scholarly journals Src activates retrograde membrane traffic through phosphorylation of GBF1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Joanne Chia ◽  
Shyi-Chyi Wang ◽  
Sheena Wee ◽  
David James Gill ◽  
Felicia Tay ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Small Gtpase ◽  
Protein Glycosylation ◽  
Intramolecular Rearrangement ◽  
Wide Spread ◽  
Src Tyrosine Kinase ◽  
Tyrosine Residues ◽  
C Terminus ◽  
Tubular Transport ◽  
Sec7 Domain

The Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP Exchange factor (GEF) GBF1 and small GTPase Arf1. Here we show that Src induces the formation of tubular transport carriers containing GALNTs. The kinase phosphorylates GBF1 on 10 tyrosine residues; two of them, Y876 and Y898 are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes GBF1 binding to the GTPase; molecular modeling suggests partial melting of the Sec7 domain and intramolecular rearrangement. GBF1 mutants defective for these rearrangements prevent binding, carrier formation and GALNTs relocation, while phosphomimetic GBF1 mutants induce tubules. In sum, Src promotes GALNTs relocation by promoting GBF1 binding to Arf1. Based on residue conservation, similar regulation of GEF-Arf complexes by tyrosine phosphorylation could be a conserved and wide-spread mechanism.

scholarly journals Src activates retrograde membrane traffic through phosphorylation of GBF1

2020 ◽  
Author(s):  
Joanne Chia ◽  
Samuel Wang ◽  
Sheena Wee ◽  
David James Gill ◽  
Lee Violette ◽  
...  
Keyword(s):  
Partial Melting ◽  
Tyrosine Phosphorylation ◽  
Small Gtpase ◽  
Protein Glycosylation ◽  
Wide Spread ◽  
Src Tyrosine Kinase ◽  
Tyrosine Residues ◽  
C Terminus ◽  
Tubular Transport ◽  
Sec7 Domain

AbstractThe Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP Exchange factor (GEF) GBF1 and small GTPase Arf1. Here we show that Src induces the formation of tubular transport carriers containing GALNTs through the activation of a GBF1-Arf1 complex. The complex is initiated by phosphorylation on GBF1 on 10 tyrosine residues; two of them, Y876 and Y898 are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes partial melting of the Sec7 domain, favoring binding to the GTPase. Perturbation of these rearrangements prevent GALNTs relocation. In sum, Src promotes GALNTs relocation by favoring binding of GBF1 to Arf1. Regulation of a GEF-Arf axis by tyrosine phosphorylation appears to be a highly conserved and wide-spread mechanism.

scholarly journals Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation

2019 ◽  
Vol 20 (24) ◽  
pp. 6307 ◽  
Author(s):  
Aura E. Ionescu ◽  
Mihaela Mentel ◽  
Cristian V.A. Munteanu ◽  
Livia E. Sima ◽  
Eliza C. Martin ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Src Kinase ◽  
Protein Tyrosine Phosphatases ◽  
Phosphorylation Sites ◽  
Src Tyrosine Kinase ◽  
Eyes Absent ◽  
Tyrosine Residues ◽  
Proliferative Effect ◽  
Phosphorylation Pattern

Eyes absent (EYA) are non-thiol-based protein tyrosine phosphatases (PTPs) that also have transcriptional co-activator functions. Their PTP activity is involved in various pathologies. Recently, we demonstrated that Src tyrosine kinase phosphorylates human EYA3 by controlling its subcellular localization. We also found EYA3′s ability to autodephosphorylate, while raising the question if the two opposing processes could be involved in maintaining a physiologically adequate level of phosphorylation. Using native and bottom-up mass spectrometry, we performed detailed mapping and characterization of human EYA3 Src-phosphorylation sites. Thirteen tyrosine residues with different phosphorylation and autodephosphorylation kinetics were detected. Among these, Y77, 96, 237, and 508 displayed an increased resistance to autodephosphorylation. Y77 and Y96 were found to have the highest impact on the overall EYA3 phosphorylation. Using cell cycle analysis, we showed that Y77, Y96, and Y237 are involved in HEK293T proliferation. Mutation of the three tyrosine residues abolished the pro-proliferative effect of EYA3 overexpression. We have also identified a Src-induced phosphorylation pattern of EYA3 in these cells. These findings suggest that EYA3′s tyrosine phosphorylation sites are non-equivalent with their phosphorylation levels being under the control of Src-kinase activity and of EYA3′s autodephosphorylation.

