Phosphorylation of PP2Ac by PKC is a key regulatory step in the PP2A-switch-dependent AKT dephosphorylation that leads to apoptosis.

BACKGROUND: Although GqPCR activation often leads to cell survival by activating the PI3K/AKT pathway, it was previously shown that in several cell types AKT activity is reduced and leads to JNK activation and apoptosis. The mechanism of AKT inactivation in these cells involves an IGBP1-coupled PP2Ac switch that induces the dephosphorylation and inactivation of both PI3K and AKT. However, the machinery involved in the initiation of PP2A switch is not known. METHODS: We used phospho-mass spectrometry to identify the phosphorylation site of PP2Ac, and raised specific antibodies to follow the regulation of this phosphorylation. Other phosphorylations were monitored by commercial antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by a TUNEL assay as well as PARP1 cleavage using SDS-PAGE and Western blotting. RESULTS: We identified Ser24 as a phosphorylation site in PP2Ac. The phosphorylation is mediated mainly by classical PKCs (PKCα and PKCß) but not by novel PKCs (PKCδ and PKCε). By replacing the phosphorylated residue with either unphosphorylatable or phosphomimetic residues (S24A and S24E), we found that this phosphorylation event is necessary and sufficient to mediate the PP2A switch, which ultimately induces AKT inactivation, and a robust JNK-dependent apoptosis. CONCLUSION: Our results show that the PP2A switch is induced by PKC-mediated phosphorylation of Ser24-PP2Ac and that this phosphorylation leads to apoptosis upon GqPCR induction of various cells. We propose that this mechanism may provide an unexpected way to treat some cancer types or problems in the endocrine machinery.

Special Issue: MAPK Signaling Cascades in Human Health and Diseases.

In order to survive and fulfil their functions, cells of any organism need to be able to respond to a large number of extracellular factors, also termed extracellular stimuli [...].

CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions.

The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact-dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34(+) progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP-protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells.

JNK Cascade-Induced Apoptosis-A Unique Role in GqPCR Signaling.

The response of cells to extracellular signals is mediated by a variety of intracellular signaling pathways that determine stimulus-dependent cell fates. One such pathway is the cJun-N-terminal Kinase (JNK) cascade, which is mainly involved in stress-related processes. The cascade transmits its signals via a sequential activation of protein kinases, organized into three to five tiers. Proper regulation is essential for securing a proper cell fate after stimulation, and the mechanisms that regulate this cascade may involve the following: (1) Activatory or inhibitory phosphorylations, which induce or abolish signal transmission. (2) Regulatory dephosphorylation by various phosphatases. (3) Scaffold proteins that bring distinct components of the cascade in close proximity to each other. (4) Dynamic change of subcellular localization of the cascade's components. (5) Degradation of some of the components. In this review, we cover these regulatory mechanisms and emphasize the mechanism by which the JNK cascade transmits apoptotic signals. We also describe the newly discovered PP2A switch, which is an important mechanism for JNK activation that induces apoptosis downstream of the Gq protein coupled receptors. Since the JNK cascade is involved in many cellular processes that determine cell fate, addressing its regulatory mechanisms might reveal new ways to treat JNK-dependent pathologies.

GqPCR-stimulated dephosphorylation of AKT is induced by an IGBP1-mediated PP2A switch.

BACKGROUND: G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH. METHODS: Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage. RESULTS: We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation. CONCLUSION: Our results show a stimulated shift of PP2Ac from PI3K to AKT termed "PP2A switch" that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation. Video Abstract.

ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK.

The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase family. Using various stimulated rodent cells and kinase activation techniques, we identified a 46-kDa ERK. The kinetics of activation of this ERK isoform was similar to that of ERK1 and ERK2 under most but not all circumstances. We purified this isoform from rat cells followed by its cloning. The sequence of this isoform revealed that it is an alternatively spliced version of the 44-kDa ERK1 and therefore we termed it ERK1b. Interestingly, this isoform had a 26-amino acid insertion between residues 340 and 341 of ERK1, which results from Intron 7 insertion to the sequence. Examining the expression pattern, we found that ERK1b is detected mainly in rat and particularly in Ras-transformed Rat1 cells. In this cell line, ERK1b was more sensitive to extracellular stimulation than ERK1 and ERK2. Moreover, unlike ERK1 and ERK2, ERK1b had a very low binding affinity to MEK1. This low interaction led to nuclear localization of this isoform when expressed together with MEK1 under conditions in which ERK1 and ERK2 are retained in the cytoplasm. In addition, ERK1b was not coimmunoprecipitated with MEK1. We identified a new, 46-kDa ERK alternatively spliced isoform. Our results indicate that this isoform is the major one to respond to exogenous stimulation in Ras-transformed cells, probably due to its differential regulation by MAPK/ERK kinase and by phosphatases.

