scholarly journals Tyrosine Phosphorylation of Selected Secretory Carrier Membrane Proteins, SCAMP1 and SCAMP3, and Association with the EGF Receptor

1998 ◽  
Vol 9 (7) ◽  
pp. 1661-1674 ◽  
Author(s):  
Theodore T. Wu ◽  
J. David Castle
Keyword(s):  
Membrane Proteins ◽  
Egf Receptor ◽  
Tyrosine Phosphatase ◽  
Chinese Hamster ◽  
Tyrosine Residue ◽  
Cell Interior ◽  
Murine Fibroblasts ◽  
Secretory Carrier Membrane Proteins

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in “patches” at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP–EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase

1994 ◽  
Vol 14 (1) ◽  
pp. 509-517
Author(s):  
W Li ◽  
R Nishimura ◽  
A Kashishian ◽  
A G Batzer ◽  
W J Kim ◽  
...  
Keyword(s):  
Growth Factor ◽  
Intracellular Signaling ◽  
Egf Receptor ◽  
Tyrosine Phosphatase ◽  
Growth Factor Receptors ◽  
Ras Signaling ◽  
Pdgf Receptor ◽  
Phosphotyrosine Phosphatase ◽  
Src Homology

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.

scholarly journals Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

2019 ◽  
Vol 30 (12) ◽  
pp. 1369-1376 ◽  
Author(s):  
Tim N. Baldering ◽  
Marina S. Dietz ◽  
Karl Gatterdam ◽  
Christos Karathanasis ◽  
Ralph Wieneke ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Superresolution Microscopy ◽  
Superresolution Imaging ◽  
Molecular Information ◽  
Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

Callose-synthesizing enzymes as membrane proteins of Betula protoplasts secrete bundles of β-1,3-glucan hollow fibrils under Ca2+-rich and acidic culture conditions

Holzforschung ◽  
2020 ◽  
Vol 74 (8) ◽  
pp. 725-732
Author(s):  
Shintaro Matsuo ◽  
Satomi Tagawa ◽  
Yudai Matsusaki ◽  
Yuri Uchi ◽  
Tetsuo Kondo
Keyword(s):  
Membrane Proteins ◽  
Woody Plants ◽  
Culture Medium ◽  
Culture Conditions ◽  
White Birch ◽  
High Concentration ◽  
Callose Synthase ◽  
Insight Into

AbstractPreviously, it was reported that plant protoplasts isolated from Betula platyphylla (white birch) callus secreted bundles of hollow callose fibrils in acidic culture medium containing a high concentration of calcium ions (Ca2+). Here, the callose synthase was characterized from in situ and in vitro perspectives. Localization of callose synthases at the secreting site of callose fiber was indicated from in situ immunostaining observation of protoplasts. For in vitro analyses, membrane proteins were extracted from membrane fraction of protoplasts with a 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) treatment. The CHAPS extract aggregated in the presence of a high concentration of Ca2+, suggesting that Ca2+ may promote the arrangement of callose synthases in the plasma membrane. The callose synthase activity was dependent on pH and Ca2+, similar to the callose synthase of Arabidopsis thaliana. However, the synthesized fibril products were longer than those produced by callose synthases of herbaceous plants. This is the first insight into the specific properties of callose synthases of woody plants that secrete of callose hollow fibers.

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site

1993 ◽  
Vol 13 (11) ◽  
pp. 7112-7121
Author(s):  
F G Gervais ◽  
L M Chow ◽  
J M Lee ◽  
P E Branton ◽  
A Veillette
Keyword(s):  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Tyrosine Residue ◽  
Catalytic Function ◽  
Inhibitory Site ◽  
Tyrosine Protein Kinase ◽  
Src Homology ◽  
Carboxy Terminal ◽  
Nih 3T3

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.

scholarly journals Nitric oxide stimulates tyrosine phosphorylation in murine fibroblasts in the absence and presence of epidermal growth factor

1995 ◽  
Vol 305 (2) ◽  
pp. 613-619 ◽  
Author(s):  
T M S Peranovich ◽  
A M da Silva ◽  
D M Fries ◽  
A Stern ◽  
H P Monteiro
Keyword(s):  
Methylene Blue ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Cyclic Gmp ◽  
Egf Receptor ◽  
Tyrosine Phosphatase ◽  
Intact Cells ◽  
Murine Fibroblasts ◽  
Epidermal Growth

