<scp>CP</scp>hos: A program to calculate and visualize evolutionarily conserved functional phosphorylation sites

Zhao, Boyang; Pisitkun, Trairak; Hoffert, Jason D.; Knepper, Mark A.; Saeed, Fahad

doi:10.1002/pmic.201200189

Cited by 26 publications

(23 citation statements)

References 28 publications

(30 reference statements)

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“…Specifically, we compared phosphopeptides derived from stimulated wild‐type cells to phosphopeptides derived from either PKCB knockout cells or wild‐type cells treated with a PKCB blocker (10 μM ruboxistaurin) (Fig EV2A). The list of 17,700 phosphopeptides that could be reproducibly identified between biological replicates (Figs 1D and EV2A and B) was pruned to 179 by selecting only peptides containing evolutionarily conserved phosphorylation sites (Zhao et al , 2012), that in addition were known or proposed secretion regulators (Materials and Methods) (Fig 1E), and that were also at least twofold more abundant in control cells compared to PKCB knockout cells or cells treated with PKCB inhibitor (Fig 1F and Appendix Table S1). …”

Section: Resultsmentioning

confidence: 99%

“…To assess the conservation of phosphorylation sites among different species, we used the standalone versions of CPhos (Zhao et al , 2012) (http://helixweb.nih.gov/CPhos) to calculate a score for each identified phosphorylation site in our phosphoproteomics data. Only phosphorylation sites with a perfect phosphorylation score (phosphorylation site score = 1) for mammals were considered.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

et al. 2016

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“…Conserved domain analysis was performed by the Batch Web CD-Search Tool (http://www.ncbi.nlm.nih.gov/Structure/ bwrpsb/bwrpsb.cgi) (18). CPhos was utilized to assess the conservation of ubiquitylated sites (http://helixweb.nih.gov/CPhos/) (19). Annotations of ubiquitylated sites were extracted from the UniProt Knowledgebase (http://www.uniprot.org/).…”

Section: Methodsmentioning

confidence: 99%

Deubiquitylation of Protein Cargo Is Not an Essential Step in Exosome Formation

Huebner

Liu

Somparn

et al. 2016

Molecular & Cellular Proteomics

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Exosomes, derived from multivesicular bodies (MVBs), contain proteins and genetic materials from their cell of origin and are secreted from various cells types, including kidney epithelial cells. In general, it is thought that protein cargo is ubiquitylated but that ubiquitin is cleaved by specific deubiquitylases during the process of cargo incorporation into MVBs. Here, we provide direct evidence that, in vivo, deubiquitylation is not essential. Ubiquitin was detected within human MVBs and urinary exosomes by electron microscopy. Of the >6000 proteins identified in human urinary exosomes was mass spectrometry, 15% were ubiquitylated with various topologies (Lys63>Lys48> Lys11>Lys6>Lys29>Lys33>Lys27). A significant preference for basic amino acids upstream of ubiquitylation sites suggests specific ubiquitylation motifs. The current studies demonstrate that, in vivo, deubiquitylation of proteins is not necessary for their incorporation into MVBs and highlight that urinary exosomes are an enriched source for studying ubiquitin modifications in physiological or disease states. Exosomes are extracellular nanovesicles (20 -100 nm) that are secreted from various cells types in the body (1). In the urinary system, exosomes are secreted from epithelial cells from all segments of the kidney tubule and urinary tract (2). Urinary exosomes contain proteins and genetic materials e.g. mRNA and miRNA, from their cell of origin that may represent physiology or pathophysiology of a variety of renal and systemic diseases (3).In general, exosomes originate in late endosomal compartments called multivesicular bodies (MVBs). An essential posttranslational modification that marks membrane proteins for incorporation into MVBs is ubiquitylation (4). Ubiquitin is a small 76 amino acid protein that requires reversible enzymatic activity of E1, an activation enzyme; E2, a conjugation enzyme; and E3, a ligation enzyme, for binding to a specific substrate. Ubiquitin acts as a signal for mediating a range of cellular functions, including protein trafficking, DNA repair, endocytosis, proteasomal and lysosomal degradation, and transcriptional regulation. The role of ubiquitin in various cellular functions can be directly related to the type of ubiquitin modifications on a specific substrate, such as monoubiquitylation, multimonoubiquitylation, and polyubiquitylation (5). Various topologies of polyubiquitin chains also play different roles in biology. For example, Lys48-linked chains can target proteins for proteasomal degradation, whereas Lys63-linked chains can target proteins for lysosomal degradation, aid in DNA repair, or play a role in transcriptional regulation (5).During the process of protein trafficking, ubiquitylated membrane protein cargo can be recognized by the endosomal-sorting complex required for transport (ESCRT) apparatus on the outer surface of MVBs. Through a cascade of protein interactions, internal luminal vesicles (ILVs) are formed inside the MVB that can be released into the extracellular environment as exosomes upon t...

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“…We also analyzed the phosphorylation sites for conservation across multiple species using the CPhos algorithm (Supplemental Figure 3). 22 Phosphorylation Regulates Podocyte-Specific and Slit Diaphragm-Associated Proteins We next generated a list of 48 phosphoproteins known to be specifically expressed in podocytes. 16 The 146 confident phosphorylation sites corresponding to these proteins are outlined in Supplemental Table 4.…”

Section: Phosphoproteomic and Proteomic Analyses Of Murine Glomerulimentioning

confidence: 99%

“…The CPhos program was used for analysis of site conservation with default settings for mouse datasets. 22 Phosphorylation enrichment analysis of PHB/Band7-like protein domains was performed as previously described. 32 For this analysis, we used 198,773 phosphorylation sites derived from previously published MS experiments across 15 species obtained from the PTMfunc database (http://www.ptmfunc.com).…”

Section: Bioinformatic Analysesmentioning

confidence: 99%

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

Rinschen

König

et al. 2014

Journal of the American Society of Nephrology

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Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulusexpressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.

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CPhos: A program to calculate and visualize evolutionarily conserved functional phosphorylation sites

Cited by 26 publications

References 28 publications

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Deubiquitylation of Protein Cargo Is Not an Essential Step in Exosome Formation

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

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