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Mouse rod photoreceptor Prkaa2 knockout phosphoproteomics

ABSTRACT: Mouse rod photoreceptors isolated from retinas of Prkaa2 rod photoreceptor specific knockout mice were processed for phosphoproteomics to elucidate downstream kinase targets. Prkaa2 is an isoform of the catalytic subunit of AMP-activated protein kinase (AMPK), an essential nutrient-sensing enzyme responsible for maintaining many metabolic processes.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Retinal Rod Cell, Retinal Rod

SUBMITTER: Tae Jun Lee

LAB HEAD: Rajendra Apte

PROVIDER: PXD045669 | Pride | 2024-06-16

REPOSITORIES: Pride

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			Action	DRS
	16288_ed02442_tc_860_P1A.mzIdentML	Mzid
	16290_ed02444_tc_860_P2A.mzIdentML	Mzid
	16291_ed02445_tc_860_P2B.mzIdentML	Mzid
	16292_ed02446_tc_860_P3A.mzIdentML	Mzid
	16293_ed02447_Tc_860_P3B.mzIdentML	Mzid

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1 - 5 of 24

Publications

Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function.

Lee Tae Jun TJ Sasaki Yo Y Ruzycki Philip A PA Ban Norimitsu N Lin Joseph B JB Wu Hung-Ting HT Santeford Andrea A Apte Rajendra S RS

JCI insight 20240116 4

AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of α, β, and γ subunits. The independent functions of the 2 isoforms of the catalytic α subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons, such as photoreceptors. Here, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, showed altered levels of cGMP, GTP, and ATP, suggesting isoform-specific ...[more]

PMID: 38227383

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Mouse rod photoreceptor Prkaa1 knockout phosphoproteomics

Project description:Mouse rod photoreceptor specific Prkaa1 knockouts were isolated from retinas and processed for phosphoproteomics to elucidate downstream kinase targets. Prkaa1 is one of the isoforms for the catalytic subunit of AMP-activated protein kinase (AMPK), an essential nutrient sensing enzyme responsible for maintaining many metabolic processes.

2024-06-16 | PXD045667 | Pride

Targetable leukemia dependency on noncanonical PI3Kγ signaling

Project description:Phosphoinositide 3-kinase gamma (PI3Kγ) is implicated as a target to repolarize tumor-associated macrophages and promote anti-tumor immune responses in solid cancers. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid, and dendritic lineages. This dependency is characterized by innate inflammatory signaling and elevation of PIK3R5, which encodes a regulatory subunit of PI3Kγ that we find stabilizes the active enzymatic complex when overexpressed. Mechanistically, we identify PAK1 kinase as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency independently of AKT. PI3Kγ inhibition dephosphorylates PAK1, activates a transcriptional network of NFκB-related tumor suppressor genes, and impairs mitochondrial oxidative phosphorylation. We find that treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukemias with activated PIK3R5, either at baseline or by exogenous inflammatory stimulation. Notably, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukemia xenografts with low baseline PIK3R5 expression, as residual leukemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Taken together, our study reveals acute leukemia dependency on a noncanonical PI3Kγ signaling pathway amenable to near-term evaluation in patients using inhibitors already in clinical development.

2024-03-19 | PXD045702 | Pride

Investigation of Proteomic and Phosphoproteomic Responses to Signaling Network Perturbations Reveals Functional Pathway Organizations in Yeast

Project description:Governance of protein phosphorylation by kinases and phosphatases constitutes an essential regulatory network in eukaryotic cells. Network dysregulation leads to severe consequences and is often a key factor in disease pathogenesis. Previous studies revealed multiple roles for protein phosphorylation and pathway structures in cellular functions from different perspectives. We sought to understand the roles of kinases and phosphatases from a protein homeostasis point of view. Using a streamlined tandem mass tag (SL-TMT) strategy, we systematically measured proteomic and phosphoproteomic responses to perturbations of phosphorylation signaling networks in yeast deletion strains. Ours results emphasize the requirement for protein normalization for more complete interpretation of phosphorylation data. Functional relationships between kinases and phosphatases were characterized at both proteome and phosphoproteome levels in three ways: 1) gene ontology enrichment analysis, 2) ∆gene-∆gene correlation networks and 3) molecule covariance networks. This resource illuminates kinase and phosphatase functions and pathway organizations.

2019-11-15 | PXD015575 | Pride

HEK293T cell nuclear & cytosolic phospho-proteome remodelling upon amino acid withdrawal. Related to Supplementary Table 4 from An et al (2020)

Project description:HEK293T Phospho proteome cytosolic vs nuclear fractionation upon short (3h) amino acids withdrawal.

