Kinomics E-Book

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 716

Veröffentlichungsjahr: 2015

Beschreibung

Authored by the world's leading kinase experts, this is a comprehensive introduction to current knowledge and practice within this emerging field.
Following an overview of the major players and pathways that define the kinome, the major part of this work is devoted to current strategies of kinome investigation and manipulation. As such, kinase engineering, peptide substrate engineering, co-substrate design and kinase inhibitor design are discussed in detail, and their potential applications in kinome analysis and kinome-based pharmacotherapy are shown.
The result is a toolbox for every kinase researcher: By addressing and comparing current approaches to the study of kinase action, both novice and established researchers will benefit from the practical knowledge contained in this invaluable reference.

Leseprobe

Table of Contents

Cover

Related Titles

Title Page

Copyright

List of Contributors

Preface

Part I: Protein Kinases Cell Signaling

Chapter 1: Global Approaches to Understanding Protein Kinase Functions

1.1 A Brief History of the Structure of the Human Kinome

1.2 Why Study Protein Kinases – Their Roles in Disease

1.3 Methodology for Assessment of Protein Kinase Functions

1.4 Final Thoughts

Acknowledgments

References

Chapter 2: “Genuine” Casein Kinase (Fam20C): The Mother of the Phosphosecretome

2.1 Introduction

2.2 Early Detection of the pS-x-E Motif in Secreted Phosphoproteins

2.3 CK1 and CK2 are Not Genuine Casein Kinases

2.4 Polo-Like Kinases: Newcomers in the Club of False “Casein Kinases”

2.5 Characterization of an Orphan Enzyme: The Spectacular Performance of a Peptide Substrate

2.6 Catalytic Activity of Fam20C: Mechanistic Aspects

2.7 A Kinase in Need of Control

2.8 Outlook

Funding

References

Chapter 3: Chemical Biology of Protein Kinases

3.1 The Basis of Chemical Genetics

3.2 Protein Kinase Chemical Genetics

3.3 Applications for AS Kinases

3.4 Current Challenges

3.5 Conclusions

Acknowledgments

References

Chapter 4: Protein Kinases and Caspases: Bidirectional Interactions in Apoptosis

4.1 Introduction

4.2 Apoptosis: Caspase-Dependent Pathways

4.3 Functional Crosstalk between Protein Kinases and Caspases

4.4 Strategies to Investigate Global Crosstalk between Protein Kinases and Caspases

4.5 Implications and Future Prospects

References

Chapter 5: The Kinomics of Malaria

5.1 Introduction

5.2 The

Plasmodium

Kinome: Salient Features

5.3 Reverse Genetics of the

Plasmodium

Kinome

5.4 Lessons from Phosphoproteomics

5.5 Host Cell Kinomics in Malaria Infection

5.6 Targeting Protein Kinases in Antimalarial Drug Discovery

5.7 Concluding Remarks

References

Part II: ATP Co-substrate Design

Chapter 6: ATP Analogs in Protein Kinase Research

6.1 Base-Modified ATP Analogs

6.2 Sugar-Modified ATP Analogs

6.3 α- and β-Phosphate-Modified ATP Analogs

6.4 γ-Phosphate-Modified ATP Analogs

6.5 Conclusions

References

Chapter 7: Electrochemical Detection of Protein Kinase-Catalyzed Phosphorylations

7.1 Introduction

7.2 Conclusions

References

Part III: New Methodologies for Kinomics

Chapter 8: Phos-tag Technology for Kinomics

8.1 Introduction

8.2 Kinomics and Phosphoproteomics

8.3 Phos-tag Technology

8.4 Highly Sensitive Detection of Phosphopeptides and Phosphoproteins by the Phos-tag Biotin Method

8.5 Protein Kinase Assay with Phos-tag Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

8.6 Conclusion

References

Chapter 9: Development of Species- and Process-Specific Peptide Kinome Arrays with Priority Application to Investigations of Infectious Disease

9.1 Phosphorylation-Mediated Signal Transduction

9.2 Peptide Arrays for Kinome Analysis

9.3 Infectious Disease

9.4 Conclusions

References

Chapter 10: New Approaches to Understanding Bacterial Histidine Kinase Activity and Inhibition

10.1 Introduction to Two-Component System Signaling

10.2 Focus on Bacterial HKs

10.3 Bacterial HK Activity

10.4 Bacterial HK Inhibition

10.5 Outlook on Tools for the Study and Inhibition of Bacterial HKs

References

Chapter 11: Methods for Large-Scale Identification of Protein Kinase Substrate Networks

11.1 Introduction

11.2 Computational Prediction of Phosphorylation Sites and Protein Kinase–Substrate Relationships

11.3 The Role of Mass Spectrometry in Identifying Posttranslational Modifications

11.4 Analog-Sensitive Kinases and Other Specific Inhibitors

11.5 Array-Based Methods

11.6 Solution-Based Methods

11.7 Future Perspectives

References

Part IV: Kinase Inhibition

Chapter 12: Developing Inhibitors of STAT3: Targeting Downstream of the Kinases for Treating Disease

