*2.2.2 Regulators of signaling cascades*

*Gene Expression and Control*

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

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

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

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

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

ERK signaling (RSKs, MSKs, MNKs, and in some cases MK3/5) [1, 5].

(SGK) and p90 ribosomal S6 kinases (RSKs) [2].

act as both upstream and downstream of JNK [7].

includes MAPKAPK2,3,5, MNKs, and MSKs [1].

*2.2.1 Specificity of signaling cascades*

**22**

The amplitude, frequency, and localization of activated MAPK-activity is tightly 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 proteins and microRNAs (miRNAs).

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 mRNAs or through complementary pairing [15].
