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MapK interaction with its targets: General considerations
Activation of the various mitogen-activated protein (MAP) kinase pathways converts many different extracellular stimuli into specific cellular responses by inducing the phosphorylation of particular groups of substrates. One important determinant for substrate specificity is likely to be the amino-acid sequence surrounding the phosphorylation site; however, these sites overlap significantly between different MAP kinase family members. The idea is now emerging that specific docking sites for protein kinases are involved in the efficient binding and phosphorylation of some substrates. The MAP kinase-activated protein (MAPKAP) kinase p90(rsk) (see Drosophila S6kII) contains two kinase domains: the amino-terminal domain (D1) is required for the phosphorylation of exogenous substrates whereas the carboxy-terminal domain (D2) is involved in autophosphorylation. Association between the extracellular signal-regulated kinase (Erk) MAP kinases and p90(rsk) family members has been detected in various cell types including Xenopus oocytes, where inactive p90(rsk) is bound to the inactive form of the Erk2-like MAP kinase p42(mpk1). A new MAP kinase docking site has been identified that is located at the carboxyl terminus of p90(rsk). This docking site is required for the efficient phosphorylation and activation of p90(rsk) in vitro and in vivo and is also both necessary and sufficient for the stable and specific association with p42(mpk1). The sequence of the docking site is conserved in other MAPKAP kinases, suggesting that it might represent a new class of interaction motif that facilitates efficient and specific signal transduction by MAP kinases (Gavin, 1999).
Smad4 (see Drosophila Medea) is a major tumor suppressor currently thought to function constitutively in the transforming growth factor beta (TGF-beta)-signaling pathway. This study reports that Smad4 activity is directly regulated by the Wnt and fibroblast growth factor (FGF) pathways through GSK3 (see Drosophila Shaggy) and mitogen-activated protein kinase (MAPK) phosphorylation sites. FGF activates MAPK, which primes three sequential GSK3 phosphorylations that generate a Wnt-regulated phosphodegron bound by the ubiquitin E3 ligase beta-TrCP (see Drosophila Slmb). In the presence of FGF, Wnt potentiates TGF-beta signaling by preventing Smad4 GSK3 phosphorylations that inhibit a transcriptional activation domain located in the linker region. When MAPK is not activated, the Wnt and TGF-beta signaling pathways remain insulated from each other. In Xenopus embryos, these Smad4 phosphorylations regulate germ-layer specification and Spemann organizer formation. The results show that three major signaling pathways critical in development and cancer are integrated at the level of Smad4 (Demagny, 2014: PubMed).
MapK targets Creb, Elk-1, and other transcription factors
A signaling pathway has been elucidated whereby growth factors activate the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB), a critical regulator of immediate early gene transcription. Growth factor-stimulated CREB phosphorylation at serine-133 is mediated by the RAS-mitogen-activated protein kinase (MAPK) pathway. MAPK activates CREB kinase, which in turn phosphorylates and activates CREB. Purification, sequencing, and biochemical characterization of CREB kinase reveals that it is identical to a member of the pp90(RSK) family, RSK2. RSK2 mediates growth factor induction of CREB serine-133 phosphorylation both in vitro and in vivo. These findings identify a cellular function for RSK2 and define a mechanism whereby growth factor signals mediated by RAS and MAPK are transmitted to the nucleus to activate gene expression (Xing, 1996).
The ETS transcription factors perform distinct biological functions despite conserving a highly similar DNA-binding domain. One distinguishing property of a subset of ETS proteins is a conserved region of 80 amino acids termed the Pointed (PNT) domain. Using enzyme kinetics it has been determined that the Ets-1 PNT domain contains an ERK2 docking site. The docking site enhances the efficiency of phosphorylation of a mitogen-activated protein kinase (MAPK) site N-terminal to the PNT domain. The site enhances ERK2 binding rather than catalysis. Three hydrophobic residues are involved in docking, and the previously determined NMR structure indicates that these residues are clustered on the surface of the Ets-1 PNT domain. The docking site function is conserved in the PNT domain of the highly related Ets-2 but not in the ets family member GABPalpha. Ablation of the docking site in Ets-1 and Ets-2 prevents Ras pathway-mediated enhancement of the transactivation function of these proteins. This study provides structural insight into the function of a MAPK docking site and describes a unique activity for the PNT domain among a subset of ets family members. The Drosophila protein Pnt-P2 is encoded by a gene that is an apparent ortholog of the genes encoding Ets-1 and Ets-2. Pnt-P2 conserves the Ras/MAPK-targeted phosphoacceptor found N-terminal to the PNT domain in Ets-1 and Ets-2. Additionally, the PNT domain of Pnt-P2 contains sequence closely resembling the ERK2 docking site of Ets-1 and Ets-2 (L228, I230, and F234). It is likely this sequence functions as a docking site for Rolled, the Drosophila MAPK related to ERK2 (Seidel, 2002).