12.1 Introduction

12.2 STAT3 Structure and Signaling

12.3 Methods for Directly Inhibiting STAT3

12.4 Conclusion

References

Chapter 13: Metal Compounds as Kinase and Phosphatase Inhibitors in Drug Development: The Role of the Metal and Ligands

13.1 Introduction

13.2 Kinase Inhibitors: From Ideal 3D Shapes to Kinase Inhibitor-Derived Ligands in Metal Complexes

13.3 Phosphatases and Metal Compounds

13.4 Conclusions

Acknowledgments

References

Index

End User License Agreement

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Guide

Cover

Table of Contents

Preface

Part I: Protein Kinases Cell Signaling

Begin Reading

List of Illustrations

Chapter 2: “Genuine” Casein Kinase (Fam20C): The Mother of the Phosphosecretome

Figure 2.1 Remarkable contribution of “casein kinases” to the generation of the human phosphoproteome. The kinome and the FJ family of atypical kinases are shown, with highlighted protein kinases endowed with remarkable casein kinase activity

in vivo

and/or

in vitro

. The overall contribution of “casein kinases” to the generation of human phosphoproteome (drawn from Ref. [8]) is shown in the bottom right corner.

Figure 2.2 Two-sample logos of PLK2 (a), CK2 (b) , and G-CK/Fam20C (c), highlighting their common acidophilic nature.

Figure 2.3 Fam20C is almost inactive in the presence of physiological concentrations of Mg

2+

and Mn

2+

(a) and can be activated by sphingosine and sphingosine-1-phosphate, but not by ceramide (b), whose structures are shown in (c).

Chapter 3: Chemical Biology of Protein Kinases

Figure 3.1 Schematic illustrating the principle of chemical genetics for an enzyme.

Figure 3.2 Structures of ATP analogs. In native ATP, R1 is −NH

2

and R

2

is O.

Figure 3.3 View of the ATP binding region of wild-type (a) and Ile338Gly AS mutant v-src (b) with residue 338 in magenta illustrating the increase in space (the hole) generated by the mutation. Groups appended to the

N

6

position of ATP can exploit this additional space. Image generated from Protein Data Bank (PDB) entry 4LGG using PyMol.

Figure 3.4 Isolation of thiophosphoprotein substrates of an AS kinase using

p

-nitrobenzyl mesylate modification of thiols with subsequent specific immunoprecipitation.

Figure 3.5 Isolation of thiophosphoprotein substrates of an AS kinase using alkylation with immobilized iodoacetamide and subsequent oxidation.

Figure 3.6 Inhibitors of AS kinases. PP1 and staurosporine provided the scaffolds for the AS kinase inhibitors.

Chapter 4: Protein Kinases and Caspases: Bidirectional Interactions in Apoptosis

Figure 4.1 Functional consequences of interactions between protein kinases and caspases. Bidirectional crosstalk between protein kinases and caspases has the capacity to transmit both proapoptotic and antiapoptotic signals. This Figure highlights outcomes that can occur as a consequence of interactions between protein kinases and caspases including protein kinase-catalyzed phosphorylation of caspases and caspase cleavage of protein kinases.

Figure 4.2 Bidirectional crosstalk between PKCδ and caspase-3 during apoptosis. During apoptosis, PKCδ phosphorylation of caspase-3 enhances caspase-3 activity, and in turn, activated caspase-3 cleaves Src-phosphorylated (tyrosine 332) PKCδ at DMQD

329

[65, 100–103]. Caspase-3 cleavage of PKCδ occurs in the hinge region that separates the regulatory domain from the catalytic domain; consequently, caspase-3 cleavage of PKCδ increases the activity of the kinase. Increased PKCδ activity may increase phosphorylation of caspase-3 suggesting that caspase-3 cleavage of PKCδ initiates a positive feedback loop for enhanced caspase-3 activity via PKCδ phosphorylation during apoptosis. Furthermore, the catalytic fragment of PKCδ translocates to the nucleus where it targets a variety of substrates to propagate proapoptotic signaling [122, 161–166, 169].

Figure 4.3 Strategies to systematically investigate bidirectional crosstalk between protein kinases and caspases. A variety of systematic strategies have been developed to elucidate the convergence of protein kinase and caspase signaling during apoptosis. These strategies include (A) the development of biosensors for live-cell imaging to investigate the spatiotemporal dynamics of apoptotic signaling [170], the development of proteomic strategies to simultaneously evaluate the phosphoproteome and the caspase degradome such as (B) PROTOMAP [8, 82] and (C) N-terminomics [10], as well as (D) the use of computational strategies and bioinformatics to analyze the expanding databases representing the phosphoproteome and the caspase degradome.

Chapter 5: The Kinomics of Malaria

Figure 5.1 The life cycle of malaria parasites.

