Bet1p activates the v-SNARE Bos1p.

Bet1p is a type II membrane protein that is required for vesicular transport between the endoplasmic reticulum and Golgi complex in the yeast Saccharomyces cerevisiae. A domain of Bet1p, that shows potential to be involved in a coiled-coil interaction, is homologous to a region of the neuronal protein SNAP-25. Here, we used in vitro binding studies to demonstrate that Bet1p plays a role in potentiating soluble NSF attachment protein receptor (SNARE) interactions. Mutational analysis points to the coiled-coil region as necessary for Bet1p function, and circular dichroism experiments support this theory. In vitro binding studies were also used to demonstrate that a direct interaction between Bet1p and Bos1p is required for the efficient interaction of the vesicle SNARE with its SNARE target. Genetic studies suggest that the interactions of Bet1p with Bos1p are regulated by the small GTP-binding protein Ypt1p.

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The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.2099 ◽

1997 ◽

Vol 17 (4) ◽

pp. 2099-2106 ◽

Cited By ~ 164

Author(s):

R Jiang ◽

M Carlson

Keyword(s):

Protein Kinase ◽

Regulatory Domain ◽

Glucose Starvation ◽

Binding Studies ◽

Triple Mutant ◽

Yeast Saccharomyces Cerevisiae ◽

In Vitro Binding ◽

Vitro Binding ◽

Two Hybrid

The Snf1 protein kinase plays a central role in the response to glucose starvation in the yeast Saccharomyces cerevisiae. Previously, we showed that two-hybrid interaction between Snf1 and its activating subunit, Snf4, is inhibited by high levels of glucose. These findings, together with biochemical evidence that Snf1 and Snf4 remain associated in cells grown in glucose, suggested that another protein (or proteins) anchors Snf1 and Snf4 into a complex. Here, we examine the possibility that a family of proteins, comprising Sip1, Sip2, and Gal83, serves this purpose. We first show that the fraction of cellular Snf4 protein that is complexed with Snf1 is reduced in a sip1delta sip2delta gal83delta triple mutant. We then present evidence that Sip1, Sip2, and Gal83 each interact independently with both Snf1 and Snf4 via distinct domains. A conserved internal region binds to the Snf1 regulatory domain, and the conserved C-terminal ASC domain binds to Snf4. Interactions were mapped by using the two-hybrid system and were confirmed by in vitro binding studies. These findings indicate that the Sip1/Sip2/Gal83 family anchors Snf1 and Snf4 into a complex. Finally, the interaction of the yeast Sip2 protein with a plant Snf1 homolog suggests that this function is conserved in plants.

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IC-P-199: Comparing [3 H]MK-6240 and [3 H]AV-1451(T-807) In In vitro Binding Studies in Brain Tissues

Alzheimer s & Dementia ◽

10.1016/j.jalz.2016.06.230 ◽

2016 ◽

Vol 12 ◽

pp. P144-P144

Author(s):

Zhizhen Zeng ◽

Patricia J. Miller ◽

Brett M. Connolly ◽

Stacey S. O’Malley ◽

Idriss Bennacef ◽

...

Keyword(s):

Binding Studies ◽

In Vitro Binding ◽

Vitro Binding ◽

Brain Tissues

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A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0457 ◽

2006 ◽

Vol 17 (11) ◽

pp. 4720-4735 ◽

Cited By ~ 61

Author(s):

Alistair N. Hume ◽

Abul K. Tarafder ◽

José S. Ramalho ◽

Elena V. Sviderskaya ◽

Miguel C. Seabra

Keyword(s):

Coiled Coil ◽

Binding Domain ◽

Actin Binding ◽

Binding Studies ◽

Null Cell ◽

Coiled Coil Region ◽

Actin Binding Domain ◽

Transport Assay ◽

Myosin Va

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

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Lyn Physically Associates With the Erythropoietin Receptor and May Play a Role in Activation of the Stat5 Pathway

Blood ◽

10.1182/blood.v91.10.3734 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3734-3745 ◽

Cited By ~ 128

Author(s):

Hiroshi Chin ◽

Ayako Arai ◽

Hiroshi Wakao ◽

Ryuichi Kamiyama ◽

Nobuyuki Miyasaka ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Kinase Domain ◽

