CiteAb

Fig.2.B showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Fig.2.C showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Fig.2.D showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Fig.1.D showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Fig.1.A showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Fig.1.E showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Fig.1.C showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Fig.1.C showing Western Blotting from the publication: The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein.

Fig.5.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Fig.5.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Fig.5.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Fig.1.D showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.2.E showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.2.G showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.2.C showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.2.A showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.2.B showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.2.D showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.1.E showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.1.A showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.1.B showing Western Blotting from the publication: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Fig.7.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.

Fig.7.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.

40 images found

A phosphate-binding pocket in cyclin B3 is essential for XErp1/Emi2 degradation in meiosis I.

In EMBO Reports on 1 February 2025 by Schunk, R., Halder, M., et al.

To ensure the correct euploid state of embryos, it is essential that vertebrate oocytes await fertilization arrested at metaphase of meiosis II. This MII arrest is mediated by XErp1/Emi2, which inhibits the ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome). Cyclin B3 in complex with Cdk1 (cyclin-dependent kinase 1) is essential to prevent an untimely arrest of vertebrate oocytes in meiosis I by targeting XErp1/Emi2 for degradation. Yet, the molecular mechanism of XErp1/Emi2 degradation in MI is not well understood. Here, by combining TRIM-Away in oocytes with egg extract and in vitro studies, we demonstrate that a hitherto unknown phosphate-binding pocket in cyclin B3 is essential for efficient XErp1/Emi2 degradation in meiosis I. This pocket enables Cdk1/cyclin B3 to bind pre-phosphorylated XErp1/Emi2 facilitating further phosphorylation events, which ultimately target XErp1/Emi2 for degradation in a Plk1- (Polo-like kinase 1) dependent manner. Key elements of this degradative mechanism are conserved in frog and mouse. Our studies identify a novel, evolutionarily conserved determinant of Cdk/cyclin substrate specificity essential to prevent an untimely oocyte arrest at meiosis I with catastrophic consequences upon fertilization.
© 2025. The Author(s).

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Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons.

In The Journal of Cell Biology on 4 November 2024 by Zhou, C., Wu, Y. K., et al.

Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.
© 2024 Zhou et al.

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A molecular switch for stress-induced activation of retrograde mitochondrial transport

Preprint on BioRxiv : the Preprint Server for Biology on 14 September 2024 by Gladkova, C., Paez-Segala, M. G., et al.

The cellular distribution of mitochondria in response to stress and local energy needs is governed by the relative activities of kinesin and dynein. The mechanism for switching between these two opposite polarity microtubule motors remains unknown. Here, we coupled a novel cellular synthetic cargo transport assay with AlphaFold2-guided mutagenesis to identify a regulatory helix in the mitochondrial adaptor protein (TRAK) that mediates switching between kinesin- and dynein-driven transport. Differences in the helix sequence explain why two near-identical TRAK isoforms transport mitochondria in predominantly opposite directions. Phosphorylation of the regulatory helix by stress-activated kinases causes the activation of dynein and dissociation of kinesin. Our results reveal a molecular mechanism for coordinating the directional transport of mitochondria in response to intracellular signals.

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UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope.

In The Journal of Cell Biology on 1 July 2024 by Liao, Y., Andronov, L., et al.

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a role of ubiquitin-associated protein 2-like (UBAP2L) in the assembly and stability of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and facilitates the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L promotes the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress, and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact NE.
© 2024 Liao et al.

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A phosphate-binding pocket in cyclin B3 is essential for XErp1/ Emi2 degradation in meiosis I

Preprint on BioRxiv : the Preprint Server for Biology on 16 May 2024 by Demmig, R., Schäfer, M., et al.

To ensure the correct euploid state of embryos, it is essential that vertebrate oocytes await fertilization arrested at metaphase of meiosis II. This MII arrest is mediated by XErp1/Emi2, which inhibits the ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome). Cyclin B3 in complex with Cdk1 (cyclin-dependent kinase 1) is essential to prevent an untimely arrest of vertebrate oocytes in meiosis I by targeting XErp1/Emi2 for degradation. Yet, the molecular mechanism of XErp1/Emi2 degradation in MI is not well understood. Here, by combining TRIM-Away in oocytes with egg extract and in vitro studies, we demonstrate that a hitherto unknown phosphate-binding pocket in cyclin B3 is essential for efficient XErp1/Emi2 degradation in meiosis I. This pocket enables Cdk1/cyclin B3 to bind pre-phosphorylated XErp1/Emi2 facilitating further phosphorylation events, which ultimately target XErp1/Emi2 for degradation in a Plk1 (Polo-like kinase 1) dependent manner. Key elements of this degradative mechanism are conserved in frog and mouse. Our studies identify a novel, evolutionarily conserved determinant of Cdk/cyclin substrate specificity essential to prevent an untimely oocyte arrest at meiosis I with catastrophic consequences upon fertilization.