Plasmodium

sporozoites injected into the human host through the bite of an infected

Anopheles

mosquito travel via the bloodstream to the liver, where they invade a hepatocyte. Intense schizogony (repeated nuclear division in the absence of cytokinesis, with eventual ontogeny of individual progeny cells) ensues, producing several thousand merozoites that are released to the bloodstream and invade erythrocytes. Malaria pathogenesis is caused by the asexual multiplication of parasites in erythrocytes through schizogony. Some merozoites, after invasion of the host red blood cell, arrest their cell cycle and differentiate into male or female gametocytes. These sexual cells mediate transmission to the mosquito vector. Once ingested by the insect, they develop into gametes (for the male gametocyte, this involves a process called

exflagellation

, whereby eight flagellated gametes are formed from each male gametocyte). Fertilization occurs in the mosquito's midgut; the zygote develops into a motile ookinete, where meiosis occurs. The ookinete crosses the midgut epithelium and establishes an oocyst, which is the site of a new round of asexual proliferation resulting in the generation of sporozoites. These accumulate in the insect's salivary glands, where they are primed to infect a new human host during a subsequent blood meal. Protein kinases that have been demonstrated to control specific life cycle transitions, or to be involved in processes at various stages of the cycle, are indicated at the sides.

Figure 5.2 The kinome of

Plasmodium falciparum

. Phylogenetic tree of the

P. falciparum

kinome. Circular tree of all 91 eukaryotic protein kinases (ePK) in

P. falciparum

as defined by Talevich

et al.

[14]. Representative genes from human (Hs),

Arabidopsis thaliana

(At), and

Plasmodium berghei

(Pb) are indicated with labels colored gold, green, and purple, respectively. Branch and arc colors indicate kinase classification by ePK major group, with minor modifications in group assignment according to the gene tree. A gray circle on a branch indicates bootstrap support greater than 50; larger circles indicate greater bootstrap values.

Figure 5.3 Phosphosites detected by mass spectrometry on

Plasmodium

kinases. Data compiled from the published global phosphoproteomic studies indicate that many of the malaria protein kinases are themselves phosphoproteins. This suggests that parasite protein kinases (like the mammalian counterparts) are under phosphorylation-dependent regulation. These phosphorylation events might be the result of autophosphorylation or of the action of upstream protein kinases in a phosphorylation cascade. Indicated are the modes of phosphorylation identified together with specific examples. Also shown is an illustration of the kinase domain, a classic activation loop starting with a DFG and ending in APE (please note that this is illustrative and that for some of the

Plasmodium

kinases listed, the DFG/APE motifs are atypical).

Chapter 6: ATP Analogs in Protein Kinase Research

Figure 6.1 (a) General kinase reaction mechanism with the protein or peptide substrate represented as an oval. (b) The crystal structure of cAMP-dependent protein kinase (PKA) bound to Mn

2+

-ATP and a peptide inhibitor (PDB: 1ATP) is shown with kinase in violet, ATP in red, peptide inhibitor in yellow, residue to be phosphorylated on the peptide in cyan, and metal ions in green. (c) The ATP modifications discussed in this chapter are indicated in boxes with positions modified in red.

Figure 6.2 The base-modified ATP analogs discussed in this chapter. The purine base was modified by attachment of a group to the C2, C6, N6, or C8 positions and/or replacement by pyrazolopyrimidine, triazole, or imidazole rings. Sections 6.1.1, 6.1.2, 6.1.3, 6.1.4 will discuss each of these analogs, with Section 6.1.5 highlighting use of the N6-modified analogs for kinase–substrate identification.

Figure 6.3 ATP analogs substituted at the C2, C6, and C8 positions were tested as ATP-competitive inhibitors against CaMKII. The

K

i

values obtained are provided [11].

Figure 6.4 Crystal structure image of c-Src kinase (PDB: 2SRC, violet mesh) bound to the ATP analog, AMP-PNP (Section 6.3.2, compound

32

), focusing on the ATP-binding pocket. The amine on C6 (the N6 position) is boxed tightly within the pocket to prevent binding of N6-modified ATP analogs. In contrast, the base-binding pocket near C2, N7, and C8 appears more open to accommodate small modifications (arrow). The “gatekeeper” amino acids mutated to accommodate N6-modified analogs are shown in yellow (T338). Each atom of AMP-PNP is color coded (C = green, N = blue, O = red, and p = orange) with hydrogens omitted for clarity. The image was created using Pymol 1.5.0.5 (Schrodinger, LLC).

Figure 6.5 N6-modified ATP analogs discussed in this chapter. The N6 position of the purine of ATP was substituted with groups varying in size.

Figure 6.6 The structures of various C3-substituted pyrazolopyrimidine triphosphate (PPTP) analogs.

Figure 6.7 The structures of N4-(benzyl) ribavirin triphosphate (

19

, N4-benzyl-RTP) and N4-(benzyl)(5-aminoimidazole-4-carboxamide ribotide) (

20

, N4-benzyle-AICAR). Hydrogen bonding in

27

(dashed line) was speculated to constrict conformation flexibility, unlike

26

.