The deposition of amyloid beta protein (A beta) in the cerebral cortex is the pathological characteristic of Alzheimer's disease (AD); patients with AD suffer from progressive memory loss. Transgenic experiments have revealed that long-term memory is dependent on cyclic AMP-response element binding protein, CREB (See Drosophila CrebB-17A). CREB phosphorylation at serine-133 is essential for its transcriptional activity. At a concentration more than 1 microM, A beta(1-40) induces CREB phosphorylation at serine-133 in rat pheochromocytoma PC12 cells, while PD98059, a MEK1 inhibitor, inhibits A beta(1-40)-induced CREB phosphorylation at serine-133. A beta(1-40) induces phosphorylation of p44 and p42 MAP kinases (Erk1 and Erk2) at tyrosine-204. It is concluded that elevated A beta(1-40) level induces CREB phosphorylation at serine-133 via p44/42 MAP kinase-dependent pathway (Sato, 1997).
Mitogenic and stress signals result in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1 (Drosophila homolog: Pointed), which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which has not been clear. Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions (Strahl, 1996).
The phosphorylation of transcription factors by mitogen-activated protein kinases (MAP) is a pivotal event in the cellular response to the activation of MAP kinase signal transduction pathways. Mitogenic and stress stimuli activate different pathways and lead to the activation of distinct groups of target proteins. Elk-1 is targeted by three distinct MAP kinase pathways. This study demonstrates that the MAP kinase ERK2 is targeted to Elk-1 by a domain that is distinct from, and located N-terminal to, its phosphoacceptor motifs. Targeting via this domain is essential for the efficient and rapid phosphorylation of Elk-1 in vitro and full and rapid activation in vivo. Specific residues involved in ERK targeting have been identified. These data indicate that the targeting of different classes of MAP kinases to their nuclear substrates may be a common mechanism to increase the specificity and efficiency of this signal transduction pathway (Yang, 1998a).
The activation of MAP kinase (MAPK) signal transduction pathways results in the phosphorylation of transcription factors by the terminal kinases in these cascades. Different pathways are activated by mitogenic and stress stimuli, which lead to the activation of distinct groups of target proteins. The ETS-domain transcription factor Elk-1 is a substrate for three distinct classes of MAPKs. Elk-1 contains a targeting domain, the D-domain, which is distinct from the phosphoacceptor motifs and is required for efficient phosphorylation and activation by the ERK MAPKs. Members of the JNK subfamily of MAPKs are also targeted to Elk-1 by this domain. Targeting via this domain is essential for the efficient and rapid phosphorylation and activation of Elk-1 both in vitro and in vivo. The ERK and JNK MAPKs use overlapping yet distinct determinants in the D-domain for targeting to Elk-1. In contrast, members of the p38 subfamily of MAPKs are not targeted to Elk-1 via this domain. These data therefore demonstrate that different classes of MAPKs exhibit differential requirements for targeting to Elk-1 (Yang, 1998b).
