ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1

There is considerable evidence that cross-talk between the Hedgehog pathway and MAPK signaling pathways occurs in several types of cancer, and contributes to the emergence of clinical resistance to Hedgehog pathway inhibitors. Here, we demonstrate that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. We show that ERK2 phosphorylates GLI1 on three evolutionarily-conserved target sites (S102, S116 and S130) located near the high-affinity binding site for the negative regulator SUFU; furthermore, these phosphorylation events cooperate to weaken the affinity of GLI1-SUFU binding by over 25 fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily-conserved SUFU-affinity-modulating region. In cultured mammalian cells, mutant GLI1 variants containing phosphomimetic substitutions of S102, S116 and S130 displayed an increased ability to drive transcription. We conclude that of multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

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Role of Cdc42-Cla4 Interaction in the Pheromone Response of Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00102-06 ◽

2006 ◽

Vol 6 (2) ◽

pp. 317-327 ◽

Cited By ~ 17

Author(s):

Melanie Heinrich ◽

Tim Köhler ◽

Hans-Ulrich Mösch

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Map Kinase ◽

Cell Cycle Arrest ◽

Map Kinases ◽

Negative Regulator ◽

Cycle Arrest ◽

Mitogen Activated Protein ◽

Novel Alleles

ABSTRACT In Saccharomyces cerevisiae, the highly conserved Rho-type GTPase Cdc42 is essential for cell division and controls cellular development during mating and invasive growth. The role of Cdc42 in mating has been controversial, but a number of previous studies suggest that the GTPase controls the mitogen-activated protein (MAP) kinase cascade by activating the p21-activated protein kinase (PAK) Ste20. To further explore the role of Cdc42 in pheromone-stimulated signaling, we isolated novel alleles of CDC42 that confer resistance to pheromone. We find that in CDC42(V36A) and CDC42(V36A, I182T) mutant strains, the inability to undergo pheromone-induced cell cycle arrest correlates with reduced phosphorylation of the mating MAP kinases Fus3 and Kss1 and with a decrease in mating efficiency. Furthermore, Cdc42(V36A) and Cdc42(V36A, I182T) proteins show reduced interaction with the PAK Cla4 but not with Ste20. We also show that deletion of CLA4 in a CDC42(V36A, I182T) mutant strain suppresses pheromone resistance and that overexpression of CLA4 interferes with pheromone-induced cell cycle arrest and MAP kinase phosphorylation in CDC42 wild-type strains. Our data indicate that Cla4 has the potential to act as a negative regulator of the mating pathway and that this function of the PAK might be under control of Cdc42. In conclusion, our study suggests that control of pheromone signaling by Cdc42 not only depends on Ste20 but also involves interaction of the GTPase with Cla4.

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Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts.

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2517 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2517-2528 ◽

Cited By ~ 118

Author(s):

J Posada ◽

J Sanghera ◽

S Pelech ◽

R Aebersold ◽

J A Cooper

Keyword(s):

Cell Cycle ◽

Map Kinase ◽

Tyrosine Phosphorylation ◽

Oocyte Maturation ◽

Mammalian Cells ◽

Map Kinases ◽

Kinase Activity ◽

Mitotic Cell ◽

Meiotic Maturation ◽

Sea Star

Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle.

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Specific Inactivation and Nuclear Anchoring of Extracellular Signal-Regulated Kinase 2 by the Inducible Dual-Specificity Protein Phosphatase DUSP5

Molecular and Cellular Biology ◽

10.1128/mcb.25.5.1830-1845.2005 ◽

2005 ◽

Vol 25 (5) ◽

pp. 1830-1845 ◽

Cited By ~ 134

Author(s):

Margret Mandl ◽

David N. Slack ◽

Stephen M. Keyse

Keyword(s):

Map Kinase ◽

Mammalian Cells ◽

Map Kinases ◽

Nuclear Translocation ◽

P38 Map Kinase ◽

Nuclear Accumulation ◽

Extracellular Signal Regulated Kinase ◽

Amino Terminal ◽

Extracellular Signal ◽

Map Kinase Phosphatase

ABSTRACT The mechanisms which determine the nuclear accumulation and inactivation of the extracellular signal-regulated kinase 1 (ERK1) or ERK2 mitogen-activated protein (MAP) kinases are poorly understood. Here we demonstrate that DUSP5, an inducible nuclear phosphatase, interacts specifically with ERK2 via a kinase interaction motif (KIM) within its amino-terminal noncatalytic domain. This binding determines the substrate specificity of DUSP5 in vivo, as it inactivates ERK2 but not Jun N-terminal protein kinase or p38 MAP kinase. Using green fluorescent protein fusions, we identify within this same domain of DUSP5 a functional nuclear localization signal (NLS) which functions independently of the KIM. Moreover, we demonstrate that the expression of DUSP5 causes both nuclear translocation and sequestration of inactive ERK2. Nuclear anchoring is ERK2 specific and requires both interactions between the DUSP5 KIM and the common docking site of ERK2 and a functional NLS within DUSP5. Finally, the expression of a catalytically inactive mutant of DUSP5 also tethers ERK2 within the nucleus. Furthermore, this nuclear ERK2 is phosphorylated by MAP kinase kinase in response to growth factors and also activates transcription factor Elk-1. We conclude that DUSP5 is an inducible nuclear ERK-specific MAP kinase phosphatase that functions as both an inactivator of and a nuclear anchor for ERK2 in mammalian cells. In addition, our data indicate that the cytoplasm may not be an exclusive site of MAP kinase activation.