Sh2 Domain ◽

Erythropoietin Receptor ◽

Tyrosine Kinase Domain ◽

Binding Studies ◽

In Vitro Binding ◽

Vitro Binding

Abstract Protein tyrosine phosphorylation plays a crucial role in signaling from the receptor for erythropoietin (Epo), although the Epo receptor (EpoR) lacks the tyrosine kinase domain. We have previously shown that the Jak2 tyrosine kinase couples with the EpoR to transduce a growth signal. In the present study, we demonstrate that Lyn, a Src family tyrosine kinase, physically associates with the EpoR in Epo-dependent hematopoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of the EpoR and that both LynA and LynB, alternatively spliced forms of Lyn, bind with the membrane-proximal 91-amino acid region of the EpoR cytoplasmic domain. In vitro binding studies using GST-Lyn fusion proteins further showed that the Src homology (SH)-2 domain of Lyn specifically binds with the tyrosine-phosphorylated EpoR in lysate from Epo-stimulated cells, whereas the tyrosine kinase domain of Lyn binds with the unphosphorylated EpoR. Far-Western blotting and synthetic phosphopeptide competition assays further indicated that the Lyn SH2 domain directly binds to the tyrosine-phosphorylated EpoR, most likely through its interaction with phosphorylated Y-464 or Y-479 in the carboxy-terminal region of the EpoR. In vitro binding studies also demonstrated that the Lyn SH2 domain directly binds to tyrosine-phosphorylated Jak2. In vitro reconstitution experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of Stat5, mainly on Y-694, and activates the DNA-binding and transcription-activating abilities of Stat5. In agreement with this, Lyn enhanced the Stat5-dependent transcriptional activation when overexpressed in 32D/EpoR-Wt cells. In addition, Lyn was demonstrated to phosphorylate the EpoR and Stat5 on tyrosines in vitro. These results suggest that Lyn may play a role in activation of the Jak2/Stat5 and other signaling pathways by the EpoR.

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Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1

International Journal of Molecular Sciences ◽

10.3390/ijms21072400 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2400 ◽

Cited By ~ 1

Author(s):

René Stürmer ◽

Jana Reising ◽

Werner Hoffmann

Keyword(s):

Molecular Mass ◽

Complex I ◽

Size Exclusion ◽

Trefoil Factor ◽

Binding Studies ◽

In Vitro Binding ◽

Vitro Binding ◽

Exclusion Chromatography ◽

Trefoil Factor Family

The skin of the frog Xenopus laeevis is protected from microbial infections by a mucus barrier that contains frog integumentary mucins (FIM)-A.1, FIM-B.1, and FIM-C.1. These gel-forming mucins are synthesized in mucous glands consisting of ordinary mucous cells and one or more cone cells at the gland base. FIM-A.1 and FIM-C.1 are unique because their cysteine-rich domains belong to the trefoil factor family (TFF). Furthermore, FIM-A.1 is unusually short (about 400 amino acid residues). In contrast, FIM-B.1 contains cysteine-rich von Willebrand D (vWD) domains. Here, we separate skin extracts by the use of size exclusion chromatography and analyze the distribution of FIM-A.1 and FIM-C.1. Two mucin complexes were detected, i.e., a high-molecular-mass Complex I, which contains FIM-C.1 and little FIM-A.1, whereas Complex II is of lower molecular mass and contains the bulk of FIM-A.1. We purified FIM-A.1 by a combination of size-exclusion chromatography (SEC) and anion-exchange chromatography and performed first in vitro binding studies with radioactively labeled FIM-A.1. Binding of 125I-labeled FIM-A.1 to the high-molecular-mass Complex I was observed. We hypothesize that the presence of FIM-A.1 in Complex I is likely due to lectin interactions, e.g., with FIM-C.1, creating a complex mucus network.

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Physical and functional interactions between Stat5 and the tyrosine- phosphorylated receptors for erythropoietin and interleukin-3

Blood ◽

10.1182/blood.v88.12.4415.bloodjournal88124415 ◽

1996 ◽

Vol 88 (12) ◽

pp. 4415-4425 ◽

Cited By ~ 64

Author(s):