Somatostatin (SRIF) was discovered as an inhibitor of GH secretion from pituitary somatotroph cells. SRIF analogs are very effective agents used to treat neuroendocrine tumors and are now being used with increasing frequency in clinical trials to treat more aggressive malignancies. However, the cellular components mediating SRIF signal transduction remain largely unknown. The SRIF type 2 receptor (SST2) was stabily expressed in GH4 rat somatomammotroph cells, establishing a physiologically relevant model system. In this model, a SRIF analog inhibits forskolin-induced cAMP accumulation, protein kinase A activation, cAMP response element-binding protein (CREB) phosphorylation, and Pit-1/GHF-1 promoter activation in an okadaic acid-insensitive manner. Pertussis toxin inhibits the effects of the NGF analog, documenting that SST2 signaling is coupled to Gi. The inhibitory effects of the NGF analog are reversed by overexpression of protein kinase A catalytic subunit, indicating that SRIF does not act via serine/threonine phosphatases, but rather, by lowering protein kinase A activity. These data define the components of the SRIF/SST2 receptor signaling pathway and provide important mechanistic insights into how SRIF controls neuroendocrine tumors (Tentler, 1997).
In Rat-1 fibroblasts, nonmitogenic doses of lysophosphatidic acid (LPA) stimulate a transient activation of mitogen-activated protein kinase (MAPK), whereas mitogenic doses elicit a sustained response. This sustained phase of MAPK activation regulates cell fate decisions such as proliferation or differentiation, presumably by inducing a program of gene expression that is not observed in response to transient MAPK activation. The expression of members of the AP-1 transcription factor complex has been examined in response to stimulation with different doses of LPA. c-Fos, c-Jun, and JunB are induced rapidly in response to LPA stimulation, whereas Fra-1 and Fra-2 are induced after a significant lag. The expression of c-Fos is transient, whereas the expression of c-Jun, JunB, Fra-1, and Fra-2 is sustained. The early expression of c-Fos can be reconstituted with nonmitogenic doses of LPA, but the response is transient when compared to that observed with mitogenic doses. In contrast, expression of Fra-1, Fra-2, and JunB and optimal expression of c-Jun are observed only with doses of LPA, which induce sustained MAPK activation and DNA synthesis. LPA-stimulated expression of c-Fos, Fra-1, Fra-2, c-Jun, and JunB is inhibited by the MEK1 inhibitor PD098059, indicating that the Raf-MEK-MAPK cascade is required for their expression. In cells expressing a conditionally active form of Raf-1 (DeltaRaf-1:ER), selective, sustained activation of Raf-MEK-MAPK is sufficient to induce expression of Fra-1, Fra-2, and JunB but, interestingly, such activation induces little or no c-Fos or c-Jun. The induction of c-Fos observed in response to LPA is strongly inhibited by buffering the intracellular [Ca2+]. Moreover, although Raf activation or calcium ionophores induce little c-Fos expression, a synergistic induction in response to the combination of DeltaRaf-1:ER and ionomycin is observed. These results suggest that kinetically distinct phases of MAPK activation serve to regulate the expression of distinct AP-1 components, such that sustained MAPK activation is required for the induced expression of Fra-1, Fra-2, c-Jun, and JunB. However, in contrast to the case for Fra-1, Fra-2, and JunB, activation of the MAPK cascade alone is not sufficient to induce c-Fos expression, which rather requires cooperation with other signals such as Ca2+ mobilization. Finally, the identification of the Fra-1, Fra-2, c-Jun, and JunB genes as those that are selectively regulated by sustained MAPK activation, or in response to activated Raf, suggests that these genes are candidates to mediate certain effects of Ras proteins in oncogenic transformation (Cook, 1999).
Germline mutations at loci encoding either the transcription factor Microphthalmia (Mi), the cytokine receptor c-Kit, or its ligand Steel factor (S1) result in strikingly similar defects in mast cell and melanocyte development. Mi is a transcription factor of the basic, helix-loop-helix, leucine zipper type, the mutation of which causes Waardenburg Syndrome type II in humans. A biochemical link is described between Kit signaling and the activity of Mi. Stimulation of melanoma cells with S1 results in activation of MAP kinase, which in turn phosphorylates Mi at a consensus target serine. This phosphorylation upregulates Mi transactivation of the tyrosinase pigmentation gene promoter. In addition to modulating pigment production, such signaling may regulate the expression of genes essential for melanocyte survival and development. The pathway represents a new application of the general MAP kinase machinery in transducing a signal between a tissue-specific receptor at the cell surface and a tissue-specific transcription factor in the nucleus (Hemesath, 1998).
