Signaling by dual specificity kinases

Dhanasekaran, N.; Reddy, E. Premkumar

doi:10.1038/sj.onc.1202251

Cited by 263 publications

(200 citation statements)

References 83 publications

(125 reference statements)

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“…Kinase specificity typically depends on the primary amino acid sequence surrounding the target phosphorylation site (25). Protein kinases can be separated into Ser͞Thr and Tyr kinases, although dual specificity kinases exist (26). The sites detected from our nuclear preparation were exclusively Ser͞Thr.…”

Section: Data-dependent Acquisition Of Ms͞ms͞ms Spectra For Improvedmentioning

confidence: 99%

Large-scale characterization of HeLa cell nuclear phosphoproteins

Beausoleil

Jedrychowski

Schwartz

et al. 2004

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

1,307

1,329

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Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.phosphorylation ͉ mass spectrometry ͉ strong cation exchange chromatography M uch of eukaryotic protein regulation occurs when protein kinases add a phosphate moiety in an ATP-dependent manner to a Ser, Thr, or Tyr residue of a substrate protein. Not surprisingly, malfunctions in this critical cellular process have been implicated as causal factors in diseases, such as diabetes, cancer, and Alzheimer's. With Ͼ500 identified kinases and thousands of potential substrates, these proteins remain attractive drug targets. Large-scale identification of phosphorylated kinase substrates will certainly enhance our understanding of diverse biological phenomena, potentially leading to targeted intervention in any number of disease paradigms.The identification of phosphorylation sites is most robustly accomplished by MS (1, 2). With tandem MS (MS͞MS), phosphopeptides are fragmented to determine their sequence and to pinpoint the specific Ser, Thr, or Tyr modified by a protein kinase. Despite many reports of thousands of identified proteins from a single biological sample, the large-scale determination of phosphorylation sites is just emerging. To date, the three largest reported repositories of identified sites are from yeast and plant studies [383 (3),125 (4) and Ϸ200 (5)], whereas the most phosphorylation sites identified from a single human sample stands at 64 (6). Clearly, to study the rich biology relying on protein phosphorylation will require more effective methodologies.Uninformative fragmentation is a fundamental obstacle to phosphorylation site analysis, regardless of the scale of an experiment. Fragmentation of phosphopeptides by collision-induced dissociation by MS͞MS commonly results in the production of a single dominant peak corresponding to a neutral loss of phosphoric acid (H 3 PO 4 , 98 Da) from the phosphopeptide (for example, see Fig. 2B). The lack of informative fragmentation at the peptide backbone severely reduces the ability of database searching algorithms to unambiguously identify the phosphopeptide. Furthermore, when a phosphopeptide is identified, it is often ...

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Section: Data-dependent Acquisition Of Ms͞ms͞ms Spectra For Improvedmentioning

confidence: 99%

Large-scale characterization of HeLa cell nuclear phosphoproteins

Beausoleil

Jedrychowski

Schwartz

et al. 2004

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

1,307

1,329

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Section: Introductionmentioning

confidence: 99%

“…Best known among these so-called dual specificity kinases are the upstream kinases that activate members of the mitogen-activated protein kinase (MAPK) family by phosphorylating a conserved TXY motif in the activation loop [2,3]. These MAPK kinases (also called MAP2K) are unique in their absolute substrate specificity for the cognate MAPKs they activate [3]. Several members of the CMGC group, which comprises the cyclin-dependent kinases (CDKs), MAPKs, glycogen synthase kinases 3 (GSK3) and CDK-like kinases (CLKs), have also become known as dual specificity kinases [4][5][6].…”

Section: Introductionmentioning

confidence: 99%

Mechanism of dual specificity kinase activity of DYRK1A

et al. 2013

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The function of many protein kinases is controlled by the phosphorylation of a critical tyrosine residue in the activation loop. Dual specificity tyrosinephosphorylation-regulated kinases (DYRKs) autophosphorylate on this tyrosine residue but phosphorylate substrates on aliphatic amino acids. This study addresses the mechanism of dual specificity kinase activity in DYRK1A and related kinases. Tyrosine autophosphorylation of DYRK1A occurred rapidly during in vitro translation and did not depend on the non-catalytic domains or other proteins. Expression in bacteria as well as in mammalian cells revealed that tyrosine kinase activity of DYRK1A is not restricted to the co-translational autophosphorylation in the activation loop. Moreover, mature DYRK1A was still capable of tyrosine autophosphorylation. Point mutants of DYRK1A and DYRK2 lacking the activation loop tyrosine showed enhanced tyrosine kinase activity. A series of structurally diverse DYRK1A inhibitors was used to pharmacologically distinguish different conformational states of the catalytic domain that are hypothesized to account for the dual specificity kinase activity. All tested compounds inhibited substrate phosphorylation with higher potency than autophosphorylation but none of the tested inhibitors differentially inhibited threonine and tyrosine kinase activity. Finally, the related cyclin-dependent kinase-like kinases (CLKs), which lack the activation loop tyrosine, autophosphorylated on tyrosine both in vitro and in living cells. We propose a model of DYRK autoactivation in which tyrosine autophosphorylation in the activation loop stabilizes a conformation of the catalytic domain with enhanced serine/threonine kinase activity without disabling tyrosine phosphorylation. The mechanism of dual specificity kinase activity probably applies to related serine/threonine kinases that depend on tyrosine autophosphorylation for maturation. Structured digital abstract• CLK1 and CLK1 phosphorylate by protein kinase assay (View interaction)• HIPK2 and HIPK2 phosphorylate by protein kinase assay (View interaction)• DYRK1A phosphorylates SF3B1 by protein kinase assay (View interaction)• DYRK1A and DYRK1A phosphorylate by protein kinase assay (View interaction)

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“…MAP kinases are regulated by MEK-mediated phosphorylation of their activation loop, a region of the catalytic domain located between conserved kinase subdomains VII and VIII in the primary structure, just below the catalytic cleft in the tertiary structure [13]. MEKs/ MKKs phosphorylate both a tyrosine and a threonine in a TXY motif found in the MAPK activation loop; for this reason, MEKs are called "dual-specificity" kinases [14]. Phosphorylation of the activation loop induces it to refold, causing subtle conformational changes which reverberate through the rest of the enzyme [15].…”

Section: Introductionmentioning

confidence: 99%

Analysis of mitogen-activated protein kinase activation and interactions with regulators and substrates

Bardwell

Shah

2006

Methods

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Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signal transduction modules in eukaryotes that are of great interest and importance. Here, we summarize some useful methods for the analysis of MAPK signaling, including methods to (1) detect MAPK activation in cells, with an emphasis on using phosphorylation-state-specific antibodies raised against mammalian phosphopeptide sequences to detect the activation of MAPKs in other species; (2) estimate the cellular concentrations of MAPKs and other proteins of interest; (3) detect and quantify the stable physical association of MAPKs with their substrates and regulators, and estimate the relevant dissociation constants; (4) delineate the MAPK-binding regions or domains of MAPK-interacting proteins, with particular emphasis on the identification and verification of MAPK-docking sites. These procedures are broadly applicable to many organisms, including both yeast and mammalian cells.

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Signaling by dual specificity kinases

Cited by 263 publications

References 83 publications

Large-scale characterization of HeLa cell nuclear phosphoproteins

Large-scale characterization of HeLa cell nuclear phosphoproteins

Mechanism of dual specificity kinase activity of DYRK1A

Analysis of mitogen-activated protein kinase activation and interactions with regulators and substrates

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