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The N and C Termini of the Splice Variants of the Human Mitogen-Activated Protein Kinase-Interacting Kinase Mnk2 Determine Activity and Localization

Molecular and Cellular Biology ◽

10.1128/mcb.23.16.5692-5705.2003 ◽

2003 ◽

Vol 23 (16) ◽

pp. 5692-5705 ◽

Cited By ~ 76

Author(s):

Gert C. Scheper ◽

Josep L. Parra ◽

Mary Wilson ◽

Barbara van Kollenburg ◽

Alfred C. O. Vertegaal ◽

...

Keyword(s):

Map Kinase ◽

Binding Site ◽

Mammalian Cells ◽

Map Kinases ◽

Splice Variants ◽

Mitogen Activated Protein Kinase ◽

Initiation Factor ◽

Mitogen Activated Protein

ABSTRACT The cap-binding eukaryotic initiation factor eIF4E is phosphorylated by the mitogen-activated protein (MAP) kinase-interacting kinases (Mnk's). Three forms of the Mnk's exist in human cells: Mnk1, Mnk2a, and Mnk2b. These last two are derived from the same gene by alternative splicing and differ only at their C termini. While Mnk2a contains a MAP kinase-binding site in this region, Mnk2b lacks such a sequence and is much less readily activated by MAP kinases in vitro. Expression of Mnk2b in mammalian cells leads to increased phosphorylation of eIF4E, showing that it acts as an eIF4E kinase in vivo. While Mnk2a is cytoplasmic, a substantial amount of Mnk2b is found in the nucleus. Both enzymes contain a stretch of basic residues in their N termini that plays a role in binding to eIF4G and functions as a nuclear localization signal. Binding of eIF4G or nuclear import appears to be regulated by the C terminus of Mnk2a. Furthermore, the MAP kinase-binding site of Mnk2a regulates nuclear entry. Within the nucleus, Mnk2b and certain variants of Mnk2a that are present in the nucleus colocalize with the promyelocytic leukemia protein PML, which also binds to eIF4E.

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Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1434 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1434-1440 ◽

Cited By ~ 43

Author(s):

S Bellier ◽

M F Dubois ◽

E Nishida ◽

G Almouzni ◽

O Bensaude

Keyword(s):

Xenopus Laevis ◽

Rna Polymerase ◽

Map Kinase ◽

Rna Polymerase Ii ◽

Oocyte Maturation ◽

Mammalian Cells ◽

Map Kinases ◽

Kinase Activity ◽

Cyclin B1 ◽

Xenopus Laevis Oocyte

Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34(cdc2)off kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells.

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Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60

eLife ◽

10.7554/elife.57591 ◽

2020 ◽

Vol 9 ◽

Author(s):

Chun Kew ◽

Wenming Huang ◽

Julia Fischer ◽

Raja Ganesan ◽

Nirmal Robinson ◽

...

Keyword(s):

Mammalian Cells ◽

Bacterial Pathogen ◽

Active Role ◽

Suppressive Effect ◽

Mapk Signaling ◽

Splicing Factor ◽

Dependent Manner ◽

Loss Of Function ◽

C Elegans ◽

Evolutionarily Conserved

Splicing is a vital cellular process that modulates important aspects of animal physiology, yet roles in regulating innate immunity are relatively unexplored. From genetic screens in C. elegans, we identified splicing factor RNP-6/PUF60 whose activity suppresses immunity, but promotes longevity, suggesting a tradeoff between these processes. Bacterial pathogen exposure affects gene expression and splicing in a rnp-6 dependent manner, and rnp-6 gain and loss-of-function activities reveal an active role in immune regulation. Another longevity promoting splicing factor, SFA-1, similarly exerts an immuno-suppressive effect, working downstream or parallel to RNP-6. RNP-6 acts through TIR-1/PMK-1/MAPK signaling to modulate immunity. The mammalian homolog, PUF60, also displays anti-inflammatory properties, and its levels swiftly decrease after bacterial infection in mammalian cells, implying a role in the host response. Altogether our findings demonstrate an evolutionarily conserved modulation of immunity by specific components of the splicing machinery.

