Phosphoproteomics Meets Chemical Genetics: Approaches for Global Mapping and Deciphering the Phosphoproteome

Protein kinases are important enzymes involved in the regulation of various cellular processes. To function properly, each protein kinase phosphorylates only a limited number of proteins among the thousands present in the cell. This provides a rapid and dynamic regulatory mechanism that controls biological functions of the proteins. Despite the importance of protein kinases, most of their substrates remain unknown. Recently, the advances in the fields of protein engineering, chemical genetics, and mass spectrometry have boosted studies on identification of bona fide substrates of protein kinases. Among the various methods in protein kinase specific substrate identification, genetically engineered protein kinases and quantitative phosphoproteomics have become promising tools. Herein, we review the current advances in the field of chemical genetics in analog-sensitive protein kinase mutants and highlight selected strategies for identifying protein kinase substrates and studying the dynamic nature of protein phosphorylation.

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EXPLICIT-Kinase: a gene expression predictor for dissecting the functions of the Arabidopsis kinome

10.1101/2021.09.15.460437 ◽

2021 ◽

Author(s):

Yuming Peng ◽

Wanzhu Zuo ◽

Yue Qin ◽

Shisong Ma

Keyword(s):

Gene Expression ◽

Protein Kinase ◽

Protein Kinases ◽

Stress Responses ◽

Expression Patterns ◽

Gene Expression Patterns ◽

The Novel ◽

Cellular Processes ◽

Bona Fide ◽

Kinase A

Protein kinases regulate virtually all cellular processes, but it remains challenging to determine the functions of all protein kinases, collectively called the kinome, in any species. We developed an approach called EXPLICIT-Kinase to predict the functions of the Arabidopsis kinome. Because the activities of many kinases can be regulated transcriptionally, their gene expression patterns provide clues to their functions. A universal gene expression predictor for Arabidopsis was constructed to predict the expression of 30,172 non-kinase genes based on the expression of 994 protein kinase genes. The model reconstituted highly accurate transcriptomes for diverse Arabidopsis samples. It identified the significant kinases as predictor kinases for predicting the expression of Arabidopsis genes and pathways. Strikingly, these predictor kinases were often known regulators of the related pathways, as exemplified by those involved in cytokinesis, tissue development, and stress responses. Comparative analyses have revealed that portions of these predictor kinases, including the novel ones, are shared and conserved between Arabidopsis and maize. The conservation between species provide additional evidence to support the novel predictor kinases as bona fide regulators of the pathways involved. Thus our approach enables the systematic dissection of the functions of the Arabidopsis kinome.

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Combining chemical genetics and proteomics to identify protein kinase substrates

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0509080102 ◽

2005 ◽

Vol 102 (50) ◽

pp. 17940-17945 ◽

Cited By ~ 90

Author(s):

N. Dephoure ◽

R. W. Howson ◽

J. D. Blethrow ◽

K. M. Shokat ◽

E. K. O'Shea

Keyword(s):

Protein Kinase ◽

Chemical Genetics ◽

Kinase Substrates

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Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Identification of a Mutant Kinase/ATP Analog Pair

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot4636 ◽

2007 ◽

Vol 2007 (8) ◽

pp. pdb.prot4636-pdb.prot4636

Author(s):

S. T. Eblen ◽

N. V. Kumar ◽

M. J. Weber

Keyword(s):

Protein Kinase ◽

Genetically Engineered ◽

Kinase Substrates ◽

Mutant Kinase ◽

Novel Protein

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Identifying and characterizing casein kinase II in human platelets

Blood ◽

10.1182/blood.v83.12.3517.3517 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3517-3523 ◽

Cited By ~ 12

Author(s):

CH Hoyt ◽

CJ Oh ◽

JB Beekman ◽

DW Litchfield ◽

KM Lerea

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Casein Kinase Ii ◽

Human Platelets ◽

Casein Kinase ◽

Immunogold Electron Microscopy ◽

Beta Subunits ◽

Specific Substrate ◽

Substrate Peptide ◽

Independent Protein

Abstract We have recently shown that inhibition of protein phosphatases in platelets causes increases in protein phosphorylations with a concomitant inhibition of platelet responses. The burst in protein phosphorylation appears to be catalyzed by messenger-independent protein kinases. The aim of the present study was to characterize the presence of broad families of protein kinases found in platelets. Lysates of control and thrombin-stimulated platelets were prepared, and proteins were separated on MONO Q fast protein liquid chromatography. In addition to the presence of histone protein kinase and tyrosine kinase activities, human platelets contain casein kinase II (CKII) activity as assessed by phosphorylation of a specific substrate peptide. Western blot analysis and immunogold electron microscopy studies further showed the presence of alpha-, alpha'-, and beta- subunits of CKII. The enzyme appears to be distributed throughout the cytosol and not secreted after thrombin treatment. Immunoprecipitation studies suggest that at least some of the holoenzymes exist as an alpha alpha' beta 2 complex. Although no activation of the enzyme was detected after thrombin treatment, our results show that CKII is a major messenger-independent protein kinase in platelets.