Calcium-Mediated Interactions Regulate the Subcellular Localization of Extracellular Signal-Regulated Kinases (ERKs).

BACKGROUND/AIMS: The subcellular localization of ERK1 and ERK2 (ERKs) in cells, which is important for proper signaling, may be regulated through protein-protein interactions. However, the proteins involved and the way they are regulated to affect localization is not entirely understood. METHODS: In order to identify the interacting proteins upon varying conditions, we used co-immunoprecipitation of ERK, active ERK and its binding CRS mutant. In addition, we examined the effect of intracellular calcium on the binding using calcium chelators and ionophores, analyzing the binding using silver stain, mass spectrometry and immunoblotting. The effect of calcium on ERK localization was examined using immunofluorescent staining and Western blotting. RESULTS: We found that inactive ERK2 interacts with a large number of proteins through its CRS/CD domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERKs' interactions influenced also the localization of ERKs, as calcium chelators enhanced nuclear translocation, while elevated calcium levels prevented it. This effect of calcium was also apparent upon the physiological lysophosphatidic acid stimulation, where ERKs translocation was delayed compared to that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERKs' translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins such as NUP153. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. CONCLUSION: Upon stimulation, ERKs are phosphorylated and released from their cytoplasmic anchors to allow shuttling into the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and therefore also their spatiotemporal regulation. Thus, calcium regulates ERKs localization, which is important for the compartmentalization of ERKs with their proper substrates, and thereby their signaling specificity.

Nuclear P38: Roles in Physiological and Pathological Processes and Regulation of Nuclear Translocation.

The p38 mitogen-activated protein kinase (p38MAPK, termed here p38) cascade is a central signaling pathway that transmits stress and other signals to various intracellular targets in the cytoplasm and nucleus. More than 150 substrates of p38α/ß have been identified, and this number is likely to increase. The phosphorylation of these substrates initiates or regulates a large number of cellular processes including transcription, translation, RNA processing and cell cycle progression, as well as degradation and the nuclear translocation of various proteins. Being such a central signaling cascade, its dysregulation is associated with many pathologies, particularly inflammation and cancer. One of the hallmarks of p38α/ß signaling is its stimulated nuclear translocation, which occurs shortly after extracellular stimulation. Although p38α/ß do not contain nuclear localization or nuclear export signals, they rapidly and robustly translocate to the nucleus, and they are exported back to the cytoplasm within minutes to hours. Here, we describe the physiological and pathological roles of p38α/ß phosphorylation, concentrating mainly on the ill-reviewed regulation of p38α/ß substrate degradation and nuclear translocation. In addition, we provide information on the p38α/ß 's substrates, concentrating mainly on the nuclear targets and their role in p38α/b functions. Finally, we also provide information on the mechanisms of nuclear p38α/b translocation and its use as a therapeutic target for p38α/ß-dependent diseases.

C-Src is Activated by the EGF Receptor in a Pathway that Mediates JNK and ERK Activation by Gonadotropin-Releasing Hormone in COS7 Cells.

The key participants in G-protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. The prototypical GPCR is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here, we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via Gαi and the EGF receptor, without the involvement of Hb-EGF or PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and PI3K. ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Beside the main pathway, the dissociated Gßγ and ß-arrestin may initiate additional (albeit minor) pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other GnRHR-bearing cells, indicating that GnRH can utilize various signaling mechanisms for MAPK activation. The unique pathway elucidated here, in which c-Src and PI3K are sequentially activated downstream of the EGF receptor, may serve as a prototype of signaling mechanisms by GnRHR and additional GPCRs in various cell types.

Beta-Like Importins Mediate the Nuclear Translocation of MAPKs.