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Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

In Cell Death Dis on 26 July 2023 by Ryder, L., Arendrup, F. S., et al.

Fig.1.D

Fig.1.D showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Nitric oxide activates p38 and JNK in a ZAK-dependent manner.a U2OS cells were treated with increasing concentrations of DEA NONOate (100 μM, 250 μM, 500 μM, 750 μM, 1000 μM – 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. b U2OS cells were treated with DEA NONOate (... more

Nitric oxide activates p38 and JNK in a ZAK-dependent manner.a U2OS cells were treated with increasing concentrations of DEA NONOate (100 μM, 250 μM, 500 μM, 750 μM, 1000 μM – 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. b U2OS cells were treated with DEA NONOate (750 μM) for the indicated times. Lysates were analyzed as in (a). c U2OS cells deleted for ZAK (ΔZAK) or ASK1 (ΔASK1) were treated with DEA NONOate (750 μM – 1 h). Lysates were analyzed as in (a). d HAP1 WT and ΔZAK cells were pretreated with ASK1 inhibitor (2 μM – 30 min) prior to treatment with DEA NONOate (750 μM – 1 h). Lysates were analyzed as in (a). e U2OS cells were pretreated with N-acetyl-L-cysteine (NAC, 10 mM – 1 h) followed by DEA NONOate treatment (750 μM – 1 h). Lysates were analyzed as in (a). f U2OS WT and ΔZAK cells were treated with S-Nitrosoglutathione (GSNO, 2 mM) for the indicated times. Lysates were analyzed as in (a). g U2OS cells were transfected either with empty vector or strep-HA-iNOS and treated with ZAK inhibitor (2 μM – 1 h). Lysates were analyzed as in (a). h U2OS WT and ΔZAK cells were transfected with empty vector or strep-HA-iNOS and supplemented with L-arginine (10 mM – 20 h) and ZAK inhibitor (2 μM – 1 h). Lysates were analyzed as in (a). less

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Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

In Cell Death Dis on 26 July 2023 by Ryder, L., Arendrup, F. S., et al.

Fig.1.A

Fig.1.A showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Nitric oxide activates p38 and JNK in a ZAK-dependent manner.a U2OS cells were treated with increasing concentrations of DEA NONOate (100 μM, 250 μM, 500 μM, 750 μM, 1000 μM – 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. b U2OS cells were treated with DEA NONOate (... more

Nitric oxide activates p38 and JNK in a ZAK-dependent manner.a U2OS cells were treated with increasing concentrations of DEA NONOate (100 μM, 250 μM, 500 μM, 750 μM, 1000 μM – 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. b U2OS cells were treated with DEA NONOate (750 μM) for the indicated times. Lysates were analyzed as in (a). c U2OS cells deleted for ZAK (ΔZAK) or ASK1 (ΔASK1) were treated with DEA NONOate (750 μM – 1 h). Lysates were analyzed as in (a). d HAP1 WT and ΔZAK cells were pretreated with ASK1 inhibitor (2 μM – 30 min) prior to treatment with DEA NONOate (750 μM – 1 h). Lysates were analyzed as in (a). e U2OS cells were pretreated with N-acetyl-L-cysteine (NAC, 10 mM – 1 h) followed by DEA NONOate treatment (750 μM – 1 h). Lysates were analyzed as in (a). f U2OS WT and ΔZAK cells were treated with S-Nitrosoglutathione (GSNO, 2 mM) for the indicated times. Lysates were analyzed as in (a). g U2OS cells were transfected either with empty vector or strep-HA-iNOS and treated with ZAK inhibitor (2 μM – 1 h). Lysates were analyzed as in (a). h U2OS WT and ΔZAK cells were transfected with empty vector or strep-HA-iNOS and supplemented with L-arginine (10 mM – 20 h) and ZAK inhibitor (2 μM – 1 h). Lysates were analyzed as in (a). less

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Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

In Cell Death Dis on 26 July 2023 by Ryder, L., Arendrup, F. S., et al.