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Phosphorylation-dependent substrate selectivity of protein kinase B (AKT1)

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012425 ◽

2020 ◽

Vol 295 (24) ◽

pp. 8120-8134

Author(s):

Nileeka Balasuriya ◽

Norman E. Davey ◽

Jared L. Johnson ◽

Huadong Liu ◽

Kyle K. Biggar ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase B ◽

Tumor Biology ◽

Peptide Libraries ◽

Substrate Selectivity ◽

Activity Data ◽

Cellular Processes ◽

Substrate Preferences ◽

Substrate Peptide ◽

Kinase Substrates

Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 “known” substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.

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The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases

Biochemical Journal ◽

10.1042/bj20051180 ◽

2005 ◽

Vol 391 (1) ◽

pp. 17-24 ◽

Cited By ~ 341

Author(s):

Alberto C. Vitari ◽

Maria Deak ◽

Nick A. Morrice ◽

Dario R. Alessi

Keyword(s):

Protein Kinase ◽

Glutamic Acid ◽

Protein Kinases ◽

Catalytic Domain ◽

Cellular Processes ◽

Basal Activity ◽

Human Genes ◽

Genes Encoding ◽

Equivalent Residue ◽

Phosphopeptide Mapping

Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed ‘STE20/SPS1-related proline/alanine-rich kinase’ (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed ‘oxidative stress response kinase-1’ (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.

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Postal code for a plant MAPK

Biochemical Journal ◽

10.1042/bj20121003 ◽

2012 ◽

Vol 446 (2) ◽

pp. e5-e7 ◽

Cited By ~ 4

Author(s):

Brian E. Ellis

Keyword(s):

Signal Transduction ◽

Protein Kinase ◽

Protein Kinases ◽

Mitogen Activated Protein Kinase ◽

Postal Code ◽

Biochemical Journal ◽

Mitogen Activated Protein ◽

Cellular Signal ◽

Efficient Route ◽

Kinase Substrates

Plants contain hundreds of protein kinases that are believed to provide cellular signal transduction services, but the identities of the proteins they are targeting are largely unknown. Using an Arabidopsis MAPK (mitogen-activated protein kinase) (MPK6) as a model, Sörensson et al. describe in this issue of the Biochemical Journal how arrayed combinatorial peptide scanning offers an efficient route to discovery of new potential kinase substrates.

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Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Phosphorylation of Substrates in Cell Lysates with Exogenous Kinase

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot4639 ◽

2007 ◽

Vol 2007 (8) ◽

pp. pdb.prot4639-pdb.prot4639

Author(s):

S. T. Eblen ◽

N. V. Kumar ◽

M. J. Weber

Keyword(s):

Protein Kinase ◽

Genetically Engineered ◽

Cell Lysates ◽

Kinase Substrates ◽

Novel Protein

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Structure and function of MK5/PRAK: the loner among the mitogen-activated protein kinase-activated protein kinases

Biological Chemistry ◽

10.1515/hsz-2013-0149 ◽

2013 ◽

Vol 394 (9) ◽

pp. 1115-1132 ◽

Cited By ~ 13

Author(s):

Ugo Moens ◽

Sergiy Kostenko

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Kinase Domain ◽

Mitogen Activated Protein Kinase ◽

Cellular Processes ◽

Mapk Pathways ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Apoptosis Gene ◽

And Function

Abstract Mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways that control pivotal cellular processes including proliferation, differentiation, survival, apoptosis, gene regulation, and motility. MAPK pathways consist of a relay of consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinases, and MAPKs. Conventional MAPKs are characterized by a conserved Thr-X-Tyr motif in the activation loop of the kinase domain, while atypical MAPKs lack this motif and do not seem to be organized into the classical three-tiered kinase cascade. One functional group of conventional and atypical MAPK substrates consists of protein kinases known as MAPK-activated protein kinases. Eleven mammalian MAPK-activated protein kinases have been identified, and they are divided into five subgroups: the ribosomal-S6-kinases RSK1-4, the MAPK-interacting kinases MNK1 and 2, the mitogen- and stress-activated kinases MSK1 and 2, the MAPK-activated protein kinases MK2 and 3, and the MAPK-activated protein kinase MK5 (also referred to as PRAK). MK5/PRAK is the only MAPK-activated protein kinase that is a substrate for both conventional and atypical MAPK, while all other MAPKAPKs are exclusively phosphorylated by conventional MAPKs. This review focuses on the structure, activation, substrates, functions, and possible implications of MK5/PRAK in malignant and nonmalignant diseases.

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