scholarly journals Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells

2007 ◽  
Vol 18 (10) ◽  
pp. 3978-3992 ◽  
Author(s):  
Asli Oztan ◽  
Mark Silvis ◽  
Ora A. Weisz ◽  
Neil A. Bradbury ◽  
Shu-Chan Hsu ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Small Gtpase ◽  
Junctional Complex ◽  
Recycling Endosomes ◽  
Permeabilized Cells ◽  
Exocyst Complex ◽  
C Terminus ◽  
Streptolysin O ◽  
Early Endosomes ◽  
Endocytic Traffic

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

scholarly journals Specific Phosphorylation of p120-Catenin Regulatory Domain Differently Modulates Its Binding to RhoA

2006 ◽  
Vol 27 (5) ◽  
pp. 1745-1757 ◽  
Author(s):  
Julio Castaño ◽  
Guiomar Solanas ◽  
David Casagolda ◽  
Imma Raurell ◽  
Patricia Villagrasa ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Regulatory Mechanism ◽  
Adherens Junction ◽  
Small Gtpase ◽  
Regulatory Domain ◽  
P120 Catenin ◽  
Tyrosine Residues ◽  
Rhoa Activity ◽  
Intracellular Proteins

ABSTRACT p120-catenin is an adherens junction-associated protein that controls E-cadherin function and stability. p120-catenin also binds intracellular proteins, such as the small GTPase RhoA. In this paper, we identify the p120-catenin N-terminal regulatory domain as the docking site for RhoA. Moreover, we demonstrate that the binding of RhoA to p120-catenin is tightly controlled by the Src family-dependent phosphorylation of p120-catenin on tyrosine residues. The phosphorylation induced by Src and Fyn tyrosine kinases on p120-catenin induces opposite effects on RhoA binding. Fyn, by phosphorylating a residue located in the regulatory domain of p120-catenin (Tyr112), inhibits the interaction of this protein with RhoA. By contrast, the phosphorylation of Tyr217 and Tyr228 by Src promotes a better affinity of p120-catenin towards RhoA. In agreement with these biochemical data, results obtained in cell lines support the important role of these phosphorylation sites in the regulation of RhoA activity by p120-catenin. Taken together, these observations uncover a new regulatory mechanism acting on p120-catenin that contributes to the fine-tuned regulation of the RhoA pathways during specific signaling events.

scholarly journals The Dense-Core Vesicle Maturation Protein CCCP-1 Binds RAB-2 and Membranes through its C-terminal Domain

2017 ◽  
Author(s):  
Jérôme Cattin-Ortolá ◽  
Irini Topalidou ◽  
Annie Dosey ◽  
Alexey J. Merz ◽  
Michael Ailion
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Dense Core ◽  
Binding Motif ◽  
Dense Core Vesicle ◽  
Terminal Domain ◽  
C Terminus

AbstractDense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.Synopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

scholarly journals Structure and Dynamics of Mono- vs. Doubly Lipidated Rab5 in Membranes

2019 ◽  
Vol 20 (19) ◽  
pp. 4773 ◽  
Author(s):  
Eileen Münzberg ◽  
Matthias Stein
Keyword(s):  
Bound States ◽  
Cysteine Residue ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Endosomal Trafficking ◽  
Membrane Composition ◽  
Insertion Depth ◽  
Rab Protein ◽  
Guanosine Diphosphate ◽  
C Terminus

The Rab5 small GTPase is a regulator of endosomal trafficking and vesicle fusion. It possesses two adjacent cysteine residues for post-translational geranylgeranylation at its C-terminus for the protein to associate with the early endosome membrane. We compare the effect of mono-lipidification of only one cysteine residue with the doubly modified, fully functional Rab protein in both guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states and in different membranes (one, three, and six-component membranes). Molecular simulations show that the mono-geranylgeranylated protein is less strongly associated with the membranes and diffuses faster than the doubly lipidated protein. The geranylgeranyl anchor membrane insertion depth is smaller and the protein–membrane distance distribution is broad and uncharacteristic for the membrane composition. The mono-geranylgeranylated protein reveals an unspecific association with the membrane and an orientation at the membrane that does not allow a nucleotide-specific recruitment of further effector proteins. This work shows that double-lipidification is critical for Rab5 to perform its physiological function and mono-geranylgeranylation renders it membrane-associated but non-functional.

