Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling

Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex. The small RNA-mediated depletion of kalirin diminished activities in cellular membranes for activating Rab11 and resulted in a shift in size of Rab11 positive structures from small to larger ones and tubulation of recycling endosomes. Our study suggests that kalirin and TRAPP form a dual GEF complex to choreograph actions of Rab11 and Rac1 at recycling endosomes.

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Hsp110 is required for spindle length control

The Journal of Cell Biology ◽

10.1083/jcb.201111105 ◽

2012 ◽

Vol 198 (4) ◽

pp. 623-636 ◽

Cited By ~ 17

Author(s):

Taras Makhnevych ◽

Philip Wong ◽

Oxana Pogoutse ◽

Franco J. Vizeacoumar ◽

Jack F. Greenblatt ◽

...

Keyword(s):

Affinity Purification ◽

Spindle Assembly ◽

S Phase ◽

Mass Spectrometry Analysis ◽

Nucleotide Exchange ◽

Spectrometry Analysis ◽

Nucleotide Exchange Factor ◽

Spindle Elongation ◽

Chaperone Complex

Systematic affinity purification combined with mass spectrometry analysis of N- and C-tagged cytoplasmic Hsp70/Hsp110 chaperones was used to identify new roles of Hsp70/Hsp110 in the cell. This allowed the mapping of a chaperone–protein network consisting of 1,227 unique interactions between the 9 chaperones and 473 proteins and highlighted roles for Hsp70/Hsp110 in 14 broad biological processes. Using this information, we uncovered an essential role for Hsp110 in spindle assembly and, more specifically, in modulating the activity of the widely conserved kinesin-5 motor Cin8. The role of Hsp110 Sse1 as a nucleotide exchange factor for the Hsp70 chaperones Ssa1/Ssa2 was found to be required for maintaining the proper distribution of kinesin-5 motors within the spindle, which was subsequently required for bipolar spindle assembly in S phase. These data suggest a model whereby the Hsp70–Hsp110 chaperone complex antagonizes Cin8 plus-end motility and prevents premature spindle elongation in S phase.

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Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

Infection and Immunity ◽

10.1128/iai.00126-12 ◽

2012 ◽

Vol 80 (12) ◽

pp. 4333-4343 ◽

Cited By ~ 32

Author(s):

Barak Hajaj ◽

Hasan Yesilkaya ◽

Rachel Benisty ◽

Maayan David ◽

Peter W. Andrew ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Mass Spectrometry Analysis ◽

Activity Levels ◽

Content Type ◽

Spectrometry Analysis ◽

Cellular Processes ◽

Thiol Peroxidase ◽

Fine Tune

ABSTRACTStreptococcus pneumoniaeis an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth,S. pneumoniaeproduces high levels of H2O2. SinceS. pneumoniaelacks catalase, the question of how it controls H2O2levels is of critical importance. Thepsalocus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase,tpxD. This study shows thattpxDencodes a functional thiol peroxidase involved in the adjustment of H2O2homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2efficiently. However,in vivoexperiments revealed that TpxD detoxifies only a fraction of the H2O2generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58undergoes selective oxidationin vivo, under conditions where H2O2is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesisin vitrowere significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2resulted intpxDupregulation, whilepsaBCAexpression was oppositely affected. However, the challenge of ΔtpxDmutants with H2O2did not affectpsaBCA, implying that TpxD is involved in the regulation of thepsaoperon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2.

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ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment

Molecular Biology of the Cell ◽

10.1091/mbc.e15-05-0278 ◽

2015 ◽

Vol 26 (23) ◽

pp. 4265-4279 ◽

Cited By ~ 5

Author(s):

Joseph C. Salem ◽

Marta M. Reviriego-Mendoza ◽

Lorraine C. Santy

Keyword(s):

Adhesion Formation ◽

Small Gtpases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Recycling Endosomes ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

And Migration ◽

Regulated Trafficking ◽

Migration Response

When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small the GTPase R-Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of α5-integrin and R-Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras–dependent effects on spreading and adhesion formation.

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Temporal Quantitative Phosphoproteomics Profiling of Interleukin-33 Signaling Network Reveals Unique Modulators of Monocyte Activation

Cells ◽

10.3390/cells11010138 ◽

2022 ◽

Vol 11 (1) ◽

pp. 138

Author(s):

Devasahayam Arokia Balaya Rex ◽

Yashwanth Subbannayya ◽

Prashant Kumar Modi ◽

Akhina Palollathil ◽

Lathika Gopalakrishnan ◽

...