Fig.1.E

Fig.1.E showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Nitric oxide activates p38 and JNK in a ZAK-dependent manner.a U2OS cells were treated with increasing concentrations of DEA NONOate (100 μM, 250 μM, 500 μM, 750 μM, 1000 μM – 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. b U2OS cells were treated with DEA NONOate (... more

Nitric oxide activates p38 and JNK in a ZAK-dependent manner.a U2OS cells were treated with increasing concentrations of DEA NONOate (100 μM, 250 μM, 500 μM, 750 μM, 1000 μM – 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. b U2OS cells were treated with DEA NONOate (750 μM) for the indicated times. Lysates were analyzed as in (a). c U2OS cells deleted for ZAK (ΔZAK) or ASK1 (ΔASK1) were treated with DEA NONOate (750 μM – 1 h). Lysates were analyzed as in (a). d HAP1 WT and ΔZAK cells were pretreated with ASK1 inhibitor (2 μM – 30 min) prior to treatment with DEA NONOate (750 μM – 1 h). Lysates were analyzed as in (a). e U2OS cells were pretreated with N-acetyl-L-cysteine (NAC, 10 mM – 1 h) followed by DEA NONOate treatment (750 μM – 1 h). Lysates were analyzed as in (a). f U2OS WT and ΔZAK cells were treated with S-Nitrosoglutathione (GSNO, 2 mM) for the indicated times. Lysates were analyzed as in (a). g U2OS cells were transfected either with empty vector or strep-HA-iNOS and treated with ZAK inhibitor (2 μM – 1 h). Lysates were analyzed as in (a). h U2OS WT and ΔZAK cells were transfected with empty vector or strep-HA-iNOS and supplemented with L-arginine (10 mM – 20 h) and ZAK inhibitor (2 μM – 1 h). Lysates were analyzed as in (a). less

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Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

In Cell Death Dis on 26 July 2023 by Ryder, L., Arendrup, F. S., et al.

Fig.1.C

Fig.1.C showing Western Blotting from the publication: Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

Nitric oxide activates p38 and JNK in a ZAK-dependent manner.a U2OS cells were treated with increasing concentrations of DEA NONOate (100 μM, 250 μM, 500 μM, 750 μM, 1000 μM – 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. b U2OS cells were treated with DEA NONOate (... more

Nitric oxide activates p38 and JNK in a ZAK-dependent manner.a U2OS cells were treated with increasing concentrations of DEA NONOate (100 μM, 250 μM, 500 μM, 750 μM, 1000 μM – 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. b U2OS cells were treated with DEA NONOate (750 μM) for the indicated times. Lysates were analyzed as in (a). c U2OS cells deleted for ZAK (ΔZAK) or ASK1 (ΔASK1) were treated with DEA NONOate (750 μM – 1 h). Lysates were analyzed as in (a). d HAP1 WT and ΔZAK cells were pretreated with ASK1 inhibitor (2 μM – 30 min) prior to treatment with DEA NONOate (750 μM – 1 h). Lysates were analyzed as in (a). e U2OS cells were pretreated with N-acetyl-L-cysteine (NAC, 10 mM – 1 h) followed by DEA NONOate treatment (750 μM – 1 h). Lysates were analyzed as in (a). f U2OS WT and ΔZAK cells were treated with S-Nitrosoglutathione (GSNO, 2 mM) for the indicated times. Lysates were analyzed as in (a). g U2OS cells were transfected either with empty vector or strep-HA-iNOS and treated with ZAK inhibitor (2 μM – 1 h). Lysates were analyzed as in (a). h U2OS WT and ΔZAK cells were transfected with empty vector or strep-HA-iNOS and supplemented with L-arginine (10 mM – 20 h) and ZAK inhibitor (2 μM – 1 h). Lysates were analyzed as in (a). less

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The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein.

In J Cell Biol on 1 May 2023 by Garner, K. E. L., Salter, A., et al.

Fig.1.C

Fig.1.C showing Western Blotting from the publication: The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein.

