Illuminating the dark phosphoproteome

Needham, Elise J.; Parker, Benjamin L.; Burykin, Timur; James, David E.; Humphrey, Sean J.

doi:10.1126/scisignal.aau8645

“…Typical kinase domains possess a well-defined fold that is similar across the available structures. Many kinases are not well studied -the so-called dark kinome 53 does not align the homologous residues between STK38 and other kinases. The same is true for the G-helix in some kinases, which may be positioned in different locations within the Cterminal domain, but retains a homologous sequence and structure, and thus is aligned differently in our MSA than in the structure alignments.…”

Section: Discussionmentioning

confidence: 99%

“…possess a well-defined fold that is similar across the available structures. Many kinases are not well studied -the so-called dark kinome53 , and it is possible to generate hypotheses about their sequence-structure-function relationships by examining their phylogenetic and structural relationships to well studied kinases. To enable this and for many other purposes, we have created a structurally-validated, multiple sequence alignment of 497 human protein kinase domains -fully annotated with gene, protein, group name, UniProt accession identifiers, and residue numbers.…”

mentioning

confidence: 99%

A Structurally-Validated Multiple Sequence Alignment of 497 Human Protein Kinase Domains

Modi

¹

,

Dunbrack

²

2019

Preprint

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Protein kinases are important in a large number of signaling pathways. Their dysregulation is involved in a number of human diseases, especially cancer. Studies on the structures of individual kinases have been used to understand the functions and phenotypes of mutations in other kinases that do not yet have experimental structures. The key factor in accurate inference by homology is an accurate sequence alignment. We present a parsimonious structure-based sequence alignment of 497 human protein kinase domains excluding atypical kinases, even those with related but somewhat different folds. Starting with a computed multiple sequence alignment, the alignment was manually refined in Jalview based on pairwise structural superposition onto a single kinase (Aurora A), followed by sequence alignment of the remaining kinases to their closest relatives with known structures. The alignment is arranged in 17 blocks of conserved regions and unaligned blocks in between that contain insertions of varying lengths present in only a subset of kinases. The aligned blocks contain well-conserved elements of secondary structure and well-known functional motifs, such as the DFG and HRD motifs. We validated the multiple sequence alignment by a pairwise, all-against-all alignment of 272 human kinases with known crystal structures. Our alignment has true-positive rate (TPR) and positive predictive value (PPV) accuracies of 97%. The remaining inaccuracy in our alignment comes from a few structures with shifted elements of secondary structure, and from the boundaries of aligned and unaligned regions, where compromises need to be made to encompass the majority of kinases. A new phylogeny of the protein kinase domains in the human genome based on our alignment indicates that 14 kinases previously labeled as "OTHER" can be confidently placed into the CAMK group. These kinases comprise the Aurora kinases, Polo kinases, ULK kinases, Calcium/calmodulin-dependent kinase kinases, and STK36.

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“…[21] Moreover,t he kinases (for example,E RK1/2 (ERK), p38) responsive to osmotic stress can be triggered by am ultitude of stimuli, including temperature,c hemical, and mechanical perturbations. [21] Moreover,t he kinases (for example,E RK1/2 (ERK), p38) responsive to osmotic stress can be triggered by am ultitude of stimuli, including temperature,c hemical, and mechanical perturbations.…”

Section: Resultsmentioning

confidence: 99%

In Situ Single‐Cell Western Blot on Adherent Cell Culture

Zhang

¹

,

Naguro

²

,

Herr

³

2019

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Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis.T oa ssayH eLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in athin layer of polyacrylamide gel. To integrate the culture,lysis,and assayworkflow,weintroduce aone-step copolymerization process that creates protein-decorated microwells.A fter single-cell culture,w el yse each cell in the microwell and perform western blotting on each resultant lysate.W eo bserve cell spreading after overnight microwellbased culture.s cWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions.W e validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.Supportinginformation, includingt he reagents, device fabrication, cell culture, cell viability and spreading assessment, osmotic stress protocols, in situ scWB procedures,a nd imaging and data analysis, and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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“…Having established the upstream cell preparation capabilities of the in situ scWB,w en ext sought to assess the downstream analytical capability by applying the western blotting function to measure osmotic-stress-induced MAP kinase phosphorylation in single cells.P hosphorylation is dynamic. [21] Moreover,t he kinases (for example,E RK1/2 (ERK), p38) responsive to osmotic stress can be triggered by am ultitude of stimuli, including temperature,c hemical, and mechanical perturbations. [22] Sample preparation, such as trypsinization and centrifugation, is thought to introduce artefacts.C onsequently,w es ought to design an integrated microfluidic device to circumvent such sample processing prior to protein analysis via in situ scWB.…”

Section: Resultsmentioning

confidence: 99%

In Situ Single‐Cell Western Blot on Adherent Cell Culture

Zhang

¹

,

Naguro

²

,

Herr

³

2019

View full text Add to dashboard Cite

Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis.T oa ssayH eLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in athin layer of polyacrylamide gel. To integrate the culture,lysis,and assayworkflow,weintroduce aone-step copolymerization process that creates protein-decorated microwells.A fter single-cell culture,w el yse each cell in the microwell and perform western blotting on each resultant lysate.W eo bserve cell spreading after overnight microwellbased culture.s cWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions.W e validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.Supportinginformation, includingt he reagents, device fabrication, cell culture, cell viability and spreading assessment, osmotic stress protocols, in situ scWB procedures,a nd imaging and data analysis, and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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Illuminating the dark phosphoproteome

Cited by 267 publications

References 208 publications

A Structurally-Validated Multiple Sequence Alignment of 497 Human Protein Kinase Domains

A Structurally-Validated Multiple Sequence Alignment of 497 Human Protein Kinase Domains

In Situ Single‐Cell Western Blot on Adherent Cell Culture

In Situ Single‐Cell Western Blot on Adherent Cell Culture

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