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Review
. 2013 Dec;12(12):3453-64.
doi: 10.1074/mcp.R113.032862. Epub 2013 Sep 13.

The Coming of Age of Phosphoproteomics--From Large Data Sets to Inference of Protein Functions

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Free PMC article
Review

The Coming of Age of Phosphoproteomics--From Large Data Sets to Inference of Protein Functions

Philippe P Roux et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Protein phosphorylation is one of the most common post-translational modifications used in signal transduction to control cell growth, proliferation, and survival in response to both intracellular and extracellular stimuli. This modification is finely coordinated by a network of kinases and phosphatases that recognize unique sequence motifs and/or mediate their functions through scaffold and adaptor proteins. Detailed information on the nature of kinase substrates and site-specific phosphoregulation is required in order for one to better understand their pathophysiological roles. Recent advances in affinity chromatography and mass spectrometry (MS) sensitivity have enabled the large-scale identification and profiling of protein phosphorylation, but appropriate follow-up experiments are required in order to ascertain the functional significance of identified phosphorylation sites. In this review, we present meaningful technical details for MS-based phosphoproteomic analyses and describe important considerations for the selection of model systems and the functional characterization of identified phosphorylation sites.

Figures

Fig. 1.
Fig. 1.
Schematic overview of MS-based phosphoproteomic approaches. Outline of cell fractionation, affinity purification, and quantitative MS analyses. Information derived from quantitative proteomics such as changes in protein and phosphopeptide abundances and variation in phosphorylation stoichiometry are outlined by a rectangle (lower right). Blue and green circles depict protein abundances from different cell extracts, and shaded red circles indicate phosphorylation stoichiometry at a single site. When profiling phosphorylation events over extended stimulation periods (>1 h), phosphoproteomic results should be normalized to account for relative changes in protein abundances.
Fig. 2.
Fig. 2.
Functional significance of protein phosphorylation. Schematic model illustrating the different roles of protein phosphorylation in mediating diverse biological outcomes. (1) Phosphorylation-dependent membrane translocation can occur when SH2 domain-containing proteins, such as Grb2 and p85, bind to tyrosine-phosphorylated receptors at the plasma membrane. (2) Phosphorylation of protein kinases within their activation loop (T-loop), such as with MEK, ERK, and RSK, is a common activation mechanism that relies on structural rearrangements within the kinase domain. (3) Phosphorylation-dependent binding of scaffold proteins such as 14-3-3 can act as a cytoplasmic anchor for certain kinase substrates such as FoxO1 when phosphorylated by Akt. (4) In some instances, protein phosphorylation can promote protein stability, such as is the case for the immediate-early gene product c-Fos. (5), (6) Phosphorylation of certain transcription factors has been shown to promote or impede DNA binding, as was demonstrated for FoxO1 and c-Fos. (7) Phosphorylation of certain proteins within sequences termed “phosphodegron” can promote their recognition by specific ubiquitin ligases and subsequent proteosomal degradation, as was shown for the transcription factor FoxO3 and the E3 ligase MDM2.
Fig. 3.
Fig. 3.
Crosstalk between protein phosphorylation and other modifications. Examples of positive and negative regulation of protein phosphorylation on different modifications are highlighted in rounded rectangles. Interplay between phosphorylation and O-GlcNAcylation on specific Ser/Thr residues is also shown.

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