BACKGROUND/AIMS: The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de-novo gene expression. However, the molecular mechanisms of this translocation is not well understood, although some studies suggest that much of this translocation may be mediated by beta-like importins (Imps). Here we undertook to study the stimulated nuclear shuttling of JNK and p38 MAPKs. METHODS: For this purpose, we used coimmunoprecipitation, proximity ligation assay, gel filtration and immunostaining to examine the mechanism of nuclear translocation of these proteins. RESULTS: We found that JNK and p38 MAPKs translocate into the nucleus in a Ran dependent, but NLS- or NTS-independent manner, unrelated to their catalytic activity. We show that this translocation involves three ß-like Imps, 3, 7 and 9. Knockdown of these Imps inhibits the nuclear translocation of the MAPKs, and thereby, phosphorylation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38α/ß bind to either Imp7 or Imp9 upon stimulated post-translational modifications of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope where Imp3 remains, while Imp7 or Imp9 escort the MAPKs into the nucleus. CONCLUSION: These results suggest that ß-like Imps are central mediators of stimulated nuclear translocation of signaling proteins, providing a central level of regulation of the induction of cellular processes such as transcription upon stimulation.

Nuclear ERK Translocation is Mediated by Protein Kinase CK2 and Accelerated by Autophosphorylation.

BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. METHODS: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. RESULTS: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. CONCLUSION: These results provide an important role of CK2 in regulating nuclear ERK activities.

The Nuclear Translocation of Mitogen-Activated Protein Kinases: Molecular Mechanisms and Use as Novel Therapeutic Target.

The mitogen-activated protein kinase (MAPK) cascades are central signaling pathways that play a central role in the regulation of most stimulated cellular processes including proliferation, differentiation, stress response and apoptosis. Currently 4 such cascades are known, each termed by its downstream MAPK components: the extracellular signal-regulated kinase 1/2 (ERK1/2), cJun-N-terminal kinase (JNK), p38 and ERK5. One of the hallmarks of these cascades is the stimulated nuclear translocation of their MAPK components using distinct mechanisms. ERK1/2 are shuttled into the nucleus by importin7, JNK and p38 by a dimer of importin3 with either importin9 or importin7, and ERK5 by importin-α/ß. Dysregulation of these cascades often results in diseases, including cancer and inflammation, as well as developmental and neurological disorders. Much effort has been invested over the years in developing inhibitors to the MAPK cascades to combat these diseases. Although some inhibitors are already in clinical use or clinical trials, their effects are hampered by development of resistance or adverse side-effects. Recently, our group developed 2 myristoylated peptides: EPE peptide, which inhibits the interaction of ERK1/2 with importin7, and PERY peptide, which prevents JNK/p38 interaction with either importin7 or importin9. These peptides block the nuclear translocation of their corresponding kinases, resulting in prevention of several cancers, while the PERY peptide also inhibits inflammation-induced diseases. These peptides provide a proof of concept for the use of the nuclear translocation of MAPKs as therapeutic targets for cancer and/or inflammation.

Nuclear ERK: Mechanism of Translocation, Substrates, and Role in Cancer.

The extracellular signal-regulated kinases 1/2 (ERK) are central signaling components that regulate stimulated cellular processes such as proliferation and differentiation. When dysregulated, these kinases participate in the induction and maintenance of various pathologies, primarily cancer. While ERK is localized in the cytoplasm of resting cells, many of its substrates are nuclear, and indeed, extracellular stimulation induces a rapid and robust nuclear translocation of ERK. Similarly to other signaling components that shuttle to the nucleus upon stimulation, ERK does not use the canonical importinmechanism of nuclear translocation. Rather, it has its own unique nuclear translocation signal (NTS) that interacts with importin7 to allow stimulated shuttling via the nuclear pores. Prevention of the nuclear translocation inhibits proliferation of B-Raf- and N/K-Ras-transformed cancers. This effect is distinct from the one achieved by catalytic Raf and MEK inhibitors used clinically, as cells treated with the translocation inhibitors develop resistance much more slowly. In this review, we describe the mechanism of ERK translocation, present all its nuclear substrates, discuss its role in cancer and compare its translocation to the translocation of other signaling components. We also present proof of principle data for the use of nuclear ERK translocation as an anti-cancer target. It is likely that the prevention of nuclear ERK translocation will eventually serve as a way to combat Ras and Raf transformed cancers with less side-effects than the currently used drugs.

Pigment Epithelium-Derived Factor and its Phosphomimetic Mutant Induce JNK-Dependent Apoptosis and P38-Mediated Migration Arrest.

BACKGROUND/AIMS: Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis, and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated, and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity, and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (EC), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level. METHODS: Here we used Western blotting, as well as in vitro binding, proliferation, apoptosis and migration assays to follow the signaling components responsible for the PEDF and EEE-PEDF effects. RESULTS: We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis, and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing stronger effect. The stronger activity of EEE-PEDF was correlated to a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found respectively regulated by differential activation of two distinct MAPK pathways, namely JNK and p38. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, while p38 leads to antimigratory effect in both EC and cancer cells. CONCLUSION: These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.