H Chin ◽

N Nakamura ◽

R Kamiyama ◽

N Miyasaka ◽

JN Ihle ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Sh2 Domain ◽

Binding Studies ◽

Interleukin 3 ◽

In Vitro Binding ◽

Vitro Binding ◽

Docking Sites ◽

Carboxy Terminal

Erythropoietin (Epo) and interleukin-3 (IL-3) stimulate activation of the Jak2 tyrosine kinase and induce tyrosine phosphorylation and activation of Stat5. In the present study, we have shown that Epo or IL-3 stimulation induces binding of Stat5 to the tyrosine-phosphorylated Epo receptor (EpoR) or IL-3 receptor beta subunit (betaIL3), respectively, in IL-3-dependent 32D cells expressing the EpoR. The binding of Stat5 to these cytokine receptors was shown to be rapid and transient, occurring within 1 minute of stimulation of cells and significantly decreasing after 5 minutes of cell treatment. In vivo binding experiments in COS cells showed that binding of Stat5 to the EpoR was mediated through the Stat5 Src homology 2 (SH2) domain. In vitro binding studies further showed that Stat5, but not other Stats examined, bound specifically to tyrosine-phosphorylated recombinant EpoR fusion proteins. In these in vivo and in vitro binding studies, Stat5 bound, albeit to a lesser degree, to truncated EpoR mutants in which all the intracellular tyrosines except Y-343 were removed. Furthermore, EpoR-derived synthetic phosphotyrosine peptides corresponding to Y-343, Y-401, Y-431, and Y-479 inhibited the in vitro binding of Stat5. When expressed in 32D cells, a mutant EpoR in which all the intracellular tyrosines were removed by carboxy-terminal truncation showed a significantly impaired ability to induce tyrosine phosphorylation of Stat5, particularly at low concentrations of Epo, but exhibited an increased sensitivity to Epo for growth signaling as compared with the wild-type EpoR. These results indicate that Stat5 specifically and transiently binds to the EpoR through the interaction between the Stat5 SH2 domain and specific phosphorylated tyrosines, including Y-343, in the EpoR cytoplasmic domain. It was implied that betaIL3 may also have similar Stat5 docking sites. The Stat5 docking sites in the EpoR were shown to facilitate specific activation of Stat5, which, however, may not be required for the EpoR-mediated growth signaling.

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IN VITRO BINDING STUDIES TO EVALUATE THE NFT-SPECIFICITY OF [3H]MK-6240 AND [3H]AV-1451 BINDING IN SUBCORTICAL REGIONS OF THE HUMAN AD BRAIN

Alzheimer s & Dementia ◽

10.1016/j.jalz.2017.06.2583 ◽

2017 ◽

Vol 13 (7) ◽

pp. P150-P151 ◽

Cited By ~ 1

Author(s):

Zhizhen Zeng ◽

Patricia J. Miller ◽

Brett M. Connolly ◽

Stacey S. O’Malley ◽

Talakad Lohith ◽

...

Keyword(s):

Binding Studies ◽

In Vitro Binding ◽

Vitro Binding ◽

Subcortical Regions

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[P4-055]: IN VITRO BINDING STUDIES TO EVALUATE THE NFT-SPECIFICITY OF [3 H]MK-6240 AND [3 H]AV-1451 BINDING IN SUBCORTICAL REGIONS OF HUMAN AD BRAIN

Alzheimer s & Dementia ◽

10.1016/j.jalz.2017.06.1920 ◽

2017 ◽

Vol 13 (7S_Part_26) ◽

pp. P1278-P1278 ◽

Cited By ~ 1

Author(s):

Zhizhen Zeng ◽

Partricia J. Miller ◽

Brett M. Connolly ◽

Stacey S. O'Malley ◽

Talakad Lohith ◽

...

Keyword(s):

Binding Studies ◽

In Vitro Binding ◽

Vitro Binding ◽

Subcortical Regions

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Requirement for Bbp1p in the Proper Mitotic Functions of Cdc5p in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e03-07-0461 ◽

2004 ◽

Vol 15 (4) ◽

pp. 1711-1723 ◽

Cited By ~ 10

Author(s):

Chong J. Park ◽

Sukgil Song ◽

Thomas H. Giddings ◽

Hyeon-Su Ro ◽

Krisada Sakchaisri ◽

...

Keyword(s):

Coiled Coil ◽

Spindle Pole ◽

Stable Complex ◽

Yeast Two Hybrid ◽

Binding Studies ◽

Interacting Protein ◽

Spindle Pole Bodies ◽

Vitro Binding ◽

Two Hybrid

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pΔC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p243–385), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pΔC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5ΔC-BBP1243–385 under CDC5 promoter control partially complemented the cdc5Δ defect. These data suggest that Bbp1pΔC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.

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