scholarly journals Ubc2, an Ortholog of the Yeast Ste50p Adaptor, Possesses a Basidiomycete-Specific Carboxy Terminal Extension Essential for Pathogenicity Independent of Pheromone Response

2008 ◽  
Vol 21 (1) ◽  
pp. 110-121 ◽  
Author(s):  
Steven J. Klosterman ◽  
Alfredo D. Martinez-Espinoza ◽  
David L. Andrews ◽  
Jeffrey R. Seay ◽  
Scott E. Gold
Keyword(s):  
Map Kinase ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Filamentous Growth ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Sh3 Domains ◽  
Additional Signal ◽  
C Terminus ◽  
Mitogen Activated Protein

Proteins involved in the mitogen-activated protein (MAP) kinase pathway controlling mating, morphogenesis, and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing sterile α motif (SAM) and ras association (RA) domains typical of Ste50-like adaptor proteins found in the fungal phylum Ascomycota, Ubc2 also contains two C-terminal SH3 domains. Yeast two-hybrid assays indicated that Ubc2 interacts with the MAP kinase-kinase kinase Ubc4 via the SAM domains at each of their respective N-termini. Site-directed mutagenesis of ubc2 and complementation analyses revealed that the SAM and RA domains of Ubc2 are essential for filamentous growth. These data support a role for the ascomycete-like N-terminus of Ubc2 in regulating pheromone-responsive mating and morphogenesis analogous to the role of Ste50p in Saccharomyces cerevisiae. In contrast, C-terminal deletion mutants were fully capable of filamentous growth and mating. However, surprisingly, these strains were nonpathogenic. Further, directed mutagenesis of the C-terminus revealed that both SH3 domains are required for pathogenicity. These results suggest that the Basidiomycota have retained the mating and morphogenetic functions of Ste50-type proteins in the N-terminal half of their Ubc2-type adaptors but, additionally, have integrated C-terminal SH3 domains that are critical for additional signal transduction mechanisms, including those that lead to pathogenesis.

scholarly journals ASC filament formation serves as a signal amplification mechanism for inflammasomes

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Mathias S. Dick ◽  
Lorenzo Sborgi ◽  
Sebastian Rühl ◽  
Sebastian Hiller ◽  
Petr Broz
Keyword(s):  
Signal Amplification ◽  
Cytokine Production ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Inflammasome Activation ◽  
Pyrin Domain ◽  
Efficient Processing ◽  
Amplification Mechanism

Abstract A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD). Here we show that assembly of the ASC speck involves oligomerization of ASCPYD into filaments and cross-linking of these filaments by ASCCARD. ASC mutants with a non-functional CARD only assemble filaments but not specks, and moreover disrupt endogenous specks in primary macrophages. Systematic site-directed mutagenesis of ASCPYD is used to identify oligomerization-deficient ASC mutants and demonstrate that ASC speck formation is required for efficient processing of IL-1β, but dispensable for gasdermin-D cleavage and pyroptosis induction. Our results suggest that the oligomerization of ASC creates a multitude of potential caspase-1 activation sites, thus serving as a signal amplification mechanism for inflammasome-mediated cytokine production.

scholarly journals The Ustilago maydis ubc4 and ubc5 Genes Encode Members of a MAP Kinase Cascade Required for Filamentous Growth

2000 ◽  
Vol 13 (7) ◽  
pp. 781-786 ◽  
Author(s):  
David L. Andrews ◽  
John D. Egan ◽  
María E. Mayorga ◽  
Scott E. Gold
Keyword(s):  
Map Kinase ◽  
Ustilago Maydis ◽  
Filamentous Growth ◽  
Mitogen Activated Protein Kinase ◽  
Genetic Determinants ◽  
Map Kinase Cascade ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Suppressor Mutants ◽  
Corn Smut

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

scholarly journals Generation ofEscherichia coliintimin derivatives with differing biological activities using site‐directed mutagenesis of the intimin C‐terminus domain

1998 ◽  
Vol 29 (2) ◽  
pp. 559-570 ◽  
Author(s):  
Gad Frankel ◽  
Alan D. Philips ◽  
Michaela Novakova ◽  
Miranda Batchelor ◽  
Susan Hicks ◽  
...  
Keyword(s):  
Biological Activities ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
C Terminus

scholarly journals BWMK1, a Novel MAP Kinase Induced by Fungal Infection and Mechanical Wounding in Rice

1999 ◽  
Vol 12 (12) ◽  
pp. 1064-1073 ◽  
Author(s):  
Chaozu He ◽  
Steven Haw Tien Fong ◽  
Daichang Yang ◽  
Guo-Liang Wang
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Magnaporthe Grisea ◽  
Mechanical Wounding ◽  
C Terminus ◽  
Acid Protein ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Rice Leaves ◽  
Activation Motif

The activation of the mitogen-activated protein (MAP) kinases by different environmental stresses has been previously observed in several dicot plant species. Here, we report the isolation of a novel MAP kinase in rice that is induced during infection by the blast fungus Magnaporthe grisea or upon mechanical wounding. The gene is designated as BWMK1 for blast- and wound-induced MAP kinase. The cDNA of BWMK1 was isolated from rice leaves challenged by the blast pathogen. Transcripts of the corresponding gene accumulated in rice leaves 4 h after blast inoculation and 30 min after mechanical wounding. This gene encodes a 506 amino acid protein that contains a new dual-phosphorylation activation motif TDY and about 150 unique amino acids on its C terminus. In-gel kinase activity and immunoprecipitation assays confirmed that BWMK1 is a functional MAP kinase. These results show that BWMK1 is a new member of the plant MAP kinase family and may mediate both defense and wound signaling in rice.

