**Abstract**

A change in the transcriptional landscape is an equilibrium-breaking event important for many biological processes. Mitogen-activated protein kinase (MAPK) signaling pathways are dedicated to sensing extracellular cues and are highly conserved across eukaryotes. Modulation of gene expression in response to the extracellular environment is one of the main mechanisms by which MAPK regulates proteome homeostasis to orchestrate adaptive responses that determine cell fate. A massive body of knowledge generated from population and single-cell analyses has led to an understanding of how MAPK pathways operate. MAPKs have thus emerged as fundamental transcriptome regulators that function through a multilayered control of gene expression, a process often deregulated in disease, which therefore provides an attractive target for therapeutic strategies. Here, we summarize the current understanding of the mechanisms underlying MAPK-mediated gene expression in organisms ranging from yeast to mammals.

**Keywords:** MAP kinases, signal transduction, transcription, gene expression, chromatin

## **1. Introduction**

The intracellular matrix is physically separated from the dynamic extracellular environment; however, their functions are intimately coordinated in order to ensure cell adaptation and survival. Mitogen-activated protein kinase (MAPK) cascades sense and integrate extracellular cues through sequential activation of protein kinases. These highly conserved transduction pathways are involved in a myriad of fundamental cellular processes and determine cell fate. Misregulation of these signaling cascades has major consequences for numerous diseases such as cancer, diabetes, inflammatory, and immune response diseases.

About 300 genes encode signaling proteins directly involved in signal transduction, including their positive and negative regulators as well [1]. Upon cell stimulation, in order to adapt to an extracellular insult, these seemingly simple linear signaling pathways harbor the potential to target a large number of substrates of which many are involved in gene expression. In fact, MAPKs control every step studied to date of the highly dynamic process of gene expression. The overall

picture of MAPK pathway substrates and interactors is still far from complete; however, the knowledge generated over the last 20 years has allowed a more holistic understanding of the underlying mechanisms of MAPK-regulated transcription. Due to the growing interest in MAPK-biology and the sheer volume of literature available, in this chapter, we not only mainly focus on the main mammalian MAPK cascades in humans (ERK1/2, JNK, p38, and ERK5), but we also discuss the main findings regarding MAPK cascades in the model organism *Saccharomyces cerevisiae*.

## **2. MAP kinase pathways**

MAPKs mediate the transmission of extracellular information through a series of consecutive chemical reactions that lead to the activation of a terminal MAPK to orchestrate the appropriate gene expression pattern. To date, four major MAPK signaling cascades have been characterized in mammals, which are named according to their MAPK components: extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase 1 to 3 (JNK), p38 α/β/γ/δ (p38), and ERK5. Apart from these main MAPKs, several atypical MAPKs have also been described (ERK 3/4, ERK 7/8, and NLK, among others) with less well-defined functions and distinct modes of activation [2].

#### **2.1 MAPK activators**

MAPKs can be activated in two different ways: (1) ligand-dependent that requires the physical interaction of a ligand (e.g., growth factors, hormones, or cytokines) with a receptor or (2) ligand-independent that mediates the signaling of physical stressors (e.g., radiation, injury, and osmotic pressure). In general, ERK1/2 responds to proliferative and survival stimuli such as growth factors, serum, and phorbol esters and, to a lesser extent, to ligands of G protein-coupled receptors (GPCRs) or cytokines, or to osmotic stress and microtubule disorganization. ERK5 is activated by growth factors [e.g., EGF, NGF, FGF-2, and brain-derived neurotrophic factor (BDNF)] and cytokines (e.g., Leukemia inhibitory factor—LIF) as well as by some stresses such as osmotic stress and hydrogen peroxide [3]. JNKs and p38 MAPKs are functionally related and are collectively named stress-activated protein kinases (SAPKs). The JNK pathway strongly responds to cytokines, growth factor deprivation, intracellular stimuli (e.g., DNA damage, cytoskeletal changes, oxidative, and ER stress), and extracellular stresses (e.g., UV radiation and osmotic stress) and less efficiently responds to stimulation by some GPCRs, serum, and growth factors [4]. Finally, p38 signaling has been shown to be consistently activated by a wide variety of environmental stresses and inflammation but to be inconsistently activated by insulin and growth factors in certain cell types [1].