Recruitment of dynein, dynactin, and LIS1 to KASH5 in HeLa cells. (A) A stable HeLa cell line inducibly expressing GFP-KASH5 (green) was labeled with antibodies against dynein IC and LIC1, dynactin p150 and p50, LIS1, and Nde1 (magenta) and imaged on a DeltaVision microscope. Images are z-project... more

Recruitment of dynein, dynactin, and LIS1 to KASH5 in HeLa cells. (A) A stable HeLa cell line inducibly expressing GFP-KASH5 (green) was labeled with antibodies against dynein IC and LIC1, dynactin p150 and p50, LIS1, and Nde1 (magenta) and imaged on a DeltaVision microscope. Images are z-projections of deconvolved image stacks. The bottom panel shows the transient expression of GFP-N2α2 in green and labeling with anti-LIC1 in magenta, with undeconvolved wide-field images. Arrowheads point out the location of centrosomes, full arrows show creases in the NE, and asterisks mark cytoplasmic accumulations of GFP-KASH5. Thin white lines on color merge images show where a line scan plot was performed, shown on the right. Scale bars = 10 μm. (B) Schematic showing KASH5 and the constructs used. For some experiments, the GFP was replaced with an HA tag. (C) Dynein, dynactin, and LIS1 are recruited to KASH5 as shown by GFP-Trap pull-downs. HeLaM cells were transiently transfected with GFP-KASH5ΔK or GFP-N2α2ΔK. The pull-downs and inputs (1.5% of total lysate) were probed with antibodies against GFP, p150, IC, LIC1, LIC2, LIS1, BICD2, and α-tubulin. Molecular weight markers are shown. Quantitation of the blot is given in Table S1. (D) The KASH5 N-terminal EF-hand domain is sufficient to recruit dynein. Lysates of HeLaM cells expressing GFP, GFP-KASH5ΔK (GFP-K5ΔK), or GFP-KASH5 N terminus (GFP-K5ND) were isolated by GFP-trap and probed for LIC1 by immunoblotting. The input is 15% of the GFP-trap sample. The graph shows the quantitation of LIC1 levels in pull-downs (n = 3 independent experiments), normalized to GFP-K5ND. Error bars denote SD. Source data are available for this figure: SourceData F1. less

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Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

In Cells on 22 March 2023 by Johansen, V. B. I., Snieckute, G., et al.

Fig.5.D

Fig.5.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Disruption of the YLD decreases the activation potential of ZAKα. (a) Schematic of the ZAKα ΔYLD mutant protein, indicating a deleted region within the YLD domain. (b) U2OS ΔZAK cells were rescued with either wildtype (WT) ZAKα or ZAKα Δ YLD, of which two clones with different expression levels w... more

Disruption of the YLD decreases the activation potential of ZAKα. (a) Schematic of the ZAKα ΔYLD mutant protein, indicating a deleted region within the YLD domain. (b) U2OS ΔZAK cells were rescued with either wildtype (WT) ZAKα or ZAKα Δ YLD, of which two clones with different expression levels were analyzed (ΔYLD #1 and #2). Cells were induced for expression with doxycycline (Dox) and treated with anisomycin (Ani—10 μg/mL, 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. (c) Cells from (b) were irradiated with UV-B (500 J/m2—1 h) and analyzed as in (b). (d) Cells from (b) were incubated in EBSS medium (18 h) and analyzed as in (b). (e) Model of ZAKα activation by ribosome impairment, here caused by binding of anisomycin (ANI) to the peptidyl transferase center. The SAM domain inhibits activation whereas the kinase scans ribosomes (top panel—left). This inhibition is relieved upon recognition of ribotoxic stress signals by the C-terminal S and CTD domains (top panel—right). Kinase activation is signified by ZAKα autophosphorylation (P). Upon mutation of the SAM domain, sensing of ribotoxic stress by the RBR region (S + CTD) is not required for ZAKα activation (middle and bottom panels). ZAKα domains are color-coded as in (a). LZ, Leucine Zipper; SAM, Sterile Alpha Motif; YLD, YEATS-Like Domain; S, Sensor Domain; CTD, C-Terminal Domain; RBR, Ribosome Binding Region; and p-, phosphorylated (activated) form of kinase. less

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Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

In Cells on 22 March 2023 by Johansen, V. B. I., Snieckute, G., et al.