Figure 6.8 N6-(benzyl)-ADP (compound

28

) was used as an ATP mimic in the crystal structure analysis of as-v-Src(I338A).

Figure 6.9 The structures of MANT-ATP (compound

29

) and TNP-ATP (compound

30

). The fluorescent modification can be on either the 2′ or 3′ position of MANT-ATP.

Figure 6.10 The general structure of α- and β-phosphate-modified ATP analogs (top left), along with the structures of the three analogs most commonly studied with protein kinases.

Figure 6.11 Kinase-catalyzed labeling using γ-phosphate-modified ATP analogs where R is a thiol or a variety of other functional groups discussed in this section. Kinase-catalyzed labeling results in the transfer of the γ-phosphoryl modification to the protein substrate, which will allow monitoring of the kinase reaction or the phosphorylated product.

Figure 6.12 (a) Kinase-catalyzed thiophosphorylation with ATPγS, with the protein or peptide substrate represented as an oval. (b) Methods for selective purification of thiophosphorylated proteins and peptides, which take advantage of the intrinsic nucleophilic reactivity of the thiophosphoryl group. Reaction A shows reaction of thiophosphate groups with iodoacetamide-biotin selectively at low pH in the presence of cysteines, with subsequent purification using streptavidin affinity resin. Reaction B also employs iodoacetamide, but in this case with a solid-phase resin attached to facilitate purification, with selective elution in the presence of cysteine using peroxide. Reaction C involves a two-step strategy where initial “capping” of cysteine-containing peptides followed by iodoacetamide labeling of thiophosphate groups resulted in selective fluorophore tagging with BODIPY. Reaction D utilizes disulfide exchange to promote solid-phase resin capture, with elution selectively in the presence of cysteine using strong base. Reaction E involves substitution of a para-nitrobenzylmesylate (PNBM), with subsequent affinity purification using antibodies recognizing the para-nitrobenzylthiophosphoryl group.

Figure 6.13 The structures of ATP-biotin (compound

35

), ATP-dansyl (compound

36

), ATP-Atto-590 (compound

37

), and ATP-ferrocene (compound

38

).

Figure 6.14 FRET-based activity assays using γ-phosphate fluorophore-modified ATP analogs. (a) ATP-dansyl (Figure 6.13, compound

36

) was used in a kinase-catalyzed labeling reaction to dansylate a rhodamine-labeled kinases substrate and produce a FRET signal at 595 nm. (b) Kinase-catalyzed labeling of a peptide conjugated to a luminescent quantum dot (CdSe/ZnS) with ATP-Atto-590 (Figure 6.13, compound

37

) resulted in a FRET signal at 654 nm and a corresponding loss in luminescence intensity of the quantum dot.

Figure 6.15 (a) The structures of ATP-arylazide (compound

39

) and ATP-benzophenone (compound

40

). (b) The mechanism of phosphorylation-dependent kinase–substrate crosslinking, where kinase-catalyzed labeling in combination with UV-mediated crosslinking results in covalent conjugation of kinase and substrate.

Figure 6.16 The structures of various γ-phosphate ATP analogs containing alkyne (compounds

41–44

), alkene (compound

45

), and azide (compounds

46–49

) groups.

Figure 6.17 (a) The structure of bifunctional C8-(azido)γ-(arylazido)-ATP analog

50

. (b) The mechanism of UV-mediated kinase–substrate crosslinking with compound

50

.

Figure 6.18 Covalent conjugation of biotin to an active site lysine of protein kinases by ATP-acyl-biotin (compound

51

). These ATP analogs require active site binding, making them probes of active kinases.

Chapter 7: Electrochemical Detection of Protein Kinase-Catalyzed Phosphorylations

Figure 7.1 (a) Schematic illustration of the peptide-based biosensor for study of PKA activity by chronocoulometry. Immobilized peptide on gold electrode phosphorylated in presence of PKA and ATP. Zr

4+

ions were attached to the phosphate sites (Step I) and then bonded to DNA-AuNPs (Step II). (b) Zr

4+

ions bind phosphopeptide as well as the phosphate backbone of DNA-AuNPs. Electrochemical detection of [Ru(NH

3

)

6

]

3+

absorbed on DNA-AuNPs was carried out by chronocoulometry (Reproduced from Ref. [1] with permission from the Royal Society of Chemistry).

Figure 7.2 Differential pulse voltammograms of AuNPs reduction peak on biotinylated peptide-based SPCE as a function of p60

c-Src

concentration: (a) 0.2 U l

−1

, (b) 0.15 U l

−1

, (c) 0.1 U l

−1

, (d) 0.05 U l

−1

, and (e) 0.2 U U l

−1

at 250 μM peptide immobilized SPCE [3].

Figure 7.3 The plot of current of quinonediimine versus PKA concentration [5].

Figure 7.4 Schematic illustration of AuNPs/MWNTs biosensor. Peptide substrate was attached to the Au surface, followed by phosphorylation in the presence of ATP-S. Subsequent exposure to AuNPs/single-wall carbon nanotube (SWCNTs) and TMB produced the electrochemical signal [6].