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Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2517-2528.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2517-2528 ◽

Cited By ~ 1

Author(s):

J Posada ◽

J Sanghera ◽

S Pelech ◽

R Aebersold ◽

J A Cooper

Keyword(s):

Cell Cycle ◽

Map Kinase ◽

Tyrosine Phosphorylation ◽

Oocyte Maturation ◽

Mammalian Cells ◽

Map Kinases ◽

Kinase Activity ◽

Mitotic Cell ◽

Meiotic Maturation ◽

Sea Star

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Apoptosis and development

Essays in Biochemistry ◽

10.1042/bse0390011 ◽

2003 ◽

Vol 39 ◽

pp. 11-24 ◽

Cited By ~ 6

Author(s):

Justin V McCarthy

Keyword(s):

Drosophila Melanogaster ◽

Mammalian Cells ◽

Cell Number ◽

Model Organisms ◽

Biological Processes ◽

Nematode Caenorhabditis Elegans ◽

Apoptotic Pathway ◽

Genetic Pathways ◽

Evolutionarily Conserved ◽

Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

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Differential Effects of Toll-Like Receptor Activation and Differential Mediation by MAP Kinases of Immune Responses in Microglial Cells

Cellular and Molecular Neurobiology ◽

10.1007/s10571-021-01127-x ◽

2021 ◽

Author(s):

Jaedeok Kwon ◽

Christos Arsenis ◽

Maria Suessmilch ◽

Alison McColl ◽

Jonathan Cavanagh ◽

...

Keyword(s):

Map Kinase ◽

Microglial Activation ◽

Map Kinases ◽

Mrna Level ◽

Cell Types ◽

Receptor Activation ◽

Microglial Cells ◽

Toll Like Receptor ◽

Disease States ◽

Nitrite Production

AbstractMicroglial activation is believed to play a role in many psychiatric and neurodegenerative diseases. Based largely on evidence from other cell types, it is widely thought that MAP kinase (ERK, JNK and p38) signalling pathways contribute strongly to microglial activation following immune stimuli acting on toll-like receptor (TLR) 3 or TLR4. We report here that exposure of SimA9 mouse microglial cell line to immune mimetics stimulating TLR4 (lipopolysaccharide—LPS) or TLR7/8 (resiquimod/R848), results in marked MAP kinase activation, followed by induction of nitric oxide synthase, and various cytokines/chemokines. However, in contrast to TLR4 or TLR7/8 stimulation, very few effects of TLR3 stimulation by poly-inosine/cytidine (polyI:C) were detected. Induction of chemokines/cytokines at the mRNA level by LPS and resiquimod were, in general, only marginally affected by MAP kinase inhibition, and expression of TNF, Ccl2 and Ccl5 mRNAs, along with nitrite production, were enhanced by p38 inhibition in a stimulus-specific manner. Selective JNK inhibition enhanced Ccl2 and Ccl5 release. Many distinct responses to stimulation of TLR4 and TLR7 were observed, with JNK mediating TNF protein induction by the latter but not the former, and suppressing Ccl5 release by the former but not the latter. These data reveal complex modulation by MAP kinases of microglial responses to immune challenge, including a dampening of some responses. They demonstrate that abnormal levels of JNK or p38 signalling in microglial cells will perturb their profile of cytokine and chemokine release, potentially contributing to abnormal inflammatory patterns in CNS disease states.

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Signal-mediated localization of Candida albicans pheromone response pathway components

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkaa033 ◽

2020 ◽

Author(s):

Anna Carolina Borges Pereira Costa ◽

Raha Parvizi Omran ◽

Chris Law ◽

Vanessa Dumeaux ◽

Malcolm Whiteway

Keyword(s):

Candida Albicans ◽

Map Kinase ◽

Nuclear Localization ◽

Map Kinases ◽

Critical Role ◽

Cell Periphery ◽

Pheromone Response ◽

Pheromone Response Pathway ◽

Localization Signal ◽

In Cells

Abstract Candida albicans opaque cells release pheromones to stimulate cells of opposite mating type to activate their pheromone response pathway. Although this fungal pathogen shares orthologous proteins involved in the process with Saccharomyces cerevisiae, the pathway in each organism has unique characteristics. We have used GFP-tagged fusion proteins to investigate the localization of the scaffold protein Cst5, as well as the MAP kinases Cek1 and Cek2, during pheromone response in C. albicans. In wild-type cells, pheromone treatment directed Cst5-GFP to surface puncta concentrated at the tips of mating projections. These puncta failed to form in cells defective in either the Gα or β subunits. However, they still formed in response to pheromone in cells missing Ste11, but with the puncta distributed around the cell periphery in the absence of mating projections. These puncta were absent from hst7Δ/Δ cells, but could be detected in the ste11Δ/Δ hst7Δ/Δ double mutant. Cek2-GFP showed a strong nuclear localization late in the response, consistent with a role in adaptation, while Cek1-GFP showed a weaker, but early increase in nuclear localization after pheromone treatment. Activation loop phosphorylation of both Cek1 and Cek2 required the presence of Ste11. In contrast to Cek2-GFP, which showed no localization signal in ste11Δ/Δ cells, Cek1-GFP showed enhanced nuclear localization that was pheromone independent in the ste11Δ/Δ mutant. The results are consistent with CaSte11 facilitating Hst7-mediated MAP kinase phosphorylation and also playing a potentially critical role in both MAP kinase and Cst5 scaffold localization.

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