pathways. Enzyme P38MAPKβ interacts directly and selectively with HDAC3 enzyme. This deacetylase decreases the P38MAPKβ phosphorylation state and inhibits the activity of the P38MAPKβ-dependent transcription factor Activating transcription factor ATF2 [615].¹⁴⁶ Consequently, HDAC3 represses TNF gene expression, especially in monocytes and macrophages.

Enzyme P38MAPKγ¹⁴⁷ behaves as a stress kinase that can function independently of phosphorylation [616]. It intervenes in cell differentiation. In skeletal muscles, where it is highly expressed, it supports fusion capacity of myoblasts, thereby promoting formation from myoblasts of myotubes. Alone or together with P38MAPKα, it also acts in erythroid differentiation.

In addition, P38MAPKγ contributes to the regulation of the stress response and cell cycle. In response to hypoxia, P38MAPKγ is phosphorylated (Thr183 and Tyr185) [616]. It is activated by dual-specificity kinases MAP2K3 and MAP2K6 enzymes, as well as MAP3K8 and RhoA and Rit GTPases. Moreover, MAP3K15 (or MLK7) stimulates the MAP2K6– P38MAPKγ axis. On the other hand, it is dephosphorylated by MAPK phosphatase MKP1 (DuSP1) and cytoplasmic PTPn3 phosphatase [616].

Enzyme P38MAPKγ is activated by various cytokines, such as IL1 and TNFα, and stress types (e.g., hypoxia and osmotic stress) [616].

Enzyme P38MAPKγ efficiently phosphorylates ATF2, ELk1, ELk4, Fos, and P53 (Ser33), but weakly MEF2a, MEF2c, and MEF2d transcription factors [616].

It also phosphorylates microtubule-associated protein Tau and BTK-associated SH3 domain-binding protein SH3BP5, a mitochondrial JNK-interacting protein. Protein P38MAPKγ can also interact with estrogen receptor ERα (or nuclear receptor NR3a1) [616].

Enzyme P38MAPKδ¹⁴⁸ is involved in cell migration, in particular in invasion of carcinoma cells. In addition, P38MAPKδ pertains to the set of 13 lung-prominent gene products with vitamin-D-dependent calcium-binding protein S100g, solute carrier family transporter SLC29a1, corticotropin-releasing hormone receptor CRHR1, and lipocalin-2, a growth factor and component of the innate immune response to bacterial infection [617].