#### **2.2 Modular architecture of the MAPK signaling cascades**

MAPK signaling is triggered by the stimulation of different membrane receptor families (e.g., receptor tyrosine kinases (RTKs), GPCRs, cytokine receptors, Ser/ Thr kinase receptors, and membrane-bound stress sensors) that are coupled to the MAPK signaling cascades. Depending on the stimulus, the signal is transmitted downstream through small G proteins, kinases, or adaptor proteins that are the immediate upstream activators of the conventional MAPK signaling cascades.

A major and highly conserved feature of MAPK pathways is their central three-tiered core signaling module of sequentially activating kinases (**Figure 1**). In the first tier, a Ser/Thr kinase MAPKKK (MAP3K) is activated by the effectors

**21**

**Figure 1.**

*control of gene expression are indicated.*

*Shaping the Transcriptional Landscape through MAPK Signaling*

mentioned above. This MAPKKK then phosphorylates and activates a MAPKK (MAP2K) in the second tier; the MAPKK is a dual specificity kinase that phosphorylates both threonine and tyrosine within a conserved Thr-Xaa-Tyr motif in its substrate. Finally, there is a terminal Ser/Thr MAPK in the third tier, which, upon activation, phosphorylates a huge number of cytoplasmic and nuclear substrates on consensus Ser/Thr-Pro sites. Although not always present, other kinases are involved in MAPK signal transduction. One such kinase is the MAP4K that phosphorylates and activates the MAP3K; downstream MAPK-activated protein kinases

*Conceptual representation of the three core components of a MAPK signaling cascade. A typical MAPK cascade is composed of three consecutively activated tiers of kinases: MAP3Ks, MAP2Ks, and MAPKs. Kinases are grouped by layers according to their position in the signaling cascade. Arrows link components of different layers representing activation pathways. The core modules of mammalian (left) and yeast (right) signaling pathways are shown. Output responses resulting from MAPK activation through substrate phosphorylation and* 

The first MAPK pathway identified was ERK1 whose activation depends on the dimerization and autophosphorylation of the ligand-activated tyrosine kinase receptors (RTKs and GPCRs). These ligand-induced chemical and conformational receptor changes trigger recruitment of the adaptor proteins Shc and Grb2, a guanine exchange factor (SOS), and the small GTPase (Ras) to the plasma membrane.

(MAPKAPKs) contribute to the spread of the signal transduction.

*DOI: http://dx.doi.org/10.5772/intechopen.80634*

*Shaping the Transcriptional Landscape through MAPK Signaling DOI: http://dx.doi.org/10.5772/intechopen.80634*

#### **Figure 1.**

*Gene Expression and Control*

**2. MAP kinase pathways**

modes of activation [2].

**2.1 MAPK activators**

picture of MAPK pathway substrates and interactors is still far from complete; however, the knowledge generated over the last 20 years has allowed a more holistic understanding of the underlying mechanisms of MAPK-regulated transcription. Due to the growing interest in MAPK-biology and the sheer volume of literature available, in this chapter, we not only mainly focus on the main mammalian MAPK cascades in humans (ERK1/2, JNK, p38, and ERK5), but we also discuss the main findings regarding MAPK cascades in the model organism *Saccharomyces cerevisiae*.