Figure 7.5 Schematic of the Ru-based detection of PKA-catalyzed phosphorylation and alkaline phosphatase (ALP) dephosphorylation on gold surface [9].

Figure 7.6 (A) Schematic illustration of the tau phosphorylation and Pin1 binding detection. (B) Plot of normalized charge transfer resistance changes as a function of concentration of Pin1 [11].

Figure 7.7 (a) Illustration of Fc–ATP electrochemical assay for detection of phosphorylation. The electrochemical signal increases upon increasing reaction variables: (b) reaction time; (c) PKC concentration; and (d) Fc–ATP concentration [12].

Figure 7.8 Structure of Fc–ATP cosubstrates with various alkyl spacer lengths. (a) Square-wave voltammograms of peptide–Au in the presence of Src protein kinase and Fc–ATP. (b) Plot of current density of Fc–phosphate–peptide–Au, associated with Fc group, as a function of Fc–ATP cosubstrate used during phosphorylation (

n

= 2 (a), 6 (b). 8 (c), and 10 (d)) [15].

Figure 7.9 Schematic diagram of Src protein kinase active site bound to longer Fc–ATP (a) and shorter Fc–ATP (b) analog [15].

Figure 7.10 (a) Illustration of hydrophilic (

3

and

4

) and hydrophobic (

5

) Fc–ATP cosubstrates. (b) SWV of Fc-phosphorylated peptide–Au by various Fc–ATP conjugates and Src protein kinase. (c) Plot of the current density as a function of protein kinase using different Fc–ATP cosubstrates (Reprinted with permission from Martic, S., Rains, M.K., Freeman, D., Kraatz, H.-B., (2011) Use of 5′-gamma-reffocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethyleneglycol) spacer in kinase-catalyzed phosphorylations,

Bioconjugate Chemistry

,

22

, 1663–1672). Copyrights (2011) American Chemical Society [16].

Figure 7.11 Schematic illustration of Fc assay for detection of Fc–STAT3 phosphorylation and monitoring STAT3–Fc–STAT3 binding as well as its inhibition. Inset: electrochemical responses for each stage by SWV [Martic, S., Rains, M.K., Haftchenary, S., Shahani, V.M., Kraskouskaya, D., Ball, D.P., Gunning, P.T., Kraatz, H.-B. (2014) Electrochemical detection of the Fc-STAT3 phosphorylation and STAT3/Fc-STAT3 dimerization and inhibition.

Mol. Biosys.

,

10

, 576–580] Reproduced by permission of the Royal Society of Chemistry.

Figure 7.12 Chemical structures of inhibitors evaluated for Src activity 14b.

Figure 7.13 (a) SWV of peptide–Au Fc-phosphorylated in the presence of Src with increasing amounts of PP2 inhibitor. (b) Plot of current densities extrapolated from SWV as a function of PP2 inhibitor concentration 14b.

Figure 7.14 Chemical structures of Ru- and Os-based inhibitors of CDK2 kinase (M = Ru, Os; R1 = Et, Me; R2 = H, Me; X = Cl, Br, I) (Reprinted with permission from (Hanif, M., Henke, H., Meier, S.M., Martic, S., Labib, M., Kandioller, W., Jakupez, M.A., Arion, V.B., Kraatz, H.-B., Keppler, B.K., Hartinger, C.G. (2010) The reactivity to biomolecules as anticancer activity determining parameters of M(II)-arene complexes of 3-hydroxy-2(1H)-pyridone,

Inorg. Chem

.

49

, 7953–7963). Copyright (2010) American chemical Society).

Figure 7.15 Electrochemical signals of Fc–ATP assay of different Ru-based CDK2 inhibitors [19].

Figure 7.16 Chemical structures of indole/quinolone-based inhibitors (

1–5

) for CK2-catalyzed phosphorylation. The electrochemical signals as a function of the inhibitor concentration 14c.

Figure 7.17 (A) Chemical structures of various STAT3 dimerization inhibitors. (B) Plot of the current density as a function of different STAT3 inhibitors (Martic, S., Rains, M.K., Haftchenary, S., Shahani, V.M., Kraskouskaya, D., Ball, D.P., Gunning, P.T., Kraatz, H.-B. (2014) Electrochemical detection of the Fc-STAT3 phosphorylation and STAT3/Fc-STAT3 dimerization and inhibition.

Mol. Biosys.

,

10

, 576–580). Reproduced by permission of the Royal Society of Chemistry).

Figure 7.18 Fc–Ab1 characterization with standard biochemical assay. (a) Detection scheme. (b) Fc-phosphorylation of caspase-3. (c) Selectivity of Fc–Ab1 for Fc-phosphorylated caspase-3 over phosphorylated caspase-3. (d) Determination of phosphorylation kinetics of Fc–ATP. (e) Determination of phosphorylation kinetics of ATP (Reprinted with permission from Martic, S., Gabriel, M., Turowec, J.P., Litchfield, D.W., and Kraatz, H.B. (2012) Versatile strategy for biochemical, electrochemical, and immunoarray detection of protein phosphorylations,

J. Am. Chem. Soc.

134

, 17036–17045. Copyright (2012) American Chemical Society.).

