PhosphoSitePlus, 2014: mutations, PTMs and recalibrations

Hornbeck, Peter; Zhang, Bin; Murray, Beth; Kornhauser, Jon M.; Latham, Vaughan M.; Skrzypek, Elźbieta

doi:10.1093/nar/gku1267

Cited by 2,755 publications

(3,198 citation statements)

References 32 publications

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“…The data also indicates that the peptide based on the pseudosubtrate of PKC␣ (number 14) binds more strongly than peptides derived from cellular substrates (Figs. 2 and 4B, numbers [1][2][3][4][5][6][7][8]. This finding is consistent with the dominant role of the pseudosubstrate in PKC autoinhibition (20).…”

Section: Resultssupporting

confidence: 83%

“…The canonical role of a protein kinase is to recognize, bind, and phosphorylate specific serine, threonine, or tyrosine residues on a substrate protein (1,2). Given that kinases are one of the largest gene families in eukaryotes and are involved in nearly every cellular function (3), significant work has focused on understanding the mechanisms that dictate substrate-kinase pairings (4).…”

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confidence: 99%

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Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Sommese¹,

Sivaramakrishnan

2016

Journal of Biological Chemistry

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The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase C␣ and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.The canonical role of a protein kinase is to recognize, bind, and phosphorylate specific serine, threonine, or tyrosine residues on a substrate protein (1, 2). Given that kinases are one of the largest gene families in eukaryotes and are involved in nearly every cellular function (3), significant work has focused on understanding the mechanisms that dictate substrate-kinase pairings (4). Although the catalytic domains of eukaryotic kinases are structurally conserved (5, 6), the local environment around the substrate binding pocket of the kinase catalytic domain varies between kinases (7). This has led to the view that phosphorylation site recognition occurs through conserved residues flanking the phospho-residue on the substrate. Linear sequence motifs have now been identified for most kinases through a combination of structural analysis and peptide libraries (4, 8). Additionally, these phospho-sites are frequently found in unstructured regions of the substrate protein that may allow for more flexible accommodation in the kinase active site (9, 10). After recognizing the substrate, the catalytic domain then transfers a phosphate group from ATP to the phosphoresidue on the substrate. Phospho-transfer is followed by release of ADP and the phosphorylated substrate to prime the kinase for another catalytic cycle (11).Crystallographic and NMR approaches have provided detailed structural information on the catalytic domains of individual kinases in complex with a variety of nucleotide analogs and inhibitors (6, 12, 13). However, the lack of defined secondary structure surrounding the phospho-motif and the inherent transient nature of the interaction has limited efforts to dissect the different states of the ...

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Section: Resultssupporting

confidence: 83%

mentioning

confidence: 99%

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Sommese¹,

Sivaramakrishnan

2016

Journal of Biological Chemistry

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“…Surprisingly, the phosphorylation sites in VAMP8 that we identified face the 3 other α‐helices of the SNARE complex (Fig 2A) (Fasshauer et al , 1998; Sutton et al , 1998; Diao et al , 2015) as part of the 16 conserved SNARE layers (Fasshauer et al , 1998) that are buried inside the SNARE complex (T47 at layer +3, S54 at layer +5, and T53 close to layer +5) (Figs 2B–D and EV2C). An additional site was listed as a putative phosphorylation site in the PhosphoSitePlus (Hornbeck et al , 2015) database (S61 at layer +7).…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

et al. 2016

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Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α‐helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

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“…Phosphosite analyses were performed with PhosphoLogo (https://hpcwebapps.cit.nih.gov/ PhosphoLogo/) (24). Kinase substrates were analyzed in comparison with phosphosite analyses (http://www.phosphosite.org/) (25).…”

Section: Functional Analysesmentioning

confidence: 99%

Angiotensin II regulates phosphorylation of actin‐associated proteins in human podocytes

et al. 2017

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Within the kidney, angiotensin II (AngII) targets different cell types in the vasculature, tubuli, and glomeruli. An important part of the renal filtration barrier is composed of podocytes with their actin-rich foot processes. In this study, we used stable isotope labeling with amino acids in cell culture coupled to mass spectrometry to characterize relative changes in the phosphoproteome of human podocytes in response to short-term treatment with AngII. In 4 replicates, we identified a total of 17,956 peptides that were traceable to 2081 distinct proteins. Bioinformatic analyses revealed that among the increasingly phosphorylated peptides are predominantly peptides that are related to actin filaments, cytoskeleton, lamellipodia, mammalian target of rapamycin, and MAPK signaling. Among others, this screening approach highlighted the increased phosphorylation of actin-bundling protein, L-plastin (LCP1). AngII-dependent phosphorylation of LCP1 in cultured podocytes was mediated by the kinases ERK, p90 ribosomal S6 kinase, PKA, or PKC. LCP1 phosphorylation increased filopodia formation. In addition, treatment with AngII led to LCP1 redistribution to the cell margins, membrane ruffling, and formation of lamellipodia. Our data highlight the importance of AngII-triggered actin cytoskeleton-associated signal transduction in

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PhosphoSitePlus, 2014: mutations, PTMs and recalibrations

Cited by 2,755 publications

References 32 publications

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Angiotensin II regulates phosphorylation of actin‐associated proteins in human podocytes

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