Keyword(s):

Inflammatory Diseases ◽

Leukocyte Adhesion ◽

Cell Cycle Checkpoints ◽

Mass Spectrometry Analysis ◽

Dna Damage And Repair ◽

Spectrometry Analysis ◽

Interleukin 33 ◽

Cellular Processes ◽

Defensive Role ◽

Signaling Modules

Interleukin-33 (IL-33), a member of the IL-1 superfamily cytokines, is an endogenous danger signal and a nuclear-associated cytokine. It is one of the essential mediators of both innate and adaptive immune responses. Aberrant IL-33 signaling has been demonstrated to play a defensive role against various infectious and inflammatory diseases. Although the signaling responses mediated by IL-33 have been previously reported, the temporal signaling dynamics are yet to be explored. To this end, we applied quantitative temporal phosphoproteomics analysis to elucidate pathways and proteins induced by IL-33 in THP-1 monocytes. Employing a TMT labeling-based quantitation and titanium dioxide (TiO2)-based phosphopeptide enrichment strategy followed by mass spectrometry analysis, we identified and quantified 9448 unique phosphopeptides corresponding to 3392 proteins that showed differential regulation. Of these, 171 protein kinases, 60 phosphatases and 178 transcription factors were regulated at different phases of IL-33 signaling. In addition to the confirmed activation of canonical signaling modules including MAPK, NFκB, PI3K/AKT modules, pathway analysis of the time-dependent phosphorylation dynamics revealed enrichment of several cellular processes, including leukocyte adhesion, response to reactive oxygen species, cell cycle checkpoints, DNA damage and repair pathways. The detailed quantitative phosphoproteomic map of IL-33 signaling will serve as a potentially useful resource to study its function in the context of inflammatory and pathological conditions.

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Rules for PP2A-controlled phosphosignalling and drug responses

10.1101/271841 ◽

2018 ◽

Cited By ~ 5

Author(s):

Otto Kauko ◽

Susumu Y. Imanishi ◽

Evgeny Kulesskiy ◽

Teemu Daniel Laajala ◽

Laxman Yetukuri ◽

...

Keyword(s):

Cancer Cell ◽

Mass Spectrometry Analysis ◽

Drug Responses ◽

Spectrometry Analysis ◽

Cellular Processes ◽

Cell Responses ◽

Systemic Analysis ◽

Phosphatase 2A ◽

Cancer Drivers ◽

Systemic Understanding

AbstractSystemic understanding of protein phosphatase 2A (PP2A)-regulated cellular processes is still at infancy. Here, we present mass-spectrometry analysis of phospho-targets (dephosphorylome) regulated by PP2A modulation. In addition to PP2A-regulated processes and targets, the data reveal important general concepts and rules related to PP2A-mediated phosphoregulation. These include the unidirectionality paradigm of regulation of phosphorylation, and differential spatial distribution of kinase-and phosphatase-dominated phosphotargets. Data also present first systemic analysis of targets of PP2A-modulating oncoproteins, CIP2A, PME-1, and SET; including targets via which PP2A may coordinately regulate activities of cancer drivers and tumor suppressors such as MYC or TP53. To validate functional utility of this dataset, PP2A dephosphorylome activity was correlated with cancer cell responses to over 300 drugs. Notably, we find that cancer therapy responses can be broadly classified based on PP2A dephosphorylome activity, both in quantitative and qualitative manner. In summary, our data characterize rules by which PP2A coordinate cancer cell phosphosignaling and drug responses. The results also may also direct the use of emerging pharmacological approaches for PP2A activity modulation in human diseases.

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Deletion of PIN4 Suppresses the Protein Transport Defects Caused by sec12-4 Mutation in Saccharomyces cerevisiae

Microbial Physiology ◽

10.1159/000509633 ◽

2020 ◽

Vol 30 (1-6) ◽

pp. 25-35

Author(s):

Akiko Murakami-Sekimata ◽

Masayuki Sekimata ◽

Natsumi Sato ◽

Yuto Hayasaka ◽

Akihiko Nakano

Keyword(s):

Protein Transport ◽

Cell Cycle Checkpoint ◽

Secretory Proteins ◽

Temperature Sensitive ◽

Nucleotide Exchange ◽

Casein Kinase I ◽

Vesicle Budding ◽

Nucleotide Exchange Factor ◽

Cycle Checkpoint ◽

Copii Vesicles

Newly synthesized secretory proteins are released into the lumen of the endoplasmic reticulum (ER). The secretory proteins are surrounded by coat protein complex II (COPII) vesicles, and transported from the ER and reach their destinations through the Golgi apparatus. Sec12p is a guanine nucleotide exchange factor for Sar1p, which initiates COPII vesicle budding from the ER. The activation of Sar1p by Sec12p and the subsequent COPII coat assembly have been well characterized, but the events that take place upstream of Sec12p remain unclear. In this study, we isolated the novel extragenic suppressor of sec12-4, PIN4/MDT1, a cell cycle checkpoint target. A yeast two-hybrid screening was used to identify Pin4/Mdt1p as a binding partner of the casein kinase I isoform Hrr25p, which we have previously identified as a modulator of Sec12p function. Deletion of PIN4 suppressed both defects of temperature-sensitive growth and the partial protein transport observed in sec12-4 mutants. The results of this study suggest that Pin4p provides novel aspects of Sec12p modulations.