Figure 7.19 (a) Western blot of cell lysate phosphorylation using ATP, (b) Western blot of cell lysate Fc-phosphorylation using Fc–ATP, and (c) relative signal intensity for cell lysate phosphorylation using ATP or Fc–ATP (Reprinted with permission from Martic, S., Gabriel, M., Turowec, J.P., Litchfield, D.W., and Kraatz, H.B. (2012) Versatile strategy for biochemical, electrochemical, and immunoarray detection of protein phosphorylations,

J. Am. Chem. Soc.

134

, 17036–17045. Copyright (2012) American Chemical Society.).

Chapter 8: Phos-tag Technology for Kinomics

Figure 8.1 Illustration of enhanced chemiluminescence (ECL) detection of phosphopeptides on a peptide microarray or of phosphoproteins on a protein-blotting membrane by using Phos-tag biotin. The phosphorylated targets are probed by using the complex of Phos-tag biotin with HRP-conjugated streptavidin, and then the Phos-tag-bound targets are detected by an ECL system.

Figure 8.2 High-throughput profiling of kinome activities by using a peptide microarray system. (a) Profiling of tyrosine kinase activities involved in the EGF signaling pathway of A431 cells. The images of detections using the lysates before (−) and after (+) EGF stimulation are shown in the left-hand and center panels, respectively. These two images are superimposed in the right-hand panel. (b) Profiling of tyrosine kinase activities with Src kinase inhibitor I involved in the treatment of EGF-stimulated A431 cells. The images from lysate samples before (−) and after (+) treatment with the inhibitor are shown in the left-hand and center panels, respectively. These two images are superimposed in the right-hand panel.

Figure 8.3 Detection of phosphoproteins on a protein-blotting membrane by using the complex of Phos-tag biotin with HRP-conjugated streptavidin. Results are shown for 10 samples of various lysates (lanes 1–10, 5 µg proteins/lane) and 2 molecular weight markers (lanes M1 and M2). Lanes 1–10 correspond to (1) lysate of human epithelial carcinoma A431 cells, (2) AP-treated lysate of A431 cells, (3) lysate of EGF-stimulated A431 cells, (4) AP-treated lysate of EGF-stimulated A431 cells, (5) lysate of pervanadate-stimulated A431 cells, (6) AP-treated lysate of pervanadate-stimulated A431 cells, (7) lysate of human cervical cancer HeLa cells, (8) AP-treated lysate of HeLa cells, (9) lysate of PMA-stimulated HeLa cells, and (10) AP-treated lysate of PMA-stimulated HeLa cells.

Figure 8.4 The principle of Phos-tag SDS-PAGE.

Figure 8.5

In vitro

Abl kinase assay by Mn

2+

–Phos-tag SDS-PAGE. (a) The phosphorylation of Abltide-GST (0.10 µg protein/lane) by Abl was monitored for 0–60 min by means of normal SDS-PAGE (12.5% (w/v) polyacrylamide) and by Mn

2+

–Phos-tag SDS-PAGE (12.5% (w/v) polyacrylamide containing 100 μM Mn

2+

–Phos-tag). The Mn

2+

–Phos-tag SDS-PAGE gel was analyzed by subsequent immunoblotting with an anti-pTyr antibody. (b) Quantitative analyses of phosphorylated and nonphosphorylated Abltide-GST observed in the CBB-stained Mn

2+

–Phos-tag SDS-PAGE gel (center panel of a).

Figure 8.6 Simultaneous detection of the activation/inactivation of ERKs by using Zn

2+

–Phos-tag SDS-PAGE. (a) A431 whole lysate (−, 10 µg proteins) and lysate from EGF-stimulated cells (+, 10 µg proteins) were subjected to Zn

2+

–Phos-tag SDS-PAGE (8.0% (w/v) polyacrylamide and 25 μM Zn

2+

–Phos-tag) followed by Western blotting with the anti-ERK antibody. The lysate from the EGF-stimulated cells (10 µg proteins) was analyzed by 2-DE consisting of normal SDS-PAGE (8.0% (w/v) polyacrylamide) as the first dimension and Zn

2+

–Phos-tag SDS-PAGE (8.0% (w/v) polyacrylamide, 25 μM Zn

2+

–Phos-tag) as the second dimension. (b) The time course of phosphorylation of ERK after stimulation with EGF was analyzed by Zn

2+

–Phos-tag SDS-PAGE (8.0% (w/v) polyacrylamide and 25 μM Zn

2+

–Phos-tag). Each lane contained 10 µg of proteins. The gels were analyzed by Western blotting with the anti-pT

