Src activates retrograde membrane traffic through phosphorylation of GBF1

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.

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Src activates retrograde membrane traffic through phosphorylation of GBF1

eLife ◽

10.7554/elife.68678 ◽

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.

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Plasma membrane Ca2+-ATPase isoform 4b is phosphorylated on tyrosine 1176 in activated human platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613551 ◽

2003 ◽

Vol 89 (01) ◽

pp. 122-131 ◽

Cited By ~ 27

Author(s):

Tina Wan ◽

Martin Zabe ◽

William Dean

Keyword(s):

Plasma Membrane ◽

Tyrosine Phosphorylation ◽

Platelet Activation ◽

Kinase Inhibitors ◽

Human Platelets ◽

Tyrosine Residues ◽

Phosphorylation Motif ◽

C Terminus ◽

Activation Sequence

SummaryPlasma membrane Ca2+-ATPase isoform 4b (PMCA4b) is phosphorylated on a tyrosine residue during platelet activation resulting in inhibition of its ATPase activity. We now report that tyrosine 1176 (Y1176) in the carboxyl (C-) terminal domain of PMCA4b is the phosphorylated residue. Two tyrosine residues located in the C-terminus of PMCA4b, Y1122 and Y1176 can be removed by calpain-dependent cleavage. This truncation removes all of the tyrosine phosphates added to PMCA during platelet activation. Sequence analysis indicates that Y1176 is a likely substrate for focal adhesion kinase (FAK), while Y1122 is not located in a tyrosine phosphorylation motif. This is the same residue we reported earlier to be phosphorylated by Src kinase in vitro. Thus we conclude that Y1176 is the only tyrosine phosphorylated during platelet activation. Results of co-immunoprecipitation, treatment with tyrosine kinase inhibitors and integrin inhibition experiments suggest that FAK is responsible for PMCA4b tyrosine phosphorylation during platelet activation.

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Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00429.2001 ◽

2002 ◽

Vol 283 (4) ◽

pp. G893-G899 ◽

Cited By ~ 5

Author(s):

Monica C. Chen ◽

Travis E. Solomon ◽

Eduardo Perez Salazar ◽

Robert Kui ◽

Enrique Rozengurt ◽

...

Keyword(s):

Gastric Mucosa ◽

Epithelial Cells ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Tyrosine Phosphorylation ◽

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Paracellular Permeability ◽

Maximal Response ◽

Src Tyrosine Kinase

Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr416 was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 μM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr416 in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this Gs-coupled receptor in primary epithelial cells.

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Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src

Biochemical Journal ◽

10.1042/bj2990613 ◽

1994 ◽

Vol 299 (3) ◽

pp. 613-621 ◽

Cited By ~ 25

Author(s):

P W Modderman ◽

A E G K von dem Borne ◽

A Sonnenberg

Keyword(s):

Tyrosine Phosphorylation ◽

Cell Activation ◽

Secretory Granules ◽

Protein S ◽

Kinase Assay ◽

Intact Cells ◽

Tyrosine Residues ◽

Reducing Conditions ◽

Sds Page

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src.

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Suppression of src transformation by overexpression of full-length GTPase-activating protein (GAP) or of the GAP C terminus

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2819-2825.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2819-2825

Author(s):

J E DeClue ◽

K Zhang ◽

P Redford ◽

W C Vass ◽

D R Lowy

Keyword(s):

Tyrosine Phosphorylation ◽

Biochemical Analysis ◽

Full Length ◽

Gtpase Activating Protein ◽

Focus Formation ◽

Coding Sequences ◽

Transformed Cell Line ◽

C Terminus ◽

Nih 3T3

Overexpression of the full-length GTPase-activating protein (GAP) has recently been shown to suppress c-ras transformation of NIH 3T3 cells but not v-ras transformation (36). Here, we show that focus formation induced by c-src was inhibited by approximately 80% when cotransfected with a plasmid encoding full-length GAP. In a similar assay, focus formation by the activated c-src (Tyr-527 to Phe) gene was inhibited by 33%. Cotransfection of the GAP C terminus coding sequences (which encode the GTPase-accelerating domain) with c-src or c-src527F inhibited transformation more efficiently than did the full-length GAP, while the GAP N terminus coding sequences had no effect on src transformation. When cells transformed by c-ras, c-src, c-src527F, or v-src were transfected with GAP or the GAP C terminus sequence in the presence of a selectable marker, 40 to 85% of the resistant colonies were found to be morphologically revertant. The GAP C terminus induced reversion of each src-transformed cell line more efficiently than the full-length GAP, but this was not the case for reversion of c-ras transformation. Biochemical analysis of v-src revertant subclones showed that the reversion correlated with overexpression of full-length GAP or the GAP C terminus. There was no decrease in the level of pp60src expression or the level of protein-tyrosine phosphorylation in vivo. We conclude that GAP can suppress transformation by src via inhibition of endogenous ras activity, without inhibiting in vivo tyrosine phosphorylation of cellular proteins induced by pp60src, and that src may negatively regulate GAP's inhibitory action on endogenous ras.

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Tyrosine phosphorylation of P160 BCR by P210 BCR-ABL

Blood ◽

10.1182/blood.v82.4.1257.1257 ◽

1993 ◽

Vol 82 (4) ◽

pp. 1257-1263 ◽

Cited By ~ 22

Author(s):

D Lu ◽

J Liu ◽

M Campbell ◽

JQ Guo ◽

N Heisterkamp ◽

...

