Phosphorylation of the Ras nucleotide exchange factor son of sevenless by mitogen-activated protein kinase.

Semaphorins are a large family of transmembrane and secreted proteins that signal primarily through the receptor plexin. Semaphorins have been characterized in the nervous system as axon guidance cues; however, they have also been shown to control development of other cellular systems such as the vasculature and lungs. As the role of semaphorins outside of the nervous system has broadened, so has elucidation of the intracellular signalling pathways they initiate. Previously, we and others have shown that plexin-B1 activates RhoA through the binding and activation of RhoGEF (guanine nucleotide-exchange factor)/LARG (leukaemia-associated RhoGEF) in response to semaphorin 4D stimulation. In the present study, we show that semaphorin 4D activates the MAPK (mitogen-activated protein kinase) pathway. We have found that the mechanism of activation requires the C-terminus of plexin-B1 and the activation of RhoA.

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Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence

Journal of Biological Chemistry ◽

10.1074/jbc.m115.691238 ◽

2015 ◽

Vol 291 (4) ◽

pp. 1703-1718 ◽

Cited By ~ 16

Author(s):

Uybach Vo ◽

Navratna Vajpai ◽

Liz Flavell ◽

Romel Bobby ◽

Alexander L. Breeze ◽

...

Keyword(s):

Intrinsic Fluorescence ◽

The Novel ◽

Nucleotide Exchange ◽

Allosteric Site ◽

Site Specific ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Catalytic Sites ◽

Son Of Sevenless

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

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Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast

The Journal of Cell Biology ◽

10.1083/jcb.200704079 ◽

2008 ◽

Vol 181 (7) ◽

pp. 1073-1081 ◽

Cited By ~ 84

Author(s):

Nadia Vadaie ◽

Heather Dionne ◽

Darowan S. Akajagbor ◽

Seth R. Nickerson ◽

Damian J. Krysan ◽

...

Keyword(s):

Mapk Pathway ◽

Active Form ◽

Cell Polarization ◽

Mitogen Activated Protein Kinase ◽

Aspartyl Protease ◽

Nucleotide Exchange ◽

Mapk Activation ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Mitogen Activated Protein

Signaling mucins are cell adhesion molecules that activate RAS/RHO guanosine triphosphatases and their effector mitogen-activated protein kinase (MAPK) pathways. We found that the Saccharomyces cerevisiae mucin Msb2p, which functions at the head of the Cdc42p-dependent MAPK pathway that controls filamentous growth, is processed into secreted and cell-associated forms. Cleavage of the extracellular inhibitory domain of Msb2p by the aspartyl protease Yps1p generated the active form of the protein by a mechanism incorporating cellular nutritional status. Activated Msb2p functioned through the tetraspan protein Sho1p to induce MAPK activation as well as cell polarization, which involved the Cdc42p guanine nucleotide exchange factor Cdc24p. We postulate that cleavage-dependent activation is a general feature of signaling mucins, which brings to light a novel regulatory aspect of this class of signaling adhesion molecule.

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Protein Kinase A-Dependent Phosphorylation Modulates β1Pix Guanine Nucleotide Exchange Factor Activity through 14-3-3β Binding

Molecular and Cellular Biology ◽

10.1128/mcb.00898-07 ◽

2007 ◽

Vol 28 (5) ◽

pp. 1679-1687 ◽

Cited By ~ 34

Author(s):

Ahmed Chahdi ◽

Andrey Sorokin

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Guanine Nucleotide Exchange Factor ◽

Small Gtpases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Kinase A

ABSTRACT β1Pix is a guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42 which has been shown to mediate signaling pathways leading to cytoskeletal reorganization. In the present study, we show that the basal association between endogenous βPix and endogenous 14-3-3β was increased after forskolin stimulation and significantly inhibited by protein kinase A inhibitor. However, forskolin stimulation failed to increase the interaction between 14-3-3β and a β1Pix mutant that is insensitive to protein kinase A phosphorylation, β1Pix(S516A, T526A). We present evidence indicating that forskolin-induced binding of 14-3-3β to β1Pix results in inhibition of Rac1 GTP loading in 293 cells and in vitro. Furthermore, we show that deletion of 10 amino acid residues within the leucine zipper domain is sufficient to block β1Pix homodimerization and 14-3-3β binding and modulates β1Pix-GEF activity. These residues also play a crucial role in β1Pix intracellular localization. These results indicate that 14-3-3β negatively affects the GEF activity of dimeric β1Pix only. Altogether, these results provide a mechanistic insight into the role of 14-3-3β in modulating β1Pix-GEF activity.

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Central role of the exchange factor GEF-H1 in TNF-α–induced sequential activation of Rac, ADAM17/TACE, and RhoA in tubular epithelial cells

Molecular Biology of the Cell ◽

10.1091/mbc.e12-09-0661 ◽

2013 ◽

Vol 24 (7) ◽

pp. 1068-1082 ◽

Cited By ~ 25

Author(s):

Faiza Waheed ◽

Qinghong Dan ◽

Yasaman Amoozadeh ◽

Yuqian Zhang ◽

Susumu Tanimura ◽

...

Keyword(s):

Epithelial Cells ◽

Growth Factor Receptor ◽

Small Gtpase ◽

Mitogen Activated Protein Kinase ◽

Erk Pathway ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Rhoa Activation ◽

Tnf Α ◽

Mitogen Activated Protein

Transactivation of the epidermal growth factor receptor (EGFR) by tumor necrosis factor-α (TNF-α) is a key step in mediating RhoA activation and cytoskeleton and junction remodeling in the tubular epithelium. In this study we explore the mechanisms underlying TNF-α–induced EGFR activation. We show that TNF-α stimulates the TNF-α convertase enzyme (TACE/a disintegrin and metalloproteinase-17), leading to activation of the EGFR/ERK pathway. TACE activation requires the mitogen-activated protein kinase p38, which is activated through the small GTPase Rac. TNF-α stimulates both Rac and RhoA through the guanine nucleotide exchange factor (GEF)-H1 but by different mechanisms. EGFR- and ERK-dependent phosphorylation at the T678 site of GEF-H1 is a prerequisite for RhoA activation only, whereas both Rac and RhoA activation require GEF-H1 phosphorylation on S885. Of interest, GEF-H1-mediated Rac activation is upstream from the TACE/EGFR/ERK pathway and regulates T678 phosphorylation. We also show that TNF-α enhances epithelial wound healing through TACE, ERK, and GEF-H1. Taken together, our findings can explain the mechanisms leading to hierarchical activation of Rac and RhoA by TNF-α through a single GEF. This mechanism could coordinate GEF functions and fine-tune Rac and RhoA activation in epithelial cells, thereby promoting complex functions such as sheet migration.

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