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The Ran GTPase System in Fission Yeast Affects Microtubules and Cytokinesis in Cells That Are Competent for Nucleocytoplasmic Protein Transport

Molecular and Cellular Biology ◽

10.1128/mcb.22.24.8491-8505.2002 ◽

2002 ◽

Vol 22 (24) ◽

pp. 8491-8505 ◽

Cited By ~ 23

Author(s):

Sandra S. Salus ◽

Janos Demeter ◽

Shelley Sazer

Keyword(s):

Fission Yeast ◽

Protein Transport ◽

Temperature Sensitive ◽

Nucleotide Exchange ◽

Cellular Processes ◽

Nucleotide Exchange Factor ◽

Ring Structures ◽

Dependent Processes ◽

In Cells

ABSTRACT Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.

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Arabidopsis phospholipase Dδ as an initiator of cytoskeleton-mediated signalling to fundamental cellular processes

Functional Plant Biology ◽

10.1071/fp08222 ◽

2009 ◽

Vol 36 (2) ◽

pp. 190 ◽

Cited By ~ 33

Author(s):

Angela Y. Y. Ho ◽

David A. Day ◽

Melissa H. Brown ◽

Jan Marc

Keyword(s):

Potential Interaction ◽

Mass Spectrometry Analysis ◽

Actin Binding ◽

Sequence Alignments ◽

Spectrometry Analysis ◽

Cellular Processes ◽

Plant Signal Transduction ◽

Interaction Partners ◽

Cytoskeletal Rearrangements ◽

Partner Proteins

Phospholipase D (PLD), in combination with the cytoskeleton, plays a key role in plant signal transduction. One isotype of the multigene Arabidopsis PLD family, AtPLDδ, has been implicated in binding microtubules, although the molecular details of the mechanism and identities of potential interaction partners are unclear. We constructed a GFP-AtPLDδ reporter gene, stably transformed it into an Arabidopsis suspension cell line, and used epitope-tagged affinity pull-down assays to isolate a complex of co-purifying proteins. Mass spectrometry analysis of the complex revealed a set of proteins including β-tubulin, actin 7, HSP70, clathrin heavy chain, ATP synthase subunits, and a band 7–4/flotillin homologue. Sequence alignments with defined tubulin- and actin-binding regions from human HsPLD2 revealed highly homologous regions in all 12 AtPLD isotypes, suggesting direct interactions of AtPLDδ with tubulin and actin, while interactions with the remaining partners are likely to be mediated by the cytoskeleton. We propose that AtPLDδ acts through a complex of cytoskeletal and partner proteins to modulate fundamental cellular processes such as cytoskeletal rearrangements, vesicular trafficking, assembly of Golgi apparatus, mitosis and cytokinesis.

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R-Ras Regulates Exocytosis by Rgl2/Rlf-mediated Activation of RalA on Endosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e06-08-0765 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1850-1860 ◽

Cited By ~ 39

Author(s):

Akiyuki Takaya ◽

Takahiro Kamio ◽

Michitaka Masuda ◽

Naoki Mochizuki ◽

Hirofumi Sawa ◽

...

Keyword(s):

Resonance Energy Transfer ◽

Resonance Energy ◽

Small Gtpase ◽

Nucleotide Exchange ◽

Cellular Functions ◽

Recycling Endosomes ◽

Nucleotide Exchange Factor ◽

Wide Range ◽

Ras Family

R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.

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Prediction and collection of protein–metabolite interactions

Briefings in Bioinformatics ◽

10.1093/bib/bbab014 ◽

2021 ◽

Author(s):

Tianyi Zhao ◽

Jinxin Liu ◽

Xi Zeng ◽

Wei Wang ◽

Sheng Li ◽

...

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Large Scale ◽

Rapid Development ◽

Area Under The Curve ◽

Membrane Receptors ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

Cellular Processes ◽

The Rich

Abstract Interactions between proteins and small molecule metabolites play vital roles in regulating protein functions and controlling various cellular processes. The activities of metabolic enzymes, transcription factors, transporters and membrane receptors can all be mediated through protein–metabolite interactions (PMIs). Compared with the rich knowledge of protein–protein interactions, little is known about PMIs. To the best of our knowledge, no existing database has been developed for collecting PMIs. The recent rapid development of large-scale mass spectrometry analysis of biomolecules has led to the discovery of large amounts of PMIs. Therefore, we developed the PMI-DB to provide a comprehensive and accurate resource of PMIs. A total of 49 785 entries were manually collected in the PMI-DB, corresponding to 23 small molecule metabolites, 9631 proteins and 4 species. Unlike other databases that only provide positive samples, the PMI-DB provides non-interaction between proteins and metabolites, which not only reduces the experimental cost for biological experimenters but also facilitates the construction of more accurate algorithms for researchers using machine learning. To show the convenience of the PMI-DB, we developed a deep learning-based method to predict PMIs in the PMI-DB and compared it with several methods. The experimental results show that the area under the curve and area under the precision-recall curve of our method are 0.88 and 0.95, respectively. Overall, the PMI-DB provides a user-friendly interface for browsing the biological functions of metabolites/proteins of interest, and experimental techniques for identifying PMIs in different species, which provides important support for furthering the understanding of cellular processes. The PMI-DB is freely accessible at http://easybioai.com/PMIDB.

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