In the present study, utilizing anti-phosphotyrosine monoclonal antibodies, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) as sources of NO and murine fibroblasts expressing the human epidermal growth factor (EGF) receptor (HER14 cells), we showed that tyrosine phosphorylation of a set of proteins (126, 56 and 43 kDa) was stimulated when cells were incubated with either SNP or SNAP and abolished by Methylene Blue and oxyhaemoglobin. Inhibition by Methylene Blue suggested an involvement of cyclic GMP in the process, which was evidenced by the effects of 8-bromo cyclic GMP. This analogue of cyclic GMP stimulated tyrosine phosphorylation of the same set of proteins phosphorylated after incubation with the NO source. Tyrosine phosphorylation of the same set of proteins was stimulated when cells were incubated simultaneously with SNP and EGF, showing that NO also potentiates EGF-evoked tyrosine kinase activity in HER14 cells. However, stimulation of the autophosphorylation of the EGF receptor, above the levels obtained for EGF alone, was not observed under those conditions. Additionally, we investigated the effects of NO on EGF-receptor tyrosine phosphatase activities in HER14 cells. Increasing concentrations of NO correlate with a gradual inhibition of these activities in HER14 cells, either in intact cells or in cell lysates. Taken together, these observations suggest that NO modulates tyrosine phosphorylation in HER14 cells.

Three mammalian SCAMPs (secretory carrier membrane proteins) are highly related products of distinct genes having similar subcellular distributions

1997 ◽  
Vol 110 (17) ◽  
pp. 2099-2107 ◽  
Author(s):  
D.R. Singleton ◽  
T.T. Wu ◽  
J.D. Castle
Keyword(s):  
Membrane Proteins ◽  
Mammalian Species ◽  
In Vitro Transcription ◽  
Complete Analysis ◽  
Mammalian Tissues ◽  
Double Label ◽  
Secretory Carrier Membrane Proteins ◽  
Expressed Sequence ◽  
Cognate Mrnas

The primary structures of three human forms of secretory carrier membrane proteins (SCAMPs) have been deduced from full-length clones isolated from a HeLa cell cDNA library and confirmed by a combination of comparison to expressed sequence tags, microsequencing of purified protein, and in vitro transcription and translation. The structures indicated that SCAMPs are highly related products of distinct genes, and that the sequence identity of an individual SCAMP between different mammalian species is almost complete. Analysis of the distribution of SCAMPs among different mammalian tissues and cells indicates parallel expression of polypeptides and cognate mRNAs, and indicates that the three SCAMPs are usually but not always expressed together. The apparent M(r)s of two SCAMPs (1 and 2) do not vary appreciably among species, while that of the third (SCAMP3) is approximately 2 kDa larger in rodent cells than in humans. Examination of the codistribution of the three forms within individual cells using double label immunofluorescence indicates extensive colocalization of SCAMP2 and SCAMP3 with endogenous SCAMP1, however, subcellular regions enriched for a particular SCAMP are readily visible. These findings suggest that the SCAMPs may largely function at the same sites during vesicular transport rather than in separate post-Golgi recycling pathways.

scholarly journals Probing Membrane Protein Assembly into Nanodiscs by In Situ Dynamic Light Scattering: A2A Receptor as a Case Study

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 400 ◽  
Author(s):  
Rosana I. Reis ◽  
Isabel Moraes
Keyword(s):  
Light Scattering ◽  
Membrane Proteins ◽  
Dynamic Light Scattering ◽  
Membrane Protein ◽  
Cell Communication ◽  
Cell Physiology ◽  
Functional Characterisation

Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure–function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein–detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins’ reconstitution into nanodiscs. The adenosine type 2A receptor (A2AR) was used as a case study.

scholarly journals Identification of Discrete Classes of Endosome-derived Small Vesicles as a Major Cellular Pool for Recycling Membrane Proteins

2001 ◽  
Vol 12 (4) ◽  
pp. 981-995 ◽  
Author(s):  
Seong-Nam Lim ◽  
Frank Bonzelius ◽  
Seng Hui Low ◽  
Holger Wille ◽  
Thomas Weimbs ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Glucose Transporter ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Ovary Cells ◽  
Early Endosomes ◽  
Endosomal Membranes ◽  
Glucose Transporter Glut4

Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5′-[γ-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface.

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