Phosphoproteomic approaches to elucidate cellular signaling networks

Schmelzle, Katrin; White, Forest M.

doi:10.1016/j.copbio.2006.06.004

Cited by 146 publications

(102 citation statements)

References 51 publications

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“…Phosphorylation of proteins is one of the most important modulatory mechanisms of activation of a number of molecular pathways and a potential indicator of cellular proliferation, migration, and other cellular functions. [5][6][7][8][9] Thus, identification of activated pathways in tumor specimens is an essential element in efficient cancer diagnosis and treatment. [6][7][8][9][10][11] Protein phosphorylation is a dynamic balance between two opposite events: phosphorylation by highly specific kinases and dephosphorylation by less-specific phosphatases.…”

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confidence: 99%

“…[5][6][7][8][9] Thus, identification of activated pathways in tumor specimens is an essential element in efficient cancer diagnosis and treatment. [6][7][8][9][10][11] Protein phosphorylation is a dynamic balance between two opposite events: phosphorylation by highly specific kinases and dephosphorylation by less-specific phosphatases. [12][13][14] To utilize phosphoproteins as markers of cellular signaling activity and tumor proliferation, 6,8,9 it is crucial to assess their expression levels in tissue specimens that reflect the in vivo status as accurately as possible.…”

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confidence: 99%

“…[6][7][8][9][10][11] Protein phosphorylation is a dynamic balance between two opposite events: phosphorylation by highly specific kinases and dephosphorylation by less-specific phosphatases. [12][13][14] To utilize phosphoproteins as markers of cellular signaling activity and tumor proliferation, 6,8,9 it is crucial to assess their expression levels in tissue specimens that reflect the in vivo status as accurately as possible. Elevated expression levels of phospho-AKT, phospho-ERK1/ 2, phospho-mTor, and phospho-p20S6K, for example, have been described to be associated with tamoxifen resistance in postmenopausal breast cancer patients.…”

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Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time

Vassilakopoulou

Parisi

Siddiqui

et al. 2015

Laboratory Investigation

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Individualized targeted therapies for cancer patients require accurate and reproducible assessment of biomarkers to be able to plan treatment accordingly. Recent studies have shown highly variable effects of preanalytical variables on gene expression profiling and protein levels of different tissue types. Several publications have described protein degradation of tissue samples as a direct result of delay of formalin fixation of the tissue. Phosphorylated proteins are more labile and epitope degradation can happen within 30 min of cold ischemic time. To address this issue, we evaluated the change in antigenicity of a series of phosphoproteins in paraffin-embedded samples from breast tumors as a function of time to formalin fixation. A tissue microarray consisting of 93 breast cancer specimens with documented time-to-fixation was used to evaluate changes in antigenicity of 12 phosphoepitopes frequently used in research settings as a function of cold ischemic time. Analysis was performed in a quantitative manner using the AQUA technology for quantitative immunofluorescence. For each marker, least squares univariate linear regression was performed and confidence intervals were computed using bootstrapping. The majority of the epitopes tested revealed changes in expression levels with increasing time to formalin fixation. Some phosphorylated proteins, such as phospho-HSP27 and phospho-S6 RP, involved in posttranslational modification and stress response pathways increased in expression or phosphorylation levels. Others (like phospho-AKT, phosphor-ERK1/2, phospho-Tyrosine, phospho-MET, and others) are quite labile and loss of antigenicity can be reported within 1-2 h of cold ischemic time. Therefore specimen collection should be closely monitored and subjected to quality control measures to ensure accurate measurement of these epitopes. However, a few phosphoepitopes (like phospho-JAK2 and phospho-ER) are sufficiently robust for routine usage in companion diagnostic testing.

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Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time

Vassilakopoulou

Parisi

Siddiqui

et al. 2015

Laboratory Investigation

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“…For example, protein phosphorylation is involved in the control of cell division, metabolic activity, cell adhesion and migration, cell to cell communication, and signal transduction [29,30]. During spermatogenesis, protein phosphorylation has been shown to play a role in several processes, including gene transcription [31], and chromatin remodeling [32].…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-specific Kinase, TSSK-2.

Zhang

Shen

Jones

et al. 2008

Biology of Reproduction

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Mammalian SPAG16L, the orthologue of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites and the protein was confirmed to be phosphorylated in vivo. A yeast-twohybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by co-immunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their co-localization was also noted by confocal microscopy in CHO cells where they were co-expressed. TSSK2 associates with SPAG16L via its C terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.

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“…Functional assays, such as measuring migration, proliferation, and apoptosis, can give information on the downstream biological response associated with a particular phosphorylation. 4 …”

Section: The Missionmentioning

confidence: 99%

Phosphoproteomics: Miles To Go Before It’s Routine

Piggee¹

2009

Anal. Chem.

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Researchers see major technological advances but still have significant challenges to overcome.Not so long ago, the process for determining protein phosphorylation was a labor-intensive and serial process. Typically, a researcher would use 32 P-labeling coupled with 2D gel electrophoresis phosphopeptide mapping, autoradiography, band excision, and Edman sequencing. In addition to expending much time and effort, researchers had to work with radioactivity, and the method could not be multiplexed. Now in 2009, two complementary techniques for identification of phosphorylation sites, MS and microarrays, have provided significant increases in throughput. In discovery mode, researchers can identify thousands of phosphorylation sites with LC/MS n , and in profiling mode, researchers can detect thousands of phosphorylation events and predict the substrates of various protein kinases with planar, suspension, and reverse microarrays. 1 As the technology has improved and researchers have been able to glean impressive amounts of information from phosphoproteomics experiments, more labs have shown interest. "I think the field of phosphoproteomics is poised to take a big leap forward," says Timothy Veenstra of the U.S. National Cancer Institute. And the widespread enthusiasm among researchers is tangible. "It's probably the most exciting time right now in phosphoproteomics," says Daniel Figeys of the University of Ottawa. " [Phosphoproteomics] used to be kind of stuck a few years ago, where the numbers of phosphorylation sites that could be mapped were dreadfully small," he says.But mapping so many sites can be a problem if researchers cannot weed through the information to find what is important in a reasonable amount of time, as Matthias Mann of the Max Planck Institute of Biochemistry (Germany) explains. "Now we're in the paradoxical situation that we almost have too much of a good thing. So it's now possible to quantify even 10,000 phosphorylation sites in a single experiment. But in my view ... without then knowing which ones of the 10,000 are actually relevant for [a particular] biological system, then it's too much," he says.With this flood of new information about protein phosphorylation, the intricacy of the cell signaling network is becoming more apparent. "In the past with proteomics, people thought, 'Okay, one gene, one functionsone gene, one protein,'" says Albert Heck of Utrecht University (The Netherlands). "It makes you wonder and makes you humble about what we know already." Researchers are learning that the cell signaling pathways are incredibly complicated. "We think we understand sometimes how proteins are interacting with each other and how signaling networks are really working," says Emanuel Petricoin of George Mason University. "We go into these clinical trials where we think we can turn off these pathways, and you have some compensatory feedback loop that was unknown take over and actually turn on another pathway that you didn't even know was connected." Elucidating the structure and function of these ...

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Phosphoproteomic approaches to elucidate cellular signaling networks

Cited by 146 publications

References 51 publications

Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time

Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time

Phosphorylation of Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-specific Kinase, TSSK-2.

Phosphoproteomics: Miles To Go Before It’s Routine

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