202

/Y

204

antibody, and then the same blot was reprobed with the anti-ERK antibody. (c) A431 whole lysate (control, 10 µg proteins) and lysate from EGF-stimulated cells (10 µg proteins, 250 ng ml

−1

of EGF for 5 min) were subjected to Zn

2+

–Phos-tag SDS-PAGE (8.0% (w/v) polyacrylamide and 25 μM Zn

2+

–Phos-tag) followed by Western blotting with the anti-pT

202

antibody or anti-pY

204

antibody (left-hand and center panels, respectively). Lysates from EGF-stimulated cells (10 µg proteins, 250 ng ml

−1

of EGF for 5 min) pretreated with A (100 nM, 30 min), pervanadate (1.0 mM, 30 min), or PD98059 (100 μM, 60 min) were similarly analyzed with the anti-ERK antibody (right-hand panel).

Figure 8.7 Phosphate-affinity 2D DIGE analysis of cellular proteins. (a) A mixture of HeLa lysate (50 µg protein labeled with Cy3) and lysate from calyculin A-treated HeLa cells (50 µg protein labeled with Cy5) was subjected to normal SDS-PAGE (8–12% (w/v) gradient polyacrylamide gel) as the first dimension. The separated sample lane was cut into three parts corresponding to 10–30, 30–50, and 50–200 kDa, respectively, and these were subjected to Zn

2+

–Phos-tag SDS-PAGE (50 μM Zn

2+

–Phos-tag) as the second dimension. (b) Phosphorylation profiling of histone H3 using Zn

2+

–Phos-tag SDS-PAGE followed by immunoblotting analysis. HeLa whole lysate (−, 10 µg protein) and calyculin A-treated cell lysate (+, 10 µg protein) were subjected to Zn

2+

–Phos-tag SDS-PAGE (12% (w/v) polyacrylamide and 25 μM Zn

2+

–Phos-tag).

Chapter 9: Development of Species- and Process-Specific Peptide Kinome Arrays with Priority Application to Investigations of Infectious Disease

Figure 9.1 Representation of the array design and data image. (a) Array design. A representative grid of spots is presented including information of three representative spots to illustrate that each spot represents a different peptide sequence and that each spot is printed as technical replicate at multiple locations through the grid. On the final array, the grid is then repeated three times to give a total of nine technical replicates per unique peptide sequence. Dark gray spots on the edge of the grid represent negative control peptides. (b) Data image. A representative image scan of a kinome peptide array. A single grid of the array is shown.

Figure 9.2 Example of a heatmap produced by PIIKA. Rows represent peptides, while columns represent samples. Each cell represents the degree of phosphorylation of a given peptide from a given sample. The dendrogram on the left represents a clustering of the peptides based on the similarity of their phosphorylation patterns in the different samples; analogously, the upper dendrogram represents a clustering of the samples based on the similarity of their phosphorylation patterns among all of the peptides. Red cells indicate greater levels of phosphorylation, while green cells indicate lower levels.

Figure 9.3 Example of a volcano plot produced by PIIKA 2. The plot shows the fold-change value and the P value for each peptide when comparing phosphorylation levels in two arrays or treatments. Peptides with high fold-change values and statistically significant P values are colored in red (for up-phosphorylated peptides) or green (for down-phosphorylated peptides) and labeled with the identity of that peptide.

Chapter 10: New Approaches to Understanding Bacterial Histidine Kinase Activity and Inhibition

Figure 10.1 Two-component signal transduction. (a) Activation of HK results in autophosphorylation of the conserved His (P∼His). Subsequently, the phosphoryl group is transferred to the conserved Asp of the cognate RR (P∼Asp). The RR typically binds DNA to regulate gene expression.

Figure 10.2 Stability of the phosphohydroxyamino acids versus the phosphoramidate residue. The greater bond stability of P∼Ser, P∼Thr, and P∼Tyr is exhibited in the free energy of hydrolysis. P∼His is also labile under acidic conditions.

Figure 10.3 Thiosphosphorylation and B-ATPγS as an activity-based probe. (a) Thiosphosphorylation of His (tP∼His). (b) Structure of B-ATPγS. (c) Proposed modification of HK His (BtP∼His) and RR Asp (BtP∼Asp) by B-ATPγS. (d) SDS-PAGE and in-gel fluorescence analysis of HK853 labeling by B-ATPγS only when probe is added (lane 2). (e) B-ATPγS competes with ATP, AMP-PNP, and ATPγS in lanes 2, 3, and 4, respectively. (f) Transfer of BODIPY-thiophosphate from HK853 (32 kDa) to cognate RR468 (17 kDa). RR labeling is only observed when HK is present (lane 1). For each image, Coomassie gels illustrate consistent protein loading. Adapted with permission from Ref. [1]. Copyright 2012 American Chemical Society.

Figure 10.4 Future objectives for HK profiling. Ideally, a probe will be added to varying sample types. Differential HK regulation would be proportional to probe turnover and thus fluorescence in gel-based analyses. Activity-based probes could also enrich HKs from the sample for protein identification by MS.

