Combining chemical genetics and proteomics to identify protein kinase substrates

Dephoure, Noah; Howson, Russell W.; Blethrow, Justin D.; Shokat, Kevan M.; O’Shea, Erin K.

doi:10.1073/pnas.0509080102

Cited by 111 publications

(84 citation statements)

References 50 publications

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“…The analogs are very poor substrates for wild-type kinases; thus, an analog-sensitive kinase (or as-kinase) can be used to specifically radiolabel its substrates in cell extracts while preserving important aspects of biological context, such as the integrity of protein complexes. Coupling this approach with the use of libraries of genetically encoded affinity-tagged proteins has facilitated identification of low-abundance S. cerevisiae Cdk1 and Pho85 substrate proteins (12,13). However, this approach is not currently tractable in organisms where genetic libraries present technical limits.…”

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confidence: 99%

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Blethrow

Glavy²,

Morgan³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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291

290

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We describe a method for rapid identification of protein kinase substrates. Cdk1 was engineered to accept an ATP analog that allows it to uniquely label its substrates with a bio-orthogonal phosphate analog tag. A highly specific, covalent capture-andrelease methodology was developed for rapid purification of tagged peptides derived from labeled substrate proteins. Application of this approach to the discovery of Cdk1-cyclin B substrates yielded identification of >70 substrates and phosphorylation sites. Many of these sites are known to be phosphorylated in vivo, but most of the proteins have not been characterized as Cdk1-cyclin B substrates. This approach has the potential to expand our understanding of kinase-substrate connections in signaling networks.P rotein kinases regulate a vast array of biological processes through phosphorylation of protein substrates. A comprehensive map of all phosphorylation sites and kinase-substrate pairs would greatly facilitate the study of signaling networks. This goal faces two fundamental challenges. First, all protein kinases use ATP as a cofactor to phosphorylate their targets, and thus the direct substrates of a single kinase cannot be easily traced in protein mixtures containing multiple kinases. Second, phosphorylation often occurs at low stoichiometry and on low-abundance proteins. This makes substrate and phosphorylation site identification very challenging. Powerful methods have been developed to address these dual problems (1, 2). Kinase-substrate pairs can be tested in multiplexed phosphorylation assays by using immobilized arrays of purified proteins. These high-throughput chip-based assays have the added benefit of presenting low-abundance proteins at easily detectable levels and have provided a first-generation map of protein phosphorylation in Saccharomyces cerevisiae (3). However, these assays do not currently allow identification of phosphorylation sites and have not been adapted to organisms with more complex proteomes. Prediction of high-likelihood kinase substrates can sometimes be achieved by using knowledge of the substrate sequence motif preferences of individual kinases, but only when these preferences are strong (4, 5). Finally, thousands of in vivo phosphorylation sites from metazoan organisms have been identified in proteomic screens (6-9), but for most of these sites the responsible upstream kinases remain unknown.We have demonstrated a chemical and genetic solution to the common use of ATP by all kinases. Our approach relies on engineering a kinase to accept unnatural ATP analogs by modification of the ATP-binding pocket (10, 11). The analogs are very poor substrates for wild-type kinases; thus, an analog-sensitive kinase (or as-kinase) can be used to specifically radiolabel its substrates in cell extracts while preserving important aspects of biological context, such as the integrity of protein complexes. Coupling this approach with the use of libraries of genetically encoded affinity-tagged proteins has facilitated identification of low-abundanc...

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mentioning

confidence: 99%

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Blethrow

Glavy²,

Morgan³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

291

290

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“…In addition, in vitro reactions of kinases with synthetic peptide libraries have been used to define kinase specificities (4,5). Finally, protein targets for individual kinases are being defined through elegant systematic studies (6)(7)(8). Notwithstanding, only MS-based proteomics currently provides the capability to identify at once and on a massive scale kinase substrates and the specific positions of their modification (9).…”