MAPKs mediate the transmission of extracellular information through a series of consecutive chemical reactions that lead to the activation of a terminal MAPK to orchestrate the appropriate gene expression pattern. To date, four major MAPK signaling cascades have been characterized in mammals, which are named according to their MAPK components: extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase 1 to 3 (JNK), p38 α/β/γ/δ (p38), and ERK5. Apart from these main MAPKs, several atypical MAPKs have also been described (ERK 3/4, ERK 7/8, and NLK, among others) with less well-defined functions and distinct

MAPKs can be activated in two different ways: (1) ligand-dependent that requires the physical interaction of a ligand (e.g., growth factors, hormones, or cytokines) with a receptor or (2) ligand-independent that mediates the signaling of physical stressors (e.g., radiation, injury, and osmotic pressure). In general, ERK1/2 responds to proliferative and survival stimuli such as growth factors, serum, and phorbol esters and, to a lesser extent, to ligands of G protein-coupled receptors (GPCRs) or cytokines, or to osmotic stress and microtubule disorganization. ERK5 is activated by growth factors [e.g., EGF, NGF, FGF-2, and brain-derived neurotrophic factor (BDNF)] and cytokines (e.g., Leukemia inhibitory factor—LIF) as well as by some stresses such as osmotic stress and hydrogen peroxide [3]. JNKs and p38 MAPKs are functionally related and are collectively named stress-activated protein kinases (SAPKs). The JNK pathway strongly responds to cytokines, growth factor deprivation, intracellular stimuli (e.g., DNA damage, cytoskeletal changes, oxidative, and ER stress), and extracellular stresses (e.g., UV radiation and osmotic stress) and less efficiently responds to stimulation by some GPCRs, serum, and growth factors [4]. Finally, p38 signaling has been shown to be consistently activated by a wide variety of environmental stresses and inflammation but to be inconsistently activated by insulin and growth factors in certain cell types [1].

**2.2 Modular architecture of the MAPK signaling cascades**

MAPK signaling is triggered by the stimulation of different membrane receptor families (e.g., receptor tyrosine kinases (RTKs), GPCRs, cytokine receptors, Ser/ Thr kinase receptors, and membrane-bound stress sensors) that are coupled to the MAPK signaling cascades. Depending on the stimulus, the signal is transmitted downstream through small G proteins, kinases, or adaptor proteins that are the immediate upstream activators of the conventional MAPK signaling cascades. A major and highly conserved feature of MAPK pathways is their central three-tiered core signaling module of sequentially activating kinases (**Figure 1**). In the first tier, a Ser/Thr kinase MAPKKK (MAP3K) is activated by the effectors

**20**

*Conceptual representation of the three core components of a MAPK signaling cascade. A typical MAPK cascade is composed of three consecutively activated tiers of kinases: MAP3Ks, MAP2Ks, and MAPKs. Kinases are grouped by layers according to their position in the signaling cascade. Arrows link components of different layers representing activation pathways. The core modules of mammalian (left) and yeast (right) signaling pathways are shown. Output responses resulting from MAPK activation through substrate phosphorylation and control of gene expression are indicated.*

mentioned above. This MAPKKK then phosphorylates and activates a MAPKK (MAP2K) in the second tier; the MAPKK is a dual specificity kinase that phosphorylates both threonine and tyrosine within a conserved Thr-Xaa-Tyr motif in its substrate. Finally, there is a terminal Ser/Thr MAPK in the third tier, which, upon activation, phosphorylates a huge number of cytoplasmic and nuclear substrates on consensus Ser/Thr-Pro sites. Although not always present, other kinases are involved in MAPK signal transduction. One such kinase is the MAP4K that phosphorylates and activates the MAP3K; downstream MAPK-activated protein kinases (MAPKAPKs) contribute to the spread of the signal transduction.