Gq-Induced Apoptosis is Mediated by AKT Inhibition That Leads to PKC-Induced JNK Activation.

BACKGROUND/AIMS: Gq protein-coupled receptors (GqPCRs) regulate various cellular processes including mainly proliferation and differentiation. In a previous study, we found that in prostate cancer cells, the GqPCR of GnRH induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Since it was thought that GqPCR induces mainly activation of AKT, we undertook to examine how general is this phenomenon and understand its signaling. METHODS: We used various cells to follow the phosphorylation of signaling components using western blotting. RESULTS: In a screen of 21 cell lines, we found that PKC activation results in the reduction of AKT activity, which correlates nicely to JNK activation and in some cases to apoptosis. To further understand the signaling pathways involved in this stimulation, we studied in detail the SVOG-4O and αT3-1 cells. We found that PGF2α and GnRH agonist (GnRH-a) indeed induce significant Gq- and PKC- dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists on c-Src activation of the JNK cascade and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3, to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. CONCLUSION: Our results present a general mechanism that mediates a GqPCR-induced, death receptors-independent, apoptosis in physiological, as well as cancer-related systems.

Mitotic Golgi translocation of ERK1c is mediated by a PI4KIIIß-14-3-3γ shuttling complex.

Golgi fragmentation is a highly regulated process that allows division of the Golgi complex between the two daughter cells. The mitotic reorganization of the Golgi is accompanied by a temporary block in Golgi functioning, as protein transport in and out of the Golgi stops. Our group has previously demonstrated the involvement of the alternatively spliced variants ERK1c and MEK1b (ERK1 is also known as MAPK3, and MEK1 as MAP2K1) in mitotic Golgi fragmentation. We had also found that ERK1c translocates to the Golgi at the G2 to M phase transition, but the molecular mechanism underlying this recruitment remains unknown. In this study, we narrowed the translocation timing to prophase and prometaphase, and elucidated its molecular mechanism. We found that CDK1 phosphorylates Ser343 of ERK1c, thereby allowing the binding of phosphorylated ERK1c to a complex that consists of PI4KIIIß (also known as PI4KB) and the 14-3-3γ dimer (encoded by YWHAB). The stability of the complex is regulated by protein kinase D (PKD)-mediated phosphorylation of PI4KIIIß. The complex assembly induces the Golgi shuttling of ERK1c, where it is activated by MEK1b, and induces Golgi fragmentation. Our work shows that protein shuttling to the Golgi is not completely abolished at the G2 to M phase transition, thus integrating several independent Golgi-regulating processes into one coherent pathway.

Activation of Signaling Cascades by Weak Extremely Low Frequency Electromagnetic Fields.

BACKGROUND/AIMS: Results from recent studies suggest that extremely low frequency magnetic fields (ELF-MF) interfere with intracellular signaling pathways related to proliferative control. The mitogen-activated protein kinases (MAPKs), central signaling components that regulate essentially all stimulated cellular processes, include the extracellular signal-regulated kinases 1/2 (ERK1/2) that are extremely sensitive to extracellular cues. Anti-phospho-ERK antibodies serve as a readout for ERK1/2 activation and are able to detect minute changes in ERK stimulation. The objective of this study was to explore whether activation of ERK1/2 and other signaling cascades can be used as a readout for responses of a variety of cell types, both transformed and non-transformed, to ELF-MF. METHODS: We applied ELF-MF at various field strengths and time periods to eight different cell types with an exposure system housed in a tissue culture incubator and followed the phosphorylation of MAPKs and Akt by western blotting. RESULTS: We found that the phosphorylation of ERK1/2 is increased in response to ELF-MF. However, the phosphorylation of ERK1/2 is likely too low to induce ELF-MF-dependent proliferation or oncogenic transformation. The p38 MAPK was very slightly phosphorylated, but JNK or Akt were not. The effect on ERK1/2 was detected for exposures to ELF-MF strengths as low as 0.15 µT and was maximal at ∼10 µT. We also show that ERK1/2 phosphorylation is blocked by the flavoprotein inhibitor diphenyleneiodonium, indicating that the response to ELF-MF may be exerted via NADP oxidase similar to the phosphorylation of ERK1/2 in response to microwave radiation. CONCLUSIONS: Our results further indicate that cells are responsive to ELF-MF at field strengths much lower than previously suspected and that the effect may be mediated by NADP oxidase. However, the small increase in ERK1/2 phosphorylation is probably insufficient to affect proliferation and oncogenic transformation. Therefore, the results cannot be regarded as proof of the involvement of ELF-MF in cancer in general or childhood leukemia in particular.