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confidence: 99%

Large-scale phosphorylation analysis of mouse liver

Villén

Beausoleil

Gerber

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

689

775

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Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a ''dipolar'' motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation.mass spectrometry ͉ proteomics I n eukaryotes, protein phosphorylation is among the most important regulatory events in cells, guiding primary biological processes, such as cell division, growth, migration, differentiation, and intercellular communication (1, 2). Numerous efforts to study protein phosphorylation continue to provide the scientific community with an expanding phosphorylation database resource. Sequence alignment studies (3) have defined which genes encode protein kinases. In addition, in vitro reactions of kinases with synthetic peptide libraries have been used to define kinase specificities (4, 5). Finally, protein targets for individual kinases are being defined through elegant systematic studies (6-8). Notwithstanding, only MS-based proteomics currently provides the capability to identify at once and on a massive scale kinase substrates and the specific positions of their modification (9).A major goal of systems biology is to integrate all in vivo phosphorylation events into the context of an organism. Phosphorylation analysis from primary tissue, in contrast to immortalized cell lines, best represents events that are occurring in the basal physiological state of an organism even though tissues often contain heterogeneous populations of cells. In the present study we chose mouse liver as a model tissue for establishing a pipeline for large-scale phosphorylation analysis. In addition, protein phosphorylation plays a critical role in normal liver development and function; therefore, the sites obtained here could be further characterized into physiological context. Several phosphorylation-related (PI3K and Akt signaling) liver phenotypes have been reported to be related to altered lipid and glucose metabolism via insulin control (10, 11) and liver regeneration (ribosomal protein S6) (12). Previously, only two studies have examined phosphorylation sites from liver tissue with 26 (13) and 339 (14) sites.As the fie...

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“…The cAMP-dependent protein kinase (PKA) 3 is expressed in all mammalian cells and plays a major role in processes such as metabolic control, memory, growth, development, and apoptosis. Thus, it has many substrates, which usually are highly tissue specific.…”

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confidence: 99%

Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

Schauble

King

Darshi

et al. 2007

Journal of Biological Chemistry

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Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N 6 -substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr 197 and Ser 338 . Based on its kinetic properties, the engineered C-subunit preferred N 6 (benzyl)-ATP and N 6 (phenethyl)-ATP over other ATP analogs, but still retained a 30 M K m for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.The cAMP-dependent protein kinase (PKA) 3 is expressed in all mammalian cells and plays a major role in processes such as metabolic control, memory, growth, development, and apoptosis. Thus, it has many substrates, which usually are highly tissue specific. One such example is the bifunctional enzyme phosphofructokinase-2 (PFK2), which upon phosphorylation by PKA is converted to its phosphatase active mode in liver, but is stabilized in its kinase mode in heart, and is not a PKA substrate in skeletal muscle (1). Although PKA has been widely studied in most cells, the direct substrates of PKA are difficult to distinguish from downstream substrates that are phosphorylated as a result of the PKA activation. A possible way to determine whether identified substrates are modified by the kinase directly or indirectly via an intermediary kinase is to engineer the ATP-binding pocket of the kinase in question to accept a bulky ATP analog that cannot be used by the wild type kinase. This strategy was developed initially for a tyrosine kinase, v-Src, (2) and has subsequently been used for other protein kinases (3). Shah et al. (4) found that a single mutation conferred specificity for ATP analogs that have a large substituent on the nitrogen at position 6 (N 6 ) on the purine ring of ATP. Two residues that were found within 5 Å of the N 6 position of ATP were based initially on the structures of PKA and CDK2 (cyclindependent kinase 2) since the crystal structure of v-Src was not available at the time (2). In v-Src, these residues are Val 323 and Ile 338 , while the corresponding residues in PKA are Val 104 and Met 120 . Although the Val 323 mutation was found to be not important for conferring specificity in v-Src, mutation of Ile 338 to Ala did alter the specificity of v-Src and allowed the enzyme to use an orthogonal bulky ATP analog. Once the crystal structure of hematopoietic cell kinase (Hck), a Src family tyrosine kinase was available, molecular modeling was employed to explore more fully the binding pocket of Hck to find t...

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Combining chemical genetics and proteomics to identify protein kinase substrates

Cited by 111 publications

References 50 publications

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Large-scale phosphorylation analysis of mouse liver

Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

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