Trends in Genetics
Volume 22, Issue 10, October 2006, Pages 545-554
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Review
Charging it up: global analysis of protein phosphorylation

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Protein phosphorylation affects most, if not all, cellular activities in eukaryotes and is essential for cell proliferation and development. An estimated 30% of cellular proteins are phosphorylated, representing the phosphoproteome, and phosphorylation can alter a protein's function, activity, localization and stability. Recent studies for large-scale identification of phosphosites using mass spectrometry are revealing the components of the phosphoproteome. The development of new tools, such as kinase assays using modified kinases or protein microarrays, enables rapid kinase substrate identification. The dynamics of specific phosphorylation events can now be monitored using mass spectrometry, single-cell analysis of flow cytometry, or fluorescent reporters. Together, these techniques are beginning to elucidate cellular processes and pathways regulated by phosphorylation, in addition to global regulatory networks.

Introduction

Protein phosphorylation is an integral part of cell signaling and has a role in almost all cellular and developmental processes [1]. At a biochemical level, protein activity, localization, stability and interactions can be controlled by phosphorylation, and kinases act as regulators of most cellular processes (Figure 1). In yeast, flies and humans, it is predicted that protein kinases phosphorylate 30% of cellular proteins [2].

In agreement with the widespread occurrence of protein phosphorylation, a significant portion of eukaryotic protein-coding genes encode protein kinases. There are predicted to be 518 putative protein kinases in humans [3], 540 in mice [4] and 122 kinases in yeast [5], although these numbers are approximate because of genes encoding atypical kinases, and those that appear to be catalytically inactive (substitution at the conserved catalytic residues Lys30, Asp125 or Asp143) are present in most eukaryotic genomes. Nonetheless, protein kinases comprise 2% of these genomes and represent one of the largest classes of proteins.

Recent years have witnessed a revolution in the global analysis of protein phosphorylation, its dynamics and the elucidation of substrates phosphorylated by protein kinases. These studies have provided remarkable insights into signaling cascades and their organization. This article reviews these advances.

Section snippets

Global mapping of phosphorylation by mass spectrometry

Traditionally phosphorylation sites have been mapped using several approaches including Edman degradation, thin-layer chromatography of peptide fragments and mutational analysis. Although successful on a small scale, these methods are not high throughput. Recent developments in mass spectrometry are overcoming the challenges in characterizing protein phosphorylation on a large scale. Initially mass spectrometric methods were problematical: the negative charge of phosphopeptides hampers their

Comparison of phosphorylation profiles using mass spectrometry

Mass spectrometry is becoming increasingly useful in comparative analysis of cell states, using either stable-isotope-tagged amine-reactive reagents (iTRAQ) [14], stable isotope labeling by amino acids in cell culture (SILAC) [15], or through additional chemical approaches 12, 16. There are four iTRAQ reagents enabling four samples to be compared in a single analysis. Each iTRAQ consists of a protein-reactive group targeting the side chain of lysines and the N-terminal amine of peptides, a

Global identification of kinase substrates

Equally challenging as the identification of phosphoproteins is the determination of the proteins responsible for the modification. Phosphorylation of a substrate usually occurs at a consensus site containing a central S/T/Y residue that is recognized by the catalytic domain of a kinase, and in many cases the interaction is assisted through secondary docking sites on the substrate [19] or by a third protein acting as a scaffold [20]. Phosphorylation at histidine has also been observed, although

Comparison of substrate identification methods

Comparison of both protein microarrays and chemical genetics using the results generated for Cdc28 and Pho85 reveals significant overlap of results in addition to differences. Both assays show enrichment for substrates with the same localization or functional categories. Both are also successful at identifying a portion of the known in vivo substrates, with the success rate specific to the kinase tested. The modified Pho85-as–Pho80 phosphorylated 19 substrates [32]. The protein microarrays

Signaling networks: taking them apart and putting them together

Signaling networks have traditionally been constructed by investigators studying one or a few proteins at a time. The use of global approaches enables the construction of elaborate pathways and networks. Recently, Collins et al. identified 289 unambiguous phosphorylation sites on synaptic proteins using mass spectrometry. They then performed kinase assays with seven kinases – protein kinase A (PKA), protein kinase B (PKB or Akt), PKC, casein kinase 2 (CK2), p38 mitogen-activated protein kinase,

Single-cell resolution: the dynamics of phosphorylation

Phosphorylation is a dynamic and cell-specific process, a point that can be lost in the study of cell lysates. Experiments can now be designed to analyze signaling networks in single cells following stimulation in complex samples containing multiple cell types. Studies using flow cytometry to identify simultaneously cell types with antibodies to cell markers in addition to the activation of kinases and other phosphoregulated signaling proteins within fixed cells have been performed. These

Conclusion and future directions

The progress of the past five years has greatly increased the number of identified phosphoproteins and the kinases responsible for their phosphorylation, and provided a wealth of information about the networks they form. One challenge for the field remains the elucidation of the function of individual phosphorylation events. As proteomics is a new field we expect many more advances to be made both in technology and information generated. Improvements in methods using mass spectrometry are

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