SH3-mediated targeting of Wrch1/RhoU by multiple adaptor proteins

2013 ◽  
Vol 394 (3) ◽  
pp. 421-432 ◽  
Author(s):  
Sarah L. Risse ◽  
Belen Vaz ◽  
Matthew F. Burton ◽  
Pontus Aspenström ◽  
Roland P. Piekorz ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Adaptor Protein ◽  
Structural Difference ◽  
Adaptor Proteins ◽  
Sh3 Domain ◽  
High Avidity ◽  
Sh3 Domains ◽  
Pxxp Motif ◽  
The Impact

Abstract Wrch1/RhoU is an atypical member of the Rho family. A major structural difference is the extended N-terminus of Wrch1 (nWrch1) containing three putative SH3 domain-binding motifs whose specificities are unknown. To define the impact of this extended region on coupling Wrch1 to cellular signaling, we analyzed in this study nWrch1 interaction with Src homology 3 (SH3) domains of different adaptor proteins. Using sedimentation and isothermal titration calorimetric (ITC) measurements, we identified isolated SH3 domains of growth factor receptor-bound protein 2 (Grb2), noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), c-Src, chicken tumor virus no. 10 (CT 10) regulator kinase 1 (Crk1), and p120 as low-affinity Wrch1-binding partners. Interestingly, under cell-based conditions, nWrch1 bound tightly to endogenous Grb2 and Nck, but not to Crk, c-Src, or p120. Consistent with this, a very tight nWrch1 interaction with full-length Grb2 and Nck1 was confirmed in vitro by ITC measurements indicating that high avidity of the adaptor proteins can compensate for the low affinity of their SH3 domains. Peptide analysis revealed that the central PxxP motif of nWrch1, which employs a minimal consensus sequence of eight amino acids with an essential arginine next to the PxxP motif, is responsible for these interactions. Thus, novel functional insights from this study suggest that multiple upstream signals may converge on Wrch1 directly through its SH3 domain-binding properties.

scholarly journals Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase

1993 ◽  
Vol 90 (23) ◽  
pp. 10952-10956 ◽  
Author(s):  
R H Chen ◽  
C Abate ◽  
J Blenis
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Downstream Signaling ◽  
C Terminus ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.

scholarly journals Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes

2020 ◽  
Vol 295 (52) ◽  
pp. 18604-18613
Author(s):  
Jonas Schäfer ◽  
Lucas Förster ◽  
Ingo Mey ◽  
Theofilos Papadopoulos ◽  
Nils Brose ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adaptor Protein ◽  
Iminodiacetic Acid ◽  
Membrane System ◽  
Adaptor Proteins ◽  
Nickel Salt ◽  
Hybrid Membranes ◽  
C Terminus ◽  
Protein Components

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically 'open' and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.

scholarly journals Grb2 monomer–dimer equilibrium determines normal versus oncogenic function

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Zamal Ahmed ◽  
Zahra Timsah ◽  
Kin M. Suen ◽  
Nathan P. Cook ◽  
Gilbert R. Lee ◽  
...  
Keyword(s):  
Map Kinase ◽  
Cancer Progression ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Breast Cancers ◽  
Mitogen Activated Protein ◽  
Self Association

Abstract The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

scholarly journals The membrane immunoglobulin receptor utilizes a Shc/Grb2/hSOS complex for activation of the mitogen-activated protein kinase cascade in a B-cell line

1995 ◽  
Vol 307 (1) ◽  
pp. 215-223 ◽  
Author(s):  
G Kumar ◽  
S Wang ◽  
S Gupta ◽  
A Nel
Keyword(s):  
Protein Kinase ◽  
B Cell ◽  
Map Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Adaptor Protein ◽  
Immunoglobulin M ◽  
Membrane Immunoglobulin ◽  
Mitogen Activated Protein ◽  
Kinase Cascade

Ligation of membrane immunoglobulin M (mIgM) receptor in the Ramos B-cell line induced tyrosine phosphorylation of several intracellular substrates, including the adaptor protein. Shc. Phosphorylated Shc could be seen to associate with Grb2 in a complex which included hSOS. Inasmuch as hSOS is involved in p21ras activation, we also demonstrated that mIgM ligation activated a Ras-dependent kinase cascade in which sequential activation of Raf-1 and MEK-1 culminates in the activation of p42 mitogen-activated protein (MAP) kinase (ERK-2). The tumour promoter and protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA), also activated Raf-1, MEK-1, and MAP kinase in Ramos cells, but did not induce tyrosine phosphorylation of Shc or Shc/Grb2 association. Okadaic acid, another tumour promoter and serine/threonine phosphatase inhibitor, activated p42 MAP kinase without activating Raf-1 or MEK-1, suggesting the existence of a serine/threonine phosphatase which directly regulates MAP kinase activity.

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