Fig.5.C

Fig.5.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Disruption of the YLD decreases the activation potential of ZAKα. (a) Schematic of the ZAKα ΔYLD mutant protein, indicating a deleted region within the YLD domain. (b) U2OS ΔZAK cells were rescued with either wildtype (WT) ZAKα or ZAKα Δ YLD, of which two clones with different expression levels w... more

Disruption of the YLD decreases the activation potential of ZAKα. (a) Schematic of the ZAKα ΔYLD mutant protein, indicating a deleted region within the YLD domain. (b) U2OS ΔZAK cells were rescued with either wildtype (WT) ZAKα or ZAKα Δ YLD, of which two clones with different expression levels were analyzed (ΔYLD #1 and #2). Cells were induced for expression with doxycycline (Dox) and treated with anisomycin (Ani—10 μg/mL, 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. (c) Cells from (b) were irradiated with UV-B (500 J/m2—1 h) and analyzed as in (b). (d) Cells from (b) were incubated in EBSS medium (18 h) and analyzed as in (b). (e) Model of ZAKα activation by ribosome impairment, here caused by binding of anisomycin (ANI) to the peptidyl transferase center. The SAM domain inhibits activation whereas the kinase scans ribosomes (top panel—left). This inhibition is relieved upon recognition of ribotoxic stress signals by the C-terminal S and CTD domains (top panel—right). Kinase activation is signified by ZAKα autophosphorylation (P). Upon mutation of the SAM domain, sensing of ribotoxic stress by the RBR region (S + CTD) is not required for ZAKα activation (middle and bottom panels). ZAKα domains are color-coded as in (a). LZ, Leucine Zipper; SAM, Sterile Alpha Motif; YLD, YEATS-Like Domain; S, Sensor Domain; CTD, C-Terminal Domain; RBR, Ribosome Binding Region; and p-, phosphorylated (activated) form of kinase. less

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Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

In Cells on 22 March 2023 by Johansen, V. B. I., Snieckute, G., et al.

Fig.1.D

Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Redundant peptide arrays underlie functionality of the sensor domain in ZAKα. (a) Schematic of the ZAKα protein highlighting the three RGRYXXR/K motifs in the sensor (S) domain. ZAKα S domain mutants used in this study are detailed in Figure S1a. LZ, Leucine Zipper; SAM, Sterile Alpha Motif; S, S... more

Redundant peptide arrays underlie functionality of the sensor domain in ZAKα. (a) Schematic of the ZAKα protein highlighting the three RGRYXXR/K motifs in the sensor (S) domain. ZAKα S domain mutants used in this study are detailed in Figure S1a. LZ, Leucine Zipper; SAM, Sterile Alpha Motif; S, Sensor Domain; CTD, C-Terminal Domain; and RBR, Ribosome Binding Region. (b) U2OS cells deleted for ZAK (U2OS ΔZAK) were rescued with either STREP-HA-tagged wildtype (WT) ZAKα, a version of ZAKα with all arginines and lysines mutated to alanine in RGRYXXR/K motifs (R → A), or a version of R → A combined with a deletion of the CTD (R → A ΔCTD). Expression was induced by doxycycline (Dox) and cells were treated with anisomycin (Ani—10 μg/mL, 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. (c) U2OS ΔZAK cells were rescued with WT or R → A ΔCTD as in (b) and were incubated in EBSS medium (18 h). Lysates were analyzed as in (b). (d) U2OS ΔZAK cells were rescued with either WT, R → A ΔCTD, or a version of ZAKα with the corresponding lysine substitution mutant (R → K ΔCTD). Cells were induced for ZAKα expression, treated with anisomycin, and analyzed as in (b). (e) U2OS ΔZAK cells were rescued with mutants of ZAKα from (a), containing only one functional RGRYXXR/K peptide. Cells were treated and analyzed as in (b). (f) U2OS ΔZAK cells were rescued with ZAKα constructs combining R → A ΔCTD mutation with a disease-causing point mutation in the SAM domain (W347S) that is associated with constitutive ZAKα activity or a version of W347S combined with R → A ΔCTD (W347S R → A ΔCTD). Cells were treated and analyzed as in (b). p-, phosphorylated (activated) form of kinase; Tub, Tubulin. less

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Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

In Cells on 22 March 2023 by Johansen, V. B. I., Snieckute, G., et al.