The bcl-6 proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center (GC) B and T cells and required for GC formation and antibody affinity maturation. Deregulation of bcl-6 expression by chromosomal rearrangements and point mutations of the bcl-6 promoter region are implicated in the pathogenesis of B-cell lymphoma. The signals regulating bcl-6 expression are not known. Antigen receptor activation leads to BCL-6 phosphorylation by mitogen-activated protein kinase (MAPK). Phosphorylation, in turn, targets BCL-6 for rapid degradation by the ubiquitin/proteasome pathway. These findings indicate that BCL-6 expression is directly controlled by the antigen receptor via MAPK activation (Niu, 1998).
MAPK is a ubiquitous, evolutionarily conserved signal transducer that is activated by heterogeneous signals that originate from the cell membrane and are transduced to MAPK via RAS proteins. Accordingly, POZ/zinc finger proteins represent a large family of highly conserved transcription factors, including Drosophila cell fate regulators such as Tramtrack and Broad-complex, as well as human cancer-associated proteins such as BCL-6 and PLZF. These molecules have strong structural (POZ and ZF domains), as well as functional homologies since they are transcriptional repressors that control cell differentiation. Most notably, POZ/zinc finger proteins also carry possible MAPK phosphorylation sites and PEST sequences in approximately the same position as those carried by BCL-6. In Drosophila, degradation of TTK88, a POZ/zinc finger inhibitor of neural-cell differentiation, has been shown to be mediated by MAPK. Thus, degradation of POZ/zinc finger transcription factors may represent a general mechanism by which the RAS/MAPK pathway controls cell function and differentiation (Niu, 1998 and references).
Although the circadian time-keeping properties of the suprachiasmatic nuclei (SCN) require gene expression, little is known about the signal transduction pathways that initiate transcription. A brief exposure to light during the subjective night, but not during the subjective day, activates the p44/42 mitogen-activated protein kinase (MAPK) signaling cascade in the SCN. In addition, MAPK stimulation activates CREB (cAMP response element binding protein), indicating that potential downstream transcription factors are stimulated by the MAPK pathway in the SCN. Striking circadian variations are observed in MAPK activity within the SCN, suggesting that the MAPK cascade is involved in clock rhythmicity (Obrietan, 1999).
A novel mitogen- and stress-activated protein kinase (MSK1) has been identifed that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of both unstimulated and stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB (Deak, 1998).
TGFbeta can override the proliferative effects of EGF and other Ras-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Ras mutations often show a loss of TGFbeta antimitogenic responses. Oncogenic Ras inhibits TGFbeta signaling in mammary and lung epithelial cells by negatively regulating the TGFbeta mediators Smad2 and Smad3. Oncogenically activated Ras inhibits the TGFbeta-induced nuclear accumulation of Smad2 and Smad3 and Smad-dependent transcription. Ras acting via Erk MAP kinases causes phosphorylation of Smad2 and Smad3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain. These sites are separate from the TGFbeta receptor phosphorylation sites that activate Smad nuclear translocation. Mutation of these MAP kinase sites in Smad3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFbeta in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Ras, induces a less extensive phosphorylation and cytoplasmic retention of Smad2 and Smad3. These results suggest a mechanism for the counterbalanced regulation of Smad2/Smad3 by TGFbeta and Ras signals in normal cells, and for the silencing of antimitogenic TGFbeta functions by hyperactive Ras in cancer cells (Kretzschmar, 1999).
The mammalian transcription factor LSF (CP2/LBP-1c) binds cellular promoters modulated by cell growth signals. LSF-DNA-binding activity is strikingly regulated by induction of cell growth in human peripheral T lymphocytes. Within 15 min of mitogenic stimulation of these cells, the level of LSF-DNA-binding activity increases by a factor of five. The level of LSF protein in the nucleus remains constant throughout this interval. However, a rapid decrease in the electrophoretic mobility of LSF, attributable to phosphorylation, correlates with the increase in DNA-binding activity. pp44 (ERK1) phosphorylates LSF in vitro on the same residue that is phosphorylated in vivo, specifically at amino acid position 291, as indicated by mutant analysis. As direct verification of the causal relationship between phosphorylation and DNA-binding activity, treatment in vitro of LSF with phosphatase both increases the electrophoretic mobility of the protein and decreases LSF-DNA-binding activity. This modulation of LSF-DNA-binding activity as T cells progress from a resting to a replicating state reveals that LSF activity is regulated during cell growth and suggests that LSF regulates growth-responsive promoters (Volker, 1997).