The first MAPK pathway identified was ERK1 whose activation depends on the dimerization and autophosphorylation of the ligand-activated tyrosine kinase receptors (RTKs and GPCRs). These ligand-induced chemical and conformational receptor changes trigger recruitment of the adaptor proteins Shc and Grb2, a guanine exchange factor (SOS), and the small GTPase (Ras) to the plasma membrane. The interaction of these four elements leads to the homo- and hetero-dimerization of the Raf family of kinases (B- or C-Raf) that activate the MAP3K module. The MAP3K then phosphorylates MEKK1/2 (MAP2K) at two serine within their activation loop (Ser-Met-Ala-Asn-Ser). Activated MEKK1/2 in turn phosphorylates ERK1/2 (MAPK) on the tyrosine and threonine residues of the Thr-Glu-Tyr motif in their activating loop. Additionally, MAPKAPKs have been identified that propagate ERK signaling (RSKs, MSKs, MNKs, and in some cases MK3/5) [1, 5].

The least studied of the four MAPK cascades is ERK5, whose mechanisms of upstream activation may include activation of tyrosine kinase receptors, the protein tyrosine kinase c-Src, the small GTPase Ras, the adaptor protein Lad1, and the protein Ser/Thr kinase WNK1, which acts as a MAP4K [1, 3]. Activation of these signaling molecules leads to activation of the MAP3Ks (not only MEKK2/3 but also TPL2 and MLTK) to phosphorylate the two alternatively spliced MEK5 isoforms (MEK5a and MEK5b, MAP2K) at the Ser-Xaa-Ala-Xaa-Thr activation motif, leading to ERK5 activation at the Thr-Glu-Tyr motif. The ERK5 pathway also involves downstream MAPKAPKs such as the serum and glucocorticoid-activated kinase (SGK) and p90 ribosomal S6 kinases (RSKs) [2].

The signal through the JNK cascade is transmitted through adaptor proteins (TRAFs), small GTPases (Rac1, Cdc42), or Ste20-like kinases that act as MAP4Ks [6]. A large number of MAP3Ks convey the signal to the main MAP2Ks (MKK4/7) by phosphorylating the sequence Ser-Xaa-Ala-Xaa-Ser/Thr in their activation loop [4]. Ultimately, the three components of the MAPK level (JNK1–3) are activated by dual Thr/Tyr phosphorylation at the Thr-Pro-Tyr motif. As for other kinases in the JNK cascade, MAPKAPKs such as MST1 are well-defined JNK substrates that can act as both upstream and downstream of JNK [7].

Finally, p38 operates through different receptors from apoptosis-related receptors to physical sensors. The initial signal is transferred using Cdc42, Rac1, and Ste20-like kinases (shared with JNK) and results in phosphorylation of the activation loop (Ser-Xaa-Ala-Xaa-Ser/Thr) of the MAP2Ks MKK3/6 that uniquely target p38. The differences between the p38 and JNK pathways lie within the specific scaffold proteins and substrates. All p38 isoforms, either the major isoforms (p38α,β,γ,δ) or the minor isoforms generated through alternative splicing, are activated through dual phosphorylation at the Thr-Gly-Tyr motif [1]. The main p38 isoform (p38α) is constitutively expressed, while the remaining isoforms are tissue-restricted. Uniquely for a MAP kinase, p38 can be activated through MAP2Kindependent mechanisms that involve adaptors that promote p38 autophosphorylation [6]. Finally, the downstream MAPKAPK layer is partially shared with ERK and includes MAPKAPK2,3,5, MNKs, and MSKs [1].

#### *2.2.1 Specificity of signaling cascades*

The signaling proteome is composed of a limited number of genes that specifically integrate a virtually endless number of extracellular stimuli. Several strategies have evolved in order to maintain the signaling fidelity. For instance, this is achieved by the interaction of MAPKs with other components of the pathway and with substrates through docking sites composed of specific consensus motifs. Two types of docking motifs have been reported: D-motif and docking site for ERK (FXF)-motif, which ensure fidelity of signaling. D-motifs contain at least two basic residues flanking hydrophobic residues and are located opposite to the catalytic pocket in MAPKs [8]. The FXF-motif is composed of two Phe residues separated by one residue [9]. Another mechanism to gain specificity of signaling is the use of MAPK-scaffold proteins, which were first described in yeast (Ste5 and Pbs2) [10, 11]. Scaffolds are crucial for maintenance of signaling specificity