Fig.1.C

Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Redundant peptide arrays underlie functionality of the sensor domain in ZAKα. (a) Schematic of the ZAKα protein highlighting the three RGRYXXR/K motifs in the sensor (S) domain. ZAKα S domain mutants used in this study are detailed in Figure S1a. LZ, Leucine Zipper; SAM, Sterile Alpha Motif; S, S... more

Redundant peptide arrays underlie functionality of the sensor domain in ZAKα. (a) Schematic of the ZAKα protein highlighting the three RGRYXXR/K motifs in the sensor (S) domain. ZAKα S domain mutants used in this study are detailed in Figure S1a. LZ, Leucine Zipper; SAM, Sterile Alpha Motif; S, Sensor Domain; CTD, C-Terminal Domain; and RBR, Ribosome Binding Region. (b) U2OS cells deleted for ZAK (U2OS ΔZAK) were rescued with either STREP-HA-tagged wildtype (WT) ZAKα, a version of ZAKα with all arginines and lysines mutated to alanine in RGRYXXR/K motifs (R → A), or a version of R → A combined with a deletion of the CTD (R → A ΔCTD). Expression was induced by doxycycline (Dox) and cells were treated with anisomycin (Ani—10 μg/mL, 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. (c) U2OS ΔZAK cells were rescued with WT or R → A ΔCTD as in (b) and were incubated in EBSS medium (18 h). Lysates were analyzed as in (b). (d) U2OS ΔZAK cells were rescued with either WT, R → A ΔCTD, or a version of ZAKα with the corresponding lysine substitution mutant (R → K ΔCTD). Cells were induced for ZAKα expression, treated with anisomycin, and analyzed as in (b). (e) U2OS ΔZAK cells were rescued with mutants of ZAKα from (a), containing only one functional RGRYXXR/K peptide. Cells were treated and analyzed as in (b). (f) U2OS ΔZAK cells were rescued with ZAKα constructs combining R → A ΔCTD mutation with a disease-causing point mutation in the SAM domain (W347S) that is associated with constitutive ZAKα activity or a version of W347S combined with R → A ΔCTD (W347S R → A ΔCTD). Cells were treated and analyzed as in (b). p-, phosphorylated (activated) form of kinase; Tub, Tubulin. less

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Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

In Cells on 22 March 2023 by Johansen, V. B. I., Snieckute, G., et al.

Fig.5.B

Fig.5.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

Disruption of the YLD decreases the activation potential of ZAKα. (a) Schematic of the ZAKα ΔYLD mutant protein, indicating a deleted region within the YLD domain. (b) U2OS ΔZAK cells were rescued with either wildtype (WT) ZAKα or ZAKα Δ YLD, of which two clones with different expression levels w... more

Disruption of the YLD decreases the activation potential of ZAKα. (a) Schematic of the ZAKα ΔYLD mutant protein, indicating a deleted region within the YLD domain. (b) U2OS ΔZAK cells were rescued with either wildtype (WT) ZAKα or ZAKα Δ YLD, of which two clones with different expression levels were analyzed (ΔYLD #1 and #2). Cells were induced for expression with doxycycline (Dox) and treated with anisomycin (Ani—10 μg/mL, 1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. (c) Cells from (b) were irradiated with UV-B (500 J/m2—1 h) and analyzed as in (b). (d) Cells from (b) were incubated in EBSS medium (18 h) and analyzed as in (b). (e) Model of ZAKα activation by ribosome impairment, here caused by binding of anisomycin (ANI) to the peptidyl transferase center. The SAM domain inhibits activation whereas the kinase scans ribosomes (top panel—left). This inhibition is relieved upon recognition of ribotoxic stress signals by the C-terminal S and CTD domains (top panel—right). Kinase activation is signified by ZAKα autophosphorylation (P). Upon mutation of the SAM domain, sensing of ribotoxic stress by the RBR region (S + CTD) is not required for ZAKα activation (middle and bottom panels). ZAKα domains are color-coded as in (a). LZ, Leucine Zipper; SAM, Sterile Alpha Motif; YLD, YEATS-Like Domain; S, Sensor Domain; CTD, C-Terminal Domain; RBR, Ribosome Binding Region; and p-, phosphorylated (activated) form of kinase. less

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RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.

In Nat Commun on 21 March 2022 by Keren-Kaplan, T., Saric, A., et al.

Fig.7.B

Fig.7.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.