Keyword(s):

Amino Acids ◽

Tyrosine Phosphorylation ◽

Deletion Mutant ◽

Physical Interaction ◽

Tyrosine Residues ◽

Ph Chromosome ◽

Pathologic Processes ◽

Simultaneous Expression

Abstract It is well established that the chimeric BCR-ABL gene formed by joining parts of the BCR and ABL genes plays a key role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias. We report that simultaneous expression of P210 BCR-ABL and P160 BCR in simian COS-1 cells yielded stable complexes of these two proteins, and induced phosphorylation of P160 BCR on tyrosine residues in vivo. Tyrosine phosphorylation of a deletion mutant encoding 553 amino acids of BCR N- terminal sequences was also detected when it was coexpressed with P210 BCR-ABL. We propose that tyrosine phosphorylation of P160 BCR by P210 BCR-ABL and their stable physical interaction may perturb normal BCR functions and that these alterations are directly involved in the pathologic processes found in Ph chromosome-associated leukemias.

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Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1.

Molecular Biology of the Cell ◽

10.1091/mbc.4.1.49 ◽

1993 ◽

Vol 4 (1) ◽

pp. 49-57 ◽

Cited By ~ 41

Author(s):

A Kashishian ◽

J A Cooper

Keyword(s):

Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Growth Factor Receptor ◽

Mitogen Activated Protein Kinase ◽

Platelet Derived Growth Factor ◽

Phosphorylation Sites ◽

C Terminus ◽

Mitogen Activated Protein ◽

Src Homology

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.

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Regulation of the Rho Family Small GTPase Wrch-1/RhoU by C-Terminal Tyrosine Phosphorylation Requires Src

Molecular and Cellular Biology ◽

10.1128/mcb.01646-09 ◽

2010 ◽

Vol 30 (17) ◽

pp. 4324-4338 ◽

Cited By ~ 29

Author(s):

Jamie K. Alan ◽

Anastacia C. Berzat ◽

Brian J. Dewar ◽

Lee M. Graves ◽

Adrienne D. Cox

Keyword(s):

Plasma Membrane ◽

Tyrosine Phosphorylation ◽

Kinase Activity ◽

Three Dimensional ◽

Small Gtpase ◽

Cell Morphogenesis ◽

New Paradigm ◽

Nonreceptor Tyrosine Kinase ◽

Serum Stimulation ◽

Rho Family

ABSTRACT Wrch-1 is an atypical Rho family small GTPase with roles in migration, epithelial cell morphogenesis, osteoclastogenesis, and oncogenic transformation. Here, we observed rapid relocalization of Wrch-1 from the plasma membrane upon serum stimulation. Studies revealed a requirement for serum-stimulated tyrosine phosphorylation of Wrch-1 at residue Y254 within its C-terminal membrane targeting domain, mediated by the nonreceptor tyrosine kinase Src. Genetic or pharmacological loss of Src kinase activity blocked both phosphorylation and relocalization of Wrch-1. Functionally, Y254 was required for proper Wrch-1 modulation of cystogenesis in three-dimensional culture, and the phospho-deficient mutant, Y254F, was enhanced in Wrch-1-mediated anchorage-independent growth. Mechanistically, C-terminal tyrosine phosphorylation and subsequent relocalization of Wrch-1 downregulated its ability to interact with and activate its effectors by decreasing active Wrch-1-GTP, perhaps by altering proximity to a GEF or GAP. Phospho-deficient Wrch-1(Y254F) remained at the plasma membrane and GTP bound and continued to recruit and activate its effector PAK, even upon serum stimulation. In contrast, a phospho-mimetic mutant, Y254E, was constitutively endosomally localized and GDP bound and failed to recruit PAK unless mutated to be constitutively active/GAP insensitive. C-terminal tyrosine phosphorylation thus represents a new paradigm in posttranslational control of small GTPase localization, activation, and biological function.

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Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation

Molecular Biology of the Cell ◽

10.1091/mbc.e18-08-0531 ◽

2019 ◽

Vol 30 (5) ◽

pp. 566-578 ◽

Cited By ~ 10

Author(s):

Shuling Fan ◽

Caroline M. Weight ◽

Anny-Claude Luissint ◽

Roland S. Hilgarth ◽

Jennifer C. Brazil ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Barrier Function ◽

Intestinal Inflammation ◽

Kinase Inhibitor ◽

Src Kinase ◽

Small Gtpase ◽

Epithelial Barrier ◽

Barrier Dysfunction ◽

Junction Protein

Junctional adhesion molecule-A (JAM-A), an epithelial tight junction protein, plays an important role in regulating intestinal permeability through association with a scaffold signaling complex containing ZO-2, Afadin, and the small GTPase Rap2. Under inflammatory conditions, we report that the cytoplasmic tail of JAM-A is tyrosine phosphorylated (p-Y280) in association with loss of barrier function. While barely detectable Y280 phosphorylation was observed in confluent monolayers of human intestinal epithelial cells under basal conditions, exposure to cytokines TNFα, IFNγ, IL-22, or IL-17A, resulted in compromised barrier function in parallel with increased p-Y280. Phosphorylation was Src kinase dependent, and we identified Yes-1 and PTPN13 as a major kinase and phosphatase for p-JAM-A Y280, respectively. Moreover, cytokines IL-22 or IL-17A induced increased activity of Yes-1. Furthermore, the Src kinase inhibitor PP2 rescued cytokine-induced epithelial barrier defects and inhibited phosphorylation of JAM-A Y280 in vitro. Phosphorylation of JAM-A Y280 and increased permeability correlated with reduced JAM-A association with active Rap2. Finally, we observed increased phosphorylation of Y280 in colonic epithelium of individuals with ulcerative colitis and in mice with experimentally induced colitis. These findings support a novel mechanism by which tyrosine phosphorylation of JAM-A Y280 regulates epithelial barrier function during inflammation.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20246307 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6307 ◽

Cited By ~ 1

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.

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