scholarly journals Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability

2020 ◽  
Vol 295 (31) ◽  
pp. 10677-10688
Author(s):  
Po-Ju Chen ◽  
Diego Zelada ◽  
Dina Cheryne Belhasan ◽  
Mohammed Akaaboune
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tyrosine Residues ◽  
C Terminus ◽  
The Stability ◽  
High Level

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap–α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap–α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

Molecular Modeling and Virtual Screening of Molecular Inhibitors for Leptospiral Collagenase

2021 ◽  
Author(s):  
Vikram Kumar ◽  
Nagesh Srikaku ◽  
Veeranarayanan Surya Aathmanathan ◽  
Padikara K Satheeshkumar ◽  
Madanan Gopalakrishnan Madathiparambil ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Modeling ◽  
Active Site ◽  
Binding Sites ◽  
Catalytic Site ◽  
Simulation Studies ◽  
C Terminus ◽  
Ligand Binding Sites ◽  
N Terminus ◽  
The Stability

Abstract Collagenase is a virulence factor which facilitates the invasion of pathogenic Leptospira into the host. In the present study, the model of Leptopsiral collagenase was constructed by employing threading method with the crystal structure of collagenase G. Three ligand binding sites at N- terminus, catalytic site and C-terminus were predicted by Metapocket server. Among sixty seven inhibitors from the ChEBI and Zinc databases, Protohypericin is predicted as the best inhibitor since it binds at the catalytic site of Leptopsiral collagenase. Molecular dynamic simulation studies validated the stability of interaction between the active site of Leptospiral collagenase and Protohypericin. The docking and molecular simulation studies corroborated the potential of the ligand to curb leptospiral infection.

scholarly journals Analysis of insulin receptor phosphorylation sites in intact rat liver cells by two-dimensional phosphopeptide mapping. Predominance of the tris-phosphorylated form of the kinase domain after stimulation by insulin

1991 ◽  
Vol 275 (1) ◽  
pp. 15-21 ◽  
Author(s):  
T Issad ◽  
J M Tavaré ◽  
R M Denton
Keyword(s):  
Insulin Receptor ◽  
Rat Liver ◽  
Insulin Receptors ◽  
Kinase Domain ◽  
Two Dimensional ◽  
Tyrosine Residues ◽  
C Terminus ◽  
Rat Liver Cells ◽  
Wheat Germ Lectin ◽  
Dimensional Mapping

1. Insulin receptors were partially purified from rat liver by chromatography on wheat-germ-lectin-Sepharose. Incubation with [gamma-32P]ATP in the presence of insulin resulted in increased phosphorylation of the beta-subunit on both tyrosine and serine residues. Two-dimensional mapping of tryptic peptides showed that, in agreement with previous studies using preparations of receptors from other sources, the tyrosine residues involved were the three tyrosines in the kinase domain (corresponding to tyrosines 1158, 1162 and 1163 of the human receptor) plus two tyrosines close to the C-terminus (corresponding to tyrosines 1328 and 1334). 2. The effects of insulin on the phosphorylation of receptors within intact rat liver cells were determined by incubating cells in the presence of [32P]Pi for 50 min and then with or without insulin for a further 10 min. The labelled receptors were then rapidly isolated by sequential use of wheat-germ-lectin-Sepharose chromatography and immuno-isolation using a monoclonal antibody to the C-terminal end of the beta-subunit. 3. Insulin was found to increase overall phosphorylation of the receptor nearly 3-fold. Two-dimensional mapping was then carried out in combination with phosphoamino acid analysis. This revealed that the pattern of phosphorylation of the receptors in cells incubated in the absence and presence of insulin exhibited a number of marked differences from that observed in previous studies on intact cells, which had been restricted to cells expressing very high levels of insulin receptors such as certain hepatoma-derived cells or cells transfected with insulin receptor cDNA. The differences in the effects of insulin included a larger increase in the proportion of receptors being phosphorylated on the three tyrosine residues of the kinase domain, no apparent phosphorylation of the two tyrosine residues close to the C-terminus and no increase in either threonine or overall serine phosphorylation. 4. The receptors appeared to be phosphorylated on a number of different serine residues in cells incubated in the absence of insulin. Evidence for both increases and decreases in the phosphorylation of specific serine residues on addition of insulin was obtained. 5. It is concluded that care should be taken when extrapolating findings on the phosphorylation of the insulin receptor within cultured cells to more physiological situations.

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