Interaction of RUFY3 and RUFY4 with dynein-dynactin.a HEK293T cells were transfected with plasmids encoding GFP (negative control), GFP-BICD2 (positive control), RUFY3-GFP or RUFY4-GFP. Cell extracts were analyzed by immunoprecipitation (IP) with antibody to GFP, followed by immunoblotting (IB) f... more

Interaction of RUFY3 and RUFY4 with dynein-dynactin.a HEK293T cells were transfected with plasmids encoding GFP (negative control), GFP-BICD2 (positive control), RUFY3-GFP or RUFY4-GFP. Cell extracts were analyzed by immunoprecipitation (IP) with antibody to GFP, followed by immunoblotting (IB) for endogenous dynein intermediate chain (DIC) and endogenous p150Glued subunit of dynactin. Ponceau S staining shows the levels of immunoprecipitated GFP-tagged proteins. The experiment shown in this panel is one of two with similar results. b Extracts of HEK293T cells were incubated with recombinant 6His-StrepII-sfGFP (abbreviated 6His-GFP in the figure) (negative control), His6-StrepII-sfGFP-BICD225-400 (His6-GFP-BICD225-400) (positive control) or 6His-StrepII-sfGFP-RUFY3 (6His-GFP-RUFY3), pulled down (PD) with Strep-Tactin agarose, and immunoblotted for endogenous dynein intermediate chain (DIC), the endogenous p150Glued of dynactin, or GFP. The GFP used to make these constructs is a variant named sfGFP, for super-folder GFP. The experiment shown in this panel is one of two with similar results. c Glutathione-Sepharose preloaded with purified, recombinant GST (negative control), GST-DLIC1 or GST-DLIC1-CT (C-terminal domain) was incubated with purified, recombinant 6His-StrepII-sfGFP-RUFY3 (6His-GFP-RUFY3). Bound proteins were detected by immunoblotting with antibodies to GFP and GST. The positions of molecular mass markers (in kDa) in panels a-c are indicated at left. d Live-cell images of HeLa cells co-expressing RUFY3-mCherry or RUFY4-mCherry (magenta) without or with GFP-p150Glued-CC1 (green). Single-channel images are shown in grayscale. Scale bars: 10 μm. Arrows point to RUFY proteins at cell tips. This experiment is one of two with similar results. less

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RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.

In Nat Commun on 21 March 2022 by Keren-Kaplan, T., Saric, A., et al.

Fig.7.A

Fig.7.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.

Interaction of RUFY3 and RUFY4 with dynein-dynactin.a HEK293T cells were transfected with plasmids encoding GFP (negative control), GFP-BICD2 (positive control), RUFY3-GFP or RUFY4-GFP. Cell extracts were analyzed by immunoprecipitation (IP) with antibody to GFP, followed by immunoblotting (IB) f... more

Interaction of RUFY3 and RUFY4 with dynein-dynactin.a HEK293T cells were transfected with plasmids encoding GFP (negative control), GFP-BICD2 (positive control), RUFY3-GFP or RUFY4-GFP. Cell extracts were analyzed by immunoprecipitation (IP) with antibody to GFP, followed by immunoblotting (IB) for endogenous dynein intermediate chain (DIC) and endogenous p150Glued subunit of dynactin. Ponceau S staining shows the levels of immunoprecipitated GFP-tagged proteins. The experiment shown in this panel is one of two with similar results. b Extracts of HEK293T cells were incubated with recombinant 6His-StrepII-sfGFP (abbreviated 6His-GFP in the figure) (negative control), His6-StrepII-sfGFP-BICD225-400 (His6-GFP-BICD225-400) (positive control) or 6His-StrepII-sfGFP-RUFY3 (6His-GFP-RUFY3), pulled down (PD) with Strep-Tactin agarose, and immunoblotted for endogenous dynein intermediate chain (DIC), the endogenous p150Glued of dynactin, or GFP. The GFP used to make these constructs is a variant named sfGFP, for super-folder GFP. The experiment shown in this panel is one of two with similar results. c Glutathione-Sepharose preloaded with purified, recombinant GST (negative control), GST-DLIC1 or GST-DLIC1-CT (C-terminal domain) was incubated with purified, recombinant 6His-StrepII-sfGFP-RUFY3 (6His-GFP-RUFY3). Bound proteins were detected by immunoblotting with antibodies to GFP and GST. The positions of molecular mass markers (in kDa) in panels a-c are indicated at left. d Live-cell images of HeLa cells co-expressing RUFY3-mCherry or RUFY4-mCherry (magenta) without or with GFP-p150Glued-CC1 (green). Single-channel images are shown in grayscale. Scale bars: 10 μm. Arrows point to RUFY proteins at cell tips. This experiment is one of two with similar results. less

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