scholarly journals Identification of key features required for efficient S-acylation and plasma membrane targeting of sprouty-2

2020 ◽  
Vol 133 (21) ◽  
pp. jcs249664
Author(s):  
Carolina Locatelli ◽  
Kimon Lemonidis ◽  
Christine Salaun ◽  
Nicholas C. O. Tomkinson ◽  
Luke H. Chamberlain
Keyword(s):  
Plasma Membrane ◽  
High Specificity ◽  
Rich Domain ◽  
Tumour Suppressor Protein ◽  
Growth Factor Signalling ◽  
Important Regulator ◽  
Α Helix ◽  
Low Activity ◽  
Insight Into ◽  
Cysteine Rich Domain

ABSTRACTSprouty-2 is an important regulator of growth factor signalling and a tumour suppressor protein. The defining feature of this protein is a cysteine-rich domain (CRD) that contains twenty-six cysteine residues and is modified by S-acylation. In this study, we show that the CRD of sprouty-2 is differentially modified by S-acyltransferase enzymes. The high specificity/low activity zDHHC17 enzyme mediated restricted S-acylation of sprouty-2, and cysteine-265 and -268 were identified as key targets of this enzyme. In contrast, the low specificity/high activity zDHHC3 and zDHHC7 enzymes mediated more expansive modification of the sprouty-2 CRD. Nevertheless, S-acylation by all enzymes enhanced sprouty-2 expression, suggesting that S-acylation stabilises this protein. In addition, we identified two charged residues (aspartate-214 and lysine-223), present on opposite faces of a predicted α-helix in the CRD, which are essential for S-acylation of sprouty-2. Interestingly, mutations that perturbed S-acylation also led to a loss of plasma membrane localisation of sprouty-2 in PC12 cells. This study provides insight into the mechanisms and outcomes of sprouty-2 S-acylation, and highlights distinct patterns of S-acylation mediated by different classes of zDHHC enzymes.

scholarly journals Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids

2013 ◽  
Vol 33 (5) ◽  
Author(s):  
Rajesh Bhardwaj ◽  
Hans-Michael Müller ◽  
Walter Nickel ◽  
Matthias Seedorf
Keyword(s):  
Plasma Membrane ◽  
Membrane Lipids ◽  
Lipid Binding ◽  
Dependent Manner ◽  
Activation Threshold ◽  
Concentration Dependent Manner ◽  
Rich Domain ◽  
Α Helix ◽  
Distinct Features ◽  
Ca2 Channels

Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding.

GFP-Tagged Protein Domains as Tools to Study Localized Second Messenger Function in Living Vells

1998 ◽  
Vol 4 (S2) ◽  
pp. 1014-1015
Author(s):  
T. Meyer ◽  
E. Oancea ◽  
T. Stauffer ◽  
M. N. Teruel
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Calcium Concentration ◽  
Protein Domains ◽  
Living Cells ◽  
Fluorescent Imaging ◽  
Protein Domain ◽  
Rich Domain ◽  
Kinase C ◽  
Cysteine Rich Domain

We have investigated the translocation of different GFP-tagged protein domains in response to various receptor stimuli. Confocal fluorescent imaging was used to study the localization of the GFP-tagged protein domain before, during and after the receptor stimuli was applied. A minimal cysteine-rich domain from protein kinase C was shown to be a useful tool to study localized diacylglycerol changes. Furthermore, the calcium-induced plasma membrane translocation of the GFP-tagged C2-domain from PKC could be used to study receptor-mediated changes in near plasma membrane calcium concentration. In addition, different pleckstrin homology domains (PH-domains) could be employed to measure changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI4.5P2) as well as of P13,4P2and PO,4,5P3. In combination, these fluorescent translocation probes provide a new means to investigate the local and temporal control of cell signaling processes in individual living cells.