Phosphorylation of transcription factors by mitogen-activated protein kinase (MAPK) cascades links cell signaling with the control of gene expression. Growth factors induce rRNA synthesis by activating MAPK-dependent signaling cascades that target the RNA polymerase I-specific transcription initiation factor TIF-IA. Activation of TIF-IA and ribosomal gene transcription is sensitive to PD98059, indicating that TIF-IA is targeted by MAPK in vivo. Phosphopeptide mapping and mutational analysis reveals two serine residues (S633 and S649) that are phosphorylated by ERK and RSK kinases. Replacement of S649 by alanine inactivates TIF-IA, inhibits pre-rRNA synthesis, and retards cell growth. The results provide a link between growth factor signaling, ribosome production, and cell growth, and may have a major impact on the mechanism of cell transformation (Zhao, 2003).
The ETS domain transcription factor Elk-1 is a direct target of the MAP kinase pathways. Phosphorylation of the Elk-1 transcriptional activation domain by MAP kinases triggers its activation. However, Elk-1 also contains two domains with repressive activities. One of these, the R motif, appears to function by suppressing the activity of the activation domain. SUMO (see Drosophila SUMO) modification of the R motif is required for this repressive activity. A dynamic interplay exists between the activating ERK MAP kinase pathway and the repressive SUMO pathway. ERK pathway activation leads to both phosphorylation of Elk-1 and loss of SUMO conjugation and, hence, to the loss of the repressive activity of the R motif. Thus, the reciprocal regulation of the activation and repressive activities are coupled by MAP kinase modification of Elk-1 (Yang, 2003).
Notch signals are important for lymphocyte development but downstream events that follow Notch signaling are not well understood. Signaling through Notch modulates the turnover of E2A proteins including E12 and E47, which are basic helix-loop-helix proteins crucial for B and T lymphocyte development. Notch-induced degradation requires phosphorylation of E47 by p42/p44 MAP kinases. Expression of the intracellular domain of Notch1 (N1-IC) enhances the association of E47 with the SCF(Skp2) E3 ubiquitin ligase and ubiquitination of E47, followed by proteasome-mediated degradation. Furthermore, N1-IC induces E2A degradation in B and T cells in the presence of activated MAP kinases. Activation of endogenous Notch receptors by treatment of splenocytes with anti-IgM or anti-CD3 plus anti-CD28 also leads to E2A degradation, which is blocked by the inhibitors of Notch activation or proteasome function. Notch-induced E2A degradation depends on the function of its downstream effector, RBP-Jkappa, probably to activate target genes involved in the ubiquitination of E2A proteins. Thus it is proposed that Notch regulates lymphocyte differentiation by controlling E2A protein turnover (Nie, 2003).
The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. These data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates (Matsumoto, 2004).
Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades. Given that there are genetic interactions between gro and components of the EGFR pathway, whether Gro is at a crossroad between this and other pathways was tested. This study shows that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways (Hasson, 2005).
Erk1/2 activation contributes to mouse ES cell pluripotency. This study found a direct role of Erk1/2 in modulating chromatin features required for regulated developmental gene expression. Erk2 binds to specific DNA sequence motifs typically accessed by Jarid2 and PRC2. Negating Erk1/2 activation leads to increased nucleosome occupancy and decreased occupancy of PRC2 and poised RNAPII at Erk2-PRC2-targeted developmental genes. Surprisingly, Erk2-PRC2-targeted genes are specifically devoid of TFIIH, known to phosphorylate RNA polymerase II (RNAPII) at serine-5, giving rise to its initiated form. Erk2 interacts with and phosphorylates RNAPII at its serine 5 residue, which is consistent with the presence of poised RNAPII as a function of Erk1/2 activation. These findings underscore a key role for Erk1/2 activation in promoting the primed status of developmental genes in mouse ES cells and suggest that the transcription complex at developmental genes is different than the complexes formed at other genes, offering alternative pathways of regulation (Tee, 2014).
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