2020-04-30 | PXD017855 | Pride

VRK1 is required in VRK2-methylated cancers of the nervous system

Project description:Collateral lethality occurs when loss of one paralog renders cancer cells dependent on the remaining paralog. Combining genome scale CRISPR/Cas9 screens coupled with RNA-sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase Vaccinia-Related Kinase 1 (VRK1) for their survival. VRK1 dependency was inversely correlated with expression of its paralog VRK2. VRK2 knockout in VRK2-expressing cell lines sensitized cells to VRK1 knockout, and conversely, VRK2 overexpression increased cell fitness in VRK1-dependent cell lines upon suppression of VRK1. DNA methylation of the VRK2 promoter was associated with low VRK2 expression in human neuroblastomas, and adult and pediatric gliomas. Mechanistically, depletion of VRK1 reduced Barrier-to-Autointegration Factor (BAF) phosphorylation, induced abnormal chromosome segregation. and dysregulated nuclear membrane re-formation following mitosis, resulting in increased DNA damage and apoptosis. Together, these observations identify VRK1 as a synthetic lethal target in VRK2-methylated adult and pediatric gliomas and neuroblastomas.

2022-09-06 | PXD030599 | Pride

Comparison of the proteome and of the phosphoproteome of S. cerevisiae cells harvested with different strategies

Project description:The budding yeast Saccharomyces cerevisiae is a powerful model system that is widely used to investigate many cellular processes. The harvesting of yeast cells is the first step in almost every experimental procedure. Here, yeast cells are isolated from their growth medium, collected, and used for successive experiments or analysis. The two most common methods to harvest S. cerevisiae are centrifugation and filtration. Understanding, if and how centrifugation and filtration affect yeast physiology is essential with respect to downstream data interpretation. Here, we profile and compare the proteomes and the phosphoproteomes, using isobaric label-based quantitative mass spectrometry, of three common methods used to harvest S. cerevisiae cells: low-speed centrifugation, high-speed centrifugation, and filtration. Our data suggest that while the proteome was stable across the tested conditions, hundreds of phosphorylation events were different between centrifugation and filtration. Our analysis shows that under our experimental conditions, filtration may cause both cell wall and osmotic stress at higher levels compared to centrifugation, and implying harvesting method-specific stresses. Thus, considering that the basal activation levels of specific stresses may differ under certain harvesting conditions is an important, but often overlooked aspect of experimental design.

2023-12-28 | PXD044387 | Pride

APC/CCdc20 dependent degradation of Clb4 is all there is to astral microtubule stabilization

Project description:Coordination between sister chromatid separation and segregation is crucial for accurate chromosome partitioning to the offspring. Key to this process is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Based on their function, spindle microtubules are conventionally divided into three classes: kinetochore, interpolar and astral microtubules (kMTs, iMTs and aMTs, respectively). While different mechanisms have been proposed to control kMT and iMT dynamics, aMT regulation remains elusive. We here show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase to control spindle orientation and are stabilized at anaphase onset. Necessary and sufficient for this stabilization is the degradation of the mitotic cyclin Clb4 mediated by the Anaphase Promoting Complex in combination with its activator subunit Cdc20 (APC/CCdc20). Our results contribute to delineate a comprehensive picture of late mitotic events, where individual steps of the signalling cascade initiated by APC/CCdc20 activation and culminating in cohesin cleavage time the final stages of mitosis by sequentially modulating the dynamics of the three classes of spindle microtubules.

2022-10-19 | PXD028828 | Pride

SARS-CoV-2 phosphoproteomics - ACE2-A549 and Vero

Project description:Multiple coronavirus have emerged independently in the past 20 years and caused lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated. Understanding the common host factors necessary for the life cycle of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that phosphorylate a cluster of serine and threonine residues on the viral N-protein in a highly coordinated way. We showed that these kinases were critical for the viral replication cycle, suggesting that inhibitors of one or more of these protein kinases could be useful for treating patients with COVID-19 infections. These phosphorylation sites are highly conserved across coronaviruses, indicating that inhibitors of these protein kinases might be broadly effective in treating multiple viral diseases.

2023-03-11 | PXD033015 | Pride

Temporal proteomic changes induced by nicotine in human cells: A quantitative proteomics approach

Project description:Nicotine is a prominent active compound in tobacco and many smoking cessation products. Some of the biological effects of nicotine are well documented in in vitro and in vivo systems; however, nominal data are available concerning the time-dependent changes on protein and phosphorylation events in response to nicotine. Here, we profiled the proteomes of SH-SY5Y and A549 cell lines subjected to acute (15min, 1h and 4h) or chronic (24h, 48h) nicotine exposures. We used sample multiplexing (TMTpro16) and quantified more than 9,000 proteins and over 7,000 phosphorylation events per cell line. Among our findings, we determined a decrease in mitochondrial protein abundance for SH-SY5Y, while we detected alterations in several immune pathways, such as the complement system, for A549. We also explored the proposed association between smoking and SARS-CoV2. Here, we found several host proteins known to interact with viral proteins that were affected by nicotine in a time dependent manner. This dataset can be mined further to investigate the potential role of nicotine in different biological contexts.

2021-05-11 | PXD023108 | Pride

TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing

Project description:The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic breast cancer cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this dataset with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex dataset by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.

2021-04-28 | PXD024275 | Pride

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