Sprouty: a controversial role in receptor tyrosine kinase signalling pathways

2003 ◽  
Vol 31 (6) ◽  
pp. 1445-1446 ◽  
Author(s):  
X. Li ◽  
L. Wheldon ◽  
J.K. Heath
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Functional Divergence ◽  
Signalling Pathways ◽  
Signalling Molecules ◽  
Rich Domain ◽  
Growth Factor Signalling ◽  
Cysteine Rich Domain ◽  
Growth Factor Signalling Pathway

Sprouty was first identified in Drosophila as a novel antagonist of the fibroblast growth factor signalling pathway. Sprouty proteins comprise a big family, members of which are characterized by a cysteine-rich domain which confers inhibitory activity, whereas differences in the N-terminal region may be responsible for functional divergence. The role of Sprouty in RTK (receptor tyrosine kinase) signalling pathways is still controversial. Sprouty may negatively or positively regulate RTK signalling via differential interaction with different signalling molecules, and hence exert different mechanism of action.

scholarly journals KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timothy H. Tran ◽  
Albert H. Chan ◽  
Lucy C. Young ◽  
Lakshman Bindu ◽  
Chris Neale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Crystal Structures ◽  
Regulatory Region ◽  
Binding Domain ◽  
Published Data ◽  
Wild Type ◽  
Kras Mutations ◽  
Rich Domain ◽  
Structural Entity ◽  
Cysteine Rich Domain

AbstractThe first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Syndet is a novel SNAP-25 related protein expressed in many tissues

1997 ◽  
Vol 110 (4) ◽  
pp. 505-513 ◽  
Author(s):  
G. Wang ◽  
J.W. Witkin ◽  
G. Hao ◽  
V.A. Bankaitis ◽  
P.E. Scherer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurotransmitter Release ◽  
Signal Sequence ◽  
Coiled Coils ◽  
Rich Domain ◽  
The Family ◽  
Neuronal Tissues ◽  
Related Proteins ◽  
Cysteine Rich Domain ◽  
Novel Protein

SNAP-25 is a synaptosomal associated protein localized at the plasma membrane of nerve terminals. SNAP-25 associates with syntaxin 1 and vesicle-associated membrane protein-2 (VAMP-2) and is thought to form a complex essential for neurotransmitter release. We have identified syndet, a novel protein related to the family of SNAP-25 isoforms. Like SNAP-25, syndet has regions with high probability of forming coiled coils, a cysteine rich-domain, and lacks a signal sequence or transmembrane domains. Syndet is tightly bound to membranes, possibly by acylation within the cysteine-rich domain. Syndet is expressed in non-neuronal tissues. In adipocytes, syndet is found at the plasma membrane and in an intracellular compartment. The identification of syndet supports the hypothesis that multiple SNAP-25 related proteins ensure specificity of vesicle fusion at the cell surface.

scholarly journals KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

2020 ◽  
Author(s):  
Timothy H. Tran ◽  
Albert H. Chan ◽  
Lucy C. Young ◽  
Lakshman Bindu ◽  
Chris Neale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Crystal Structures ◽  
Kras Mutation ◽  
Regulatory Region ◽  
Binding Domain ◽  
Published Data ◽  
Wild Type ◽  
Rich Domain ◽  
Structural Entity ◽  
Cysteine Rich Domain

ABSTRACTA vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

scholarly journals Autoregulation of the Raf-1 serine/threonine kinase

1998 ◽  
Vol 95 (16) ◽  
pp. 9214-9219 ◽  
Author(s):  
Richard E. Cutler ◽  
Robert M. Stephens ◽  
Misty R. Saracino ◽  
Deborah K. Morrison
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Regulatory Region ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Rich Domain ◽  
Kinase C ◽  
Evolutionarily Conserved ◽  
Phosphorylation Event ◽  
Cysteine Rich Domain

The Raf-1 serine/threonine kinase is a key protein involved in the transmission of many growth and developmental signals. In this report, we show that autoinhibition mediated by the noncatalytic, N-terminal regulatory region of Raf-1 is an important mechanism regulating Raf-1 function. The inhibition of the regulatory region occurs, at least in part, through binding interactions involving the cysteine-rich domain. Events that disrupt this autoinhibition, such as mutation of the cysteine-rich domain or a mutation mimicking an activating phosphorylation event (Y340D), alleviate the repression of the regulatory region and increase Raf-1 activity. Based on the striking similarites between the autoregulation of the serine/threonine kinases protein kinase C, Byr2, and Raf-1, we propose that relief of autorepression and activation at the plasma membrane is an evolutionarily conserved mechanism of kinase regulation.