**23**

*Shaping the Transcriptional Landscape through MAPK Signaling*

as they sequester multiprotein interactions to prevent crosstalk by controlling

controlled, not only through positive and negative feedback mechanisms at the post-translational level mediated by regulatory proteins (e.g., phosphatases and kinases) but also through post-transcriptional control mediated by RNA-binding

The fastest mechanism of ablating MAPK activity is to remove one of the two activating phosphates through the activity of specific phosphatases. Their role in regulating the terminal MAPK has been extensively studied, but little is known about their effect on upstream signaling components. Phosphatase activity is mainly derived from Ser/Thr phosphatases, Tyr phosphatases, and the dual specificity phosphatases (DUSP) known as MAPK phosphatases (MKP) [1]. Based on sequence homology, substrate specificity, and subcellular localization, DUSPs can be divided into three groups: nuclear inducible (DUSP1/2/4/5), cytoplasmic and ERK-specific (DUSP6/7/9), and DUSPs with no specific cellular localization that targets JNK and p38 SAPKs (DUSP8/10/16) [4, 12]. MAPKs also exert a transcriptional control of regulatory elements such as these phosphatases and thereby generate a negative feedback loop. Another relevant type of negative feedback regulation is driven by the direct phosphorylation of different upstream components of the MAPK cascade by the MAPK itself to modulate basal [13] and stimuli-dependent signaling dynamics [5]. Additionally, scaffold proteins and other enzymatic activities either positively or negatively regulate different levels of MAPK signal transduction such as, for example, the formation of the ligand-receptor signaling complex, the intracellular modular interactions, and the degradation of the components [14]. Post-transcriptional regulation of MAPKs can also be achieved at the RNA level. RNA-binding proteins and miRNA negatively regulate MAPK gene expression by directly cleaving their

Five MAPK pathways have been well characterized in the budding yeast, *S. cerevisiae*. In vegetative cells, the four MAPKs Fus3, Kss1, Hog1, and Slt2/Mpk1 are involved in the mating-pheromone response, the filamentous-invasion pathway, the high osmolarity growth, and the cell integrity pathway, respectively. The fifth

Haploid yeast cells sense the reciprocal mating pheromones (α-factor or a-factor) through Ste2 and Ste3 GPCRs. The signal is then transmitted by GTPases to the p21-activated kinase (PAK)-like kinase Ste20, the MAPK scaffold Ste5, Cdc42, a guanine-nucleotide exchange factor (GEF), and Far1. Ste5 signals and serves as a scaffold that links the MAP4K and the MAPK signalosome (Ste11 → Ste7 → Fus3;

The high osmolarity glycerol (Hog1) MAPK, the yeast homolog of p38, is activated in response to osmotic stress as a consequence of signaling elicited from two upstream-independent mechanisms (Sln1/Sho1). The Sln1 sensor is the primary osmosensor and is a complex variation of the well-known bacterial two-component system. Upon osmostress, inactivation of the transmembrane histidine kinase Sln1 leads to the derepression and activation of the MAP3K (Ssk2/22) via Ypd1/Skk1. The Sho1 osmosensing branch is mediated by mucin-like proteins (Hkr1 and Msb2) and ultimately activates the MAP3K Ste11 through the integral transmembrane

MAPK, Smk1, is believed to play a role in spore wall assembly [16, 17].

The amplitude, frequency, and localization of activated MAPK-activity is tightly

*DOI: http://dx.doi.org/10.5772/intechopen.80634*

stability and subcellular localization.

*2.2.2 Regulators of signaling cascades*

proteins and microRNAs (miRNAs).

mRNAs or through complementary pairing [15].

**2.3 Yeast MAPK cascades**

the latter is the ERK1 homolog) [18].

as they sequester multiprotein interactions to prevent crosstalk by controlling stability and subcellular localization.