Nuclear to cytoplasmic shuttling of ERK promotes differentiation of muscle stem/progenitor cells.

The transition between the proliferation and differentiation of progenitor cells is a key step in organogenesis, and alterations in this process can lead to developmental disorders. The extracellular signal-regulated kinase 1/2 (ERK) signaling pathway is one of the most intensively studied signaling mechanisms that regulates both proliferation and differentiation. How a single molecule (e.g. ERK) can regulate two opposing cellular outcomes is still a mystery. Using both chick and mouse models, we shed light on the mechanism responsible for the switch from proliferation to differentiation of head muscle progenitors and implicate ERK subcellular localization. Manipulation of the fibroblast growth factor (FGF)-ERK signaling pathway in chick embryos in vitro and in vivo demonstrated that blockage of this pathway accelerated myogenic differentiation, whereas its activation diminished it. We next examined whether the spatial subcellular localization of ERK could act as a switch between proliferation (nuclear ERK) and differentiation (cytoplasmic ERK) of muscle progenitors. A myristoylated peptide that blocks importin 7-mediated ERK nuclear translocation induced robust myogenic differentiation of muscle progenitor/stem cells in both head and trunk. In the mouse, analysis of Sprouty mutant embryos revealed that increased ERK signaling suppressed both head and trunk myogenesis. Our findings, corroborated by mathematical modeling, suggest that ERK shuttling between the nucleus and the cytoplasm provides a switch-like transition between proliferation and differentiation of muscle progenitors.

The ERK cascade inhibitors: Towards overcoming resistance.

The RAS-ERK pathway plays a major regulatory role in various cellular processes. This pathway is hyperactivated and takes an active part in the malignant transformation of more than 85% of cancers. The hyperactivation is mainly due to oncogenic activating mutations in the pathway's components RAS, RAF and MEK, but also due to indirect mechanisms in cells transformed by other oncogenes. Various inhibitors targeting the different tiers of the cascade have been successfully developed and clinically approved, while some are still undergoing preclinical and clinical evaluation. Treatments with the clinically approved RAF and MEK inhibitors have substantially improved the clinical outcome of metastatic mutated-BRAF melanoma. However, the rapid emergence of drug resistance of initially responsive cancers and limited efficacy towards other cancers has led to only marginal patient benefit. Deciphering the molecular mechanisms underlying intrinsic or acquired resistance is a necessity in order to enhance the treatment efficacy of ERK-addicted cancers. Therefore, many studies in the past 5 years embarked on this campaign, revealing several resistance mechanisms. These include, expression of drug-resistant RAF isoforms, molecular or genetic alterations of active downstream components, overexpression of upstream components of the cascade that can reactivate ERK and other survival-related pathways. The understanding of these molecular resistance mechanisms led to further development of drugs that can overcome drug resistance, including our own effort aiming to prevent the nuclear translocation of ERK without affecting its activation. In this review we will focus on the mechanisms underlying drug resistance and efforts to develop activity-independent, more efficacious, antitumor drugs.

Specific phosphorylation and activation of ERK1c by MEK1b: a unique route in the ERK cascade.

Extracellular signal-regulated kinases (ERKs) are key signaling molecules that regulate a large number of cellular processes, including mitosis. We showed previously that ERK1c, an alternatively spliced form of ERK1, facilitates mitotic Golgi fragmentation without the involvement of ERK1 and ERK2. Here we demonstrate that activation of ERK1c is mainly mediated by mitogen-activated protein kinase (MAPK)/ERK kinase 1b (MEK1b), which is an alternatively spliced form of MEK1 that was previously considered an inactive kinase. MEK1b phosphorylation and activity are preferentially stimulated by nocodazole, to induce its specific activity toward ERK1c. MEK1/2, on the other hand, preferentially target ERK1/2 in response to growth factors, such as EGF. As previously demonstrated for ERK1c, also MEK1b expression and activity are elevated during mitosis, and thereby enhance Golgi fragmentation and mitotic rate. MEK1 activity is also increased during mitosis, but this isoform facilitates mitotic progression without affecting the Golgi architecture. These results illustrate that the ERK cascade is divided into two routes: the classic MEK1/2-ERK1/2 and the splice-variant MEK1b-ERK1c, each of which regulates distinct cellular processes and thus extends the cascade specificity.