scholarly journals Transcripts encoding the sperm surface protein tMDC II are non-functional in the human

1999 ◽  
Vol 341 (3) ◽  
pp. 771-775 ◽  
Author(s):  
Jan FRAYNE ◽  
Elizabeth A. C. DIMSEY ◽  
Jenny A. JURY ◽  
Len HALL
Keyword(s):  
Plasma Membrane ◽  
Macaca Fascicularis ◽  
Surface Protein ◽  
Polyclonal Antiserum ◽  
Human Testis ◽  
Domain Family ◽  
Western Blots ◽  
Rich Domain ◽  
Proposed Model ◽  
Cysteine Rich Domain

Five members of the MDC (metalloproteinase-like,disintegrin-like cysteine-rich domain) family of proteins (fertilin α, fertilin β, tMDC I, tMDC II and tMDC III) are expressed on the surface of macaque (Macaca fascicularis) sperm, where they have been proposed to play a role in sperm-egg binding via an interaction between their disintegrin-like domain and one or more integrins on the egg plasma membrane. Of these, two (fertilin α and tMDC I) have recently been shown to be non-functional in the human. Here we report the existence of multiple isoforms of human tMDC II transcripts in the human, all of which are also non-functional owing to the presence of deletions and in-frame termination codons, when compared with the macaque orthologue, a finding which is further supported by the lack of immunoreactivity on Western blots of human testis and sperm extracts probed with a macaque anti-tMDC II polyclonal antiserum. These results are discussed in the context of our proposed model for multiple proteins implicated in sperm-egg interactions.

scholarly journals Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

2015 ◽  
Vol 35 (11) ◽  
pp. 2007-2023 ◽  
Author(s):  
Tadasuke Tsukiyama ◽  
Akimasa Fukui ◽  
Sayuri Terai ◽  
Yoichiro Fujioka ◽  
Keisuke Shinada ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Target Genes ◽  
Pdz Domain ◽  
Ring Finger ◽  
Missense Mutations ◽  
Ring Finger Domain ◽  
Rich Domain ◽  
Insight Into ◽  
Cysteine Rich Domain

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.

Plasma membrane targeting of SNAP-25 increases its local concentration and is necessary for SNARE complex formation and regulated exocytosis

2002 ◽  
Vol 115 (16) ◽  
pp. 3341-3351 ◽  
Author(s):  
Darshan K. Koticha ◽  
Ellen E. McCarthy ◽  
Giulia Baldini
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Snare Complex ◽  
Local Concentration ◽  
Hormone Release ◽  
Regulated Exocytosis ◽  
Rich Domain ◽  
Membrane Bound ◽  
Snare Complex Formation ◽  
Cysteine Rich Domain

SNAP-25 is an integral protein of the plasma membrane involved in neurotransmission and hormone secretion. The cysteine-rich domain of SNAP-25 is essential for membrane binding and plasma-membrane targeting. However, this domain is not required for SNARE complex formation and fusion of membranes in vitro. In this paper, we describe an `intact-cell'-based system designed to compare the effect of similar amounts of membrane-bound and soluble SNAP-25 proteins on regulated exocytosis. In transfected neuroblastoma cells,Botulinum neurotoxin E (BoNT/E), a protease that cleaves SNAP-25, blocks regulated release of hormone. However, hormone release is rescued by expressing a wild-type SNAP-25 protein resistant to the toxin. BoNT/E-resistant SNAP-25 proteins lacking the cysteine-rich domain or with all the cysteines substituted by alanines do not form SNARE complexes or rescue regulated exocytosis when expressed at the same level as membrane-bound SNAP-25, which is approximately four-fold higher than the endogenous protein. We conclude that the cysteine-rich domain of SNAP-25 is essential for Ca2+-dependent hormone release because, by targeting SNAP-25 to the plasma membrane, it increases its local concentration, leading to the formation of enough SNARE complexes to support exocytosis.

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