Figure 10.5 Recent compounds identified for the inhibition of individual HKs that translated to antimicrobial activity. Signermycin B,

1

(WalK, Gram-positive bacteria); vz0825,

2

(KdpD,

V. cholerae

); waldiomycin,

3

(WalK, Gram-positive bacteria); and LED209,

4

(QseC, EHEC

E. coli

).

Figure 10.6 Compounds demonstrated to target the ATP-binding domain of individual HKs. Inhibitors exhibited efficacy in infection models.

5–8

, PhoQ (

S. flexneri

);

9–14

, VicK (

S. pneumoniae

); and

15–16

, YycG (

S. epidermidis

).

Figure 10.7 Compounds that inhibit multiple HKs through the ATP-binding domain: walkmycin C,

17

, and TEP,

18

.

Figure 10.8 Previously reported inhibitors are postulated to bind through a common scaffold. (a) Conservation shown by shading in the HK ATP-binding, rendered on HK853 in complex with ADP. The highly conserved D411 is shown binding to the N6 amine of ADP. (b) Inhibitors from the literature predicted to bind HK through the active site D411. (c) Pose of

21

in the HK853 model, forming a salt bridge with the invariant D411. Docking also suggests a stacking interaction with Y384. Adapted from Ref. [84]. Reproduced with permission from the Royal Society of Chemistry (http://pubs.rsc.org/en/content/articlelanding/2013/md/c2md20308a).

Figure 10.9 Guanidine fragment binding to HK. (a) Deconstruction of inhibitors into a guanidine-based fragment

24

for analysis of binding to HK853. Optimization with sulfonyl group,

25

, hypothesized to mimic a phosphate group. (b) Binding curves of

24

and

25

generated from STM-NMR data. Adapted from Ref. [84]. Reproduced with permission from the Royal Society of Chemistry (http://pubs.rsc.org/en/content/articlelanding/2013/md/c2md20308a).

Chapter 11: Methods for Large-Scale Identification of Protein Kinase Substrate Networks

Figure 11.1 Reagents for quantitative mass spectrometry. Stable isotopic labeling by amino acids in cell culture (SILAC) relies on isotopically labeled amino acids that are metabolically incorporated to distinguish control and treated samples. The structures of commonly utilized isotopically labeled (a) L-lysine and (b) L-arginine variants are shown compared to their naturally occurring counterparts.

13

C and

15

N isotopically labeled atoms are indicated with red and blue asterisks, respectively. (c) The conserved structure of the tandem mass tag (TMT) reagent is shown with the amine reactive, reporter, and balancing group functions indicated. The fragmentation patterns of the reporter exposed to high-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) are indicated with dashed red lines. (d) Example structures of TMT 127 and TMT 130 reagents are shown with

13

C- and

15

N-labeled atoms indicated with red and blue asterisks, respectively. Notice that TMT 127 will release a lower molecular weight fragment upon dissociation than TMT 130.

Figure 11.2 Identification of protein kinase substrates via analog-sensitive protein kinases. (a) The chemical structure of the selective Src protein kinase inhibitor PP1 as well as the PP1 C3-modified bulky derivatives NA-PP1 and NM-PP1 is shown. NA-PP1 and NM-PP1 are selective inhibitors of some analog-sensitized protein kinase isoforms. (b) Wild-type protein kinases are not sensitive to bulky protein kinase inhibitor analogs because these small molecules will not fit into the protein kinase active site. As a result, wild-type kinases can phosphorylate their substrates (blue line) in the presence of ATP and the bulky protein kinase inhibitor (red). In contrast, analog-sensitized protein kinases (as-1) can bind bulky protein kinase inhibitors due to an enlarged ATP-binding pocket. This binding event inhibits protein kinase activity and protein kinase substrate phosphorylation. As a result, selective protein kinase substrates of a protein kinase will appear less abundant in a phosphoproteomic experiment. (c) The structure of N6-benzyl-ATP-sulfhydryl-phosphate is shown. This compound can be utilized to selectively transfer sulfhydryl-phosphate to substrates (blue line) of analog-sensitized protein kinases (d), but cannot be utilized by wild-type protein kinases. As a result, specific substrates of analog-sensitized protein kinases can be selectively labeled and purified by affinity chromatography [96] or derivatization and immunoprecipitation [97].

Figure 11.3 Peptide and protein array assays of protein kinases and their substrates. Peptide arrays are generated by spotting synthesized peptides in a rational fashion on a glass slide. Protein arrays can be generated in a similar manner by spotting

in vitro

synthesized proteins on a glass slide containing immobilized Ni-NTA or glutathione. A recombinant protein kinase (red) is then incubated with the slide in the presence of radiolabeled γ-[

32

P]-ATP. The recombinant protein kinase transfers radiolabeled phosphate to cognate substrates on the slide, which is then imaged via autoradiography. The intensity of peptide or protein substrate spots is indicative of protein kinase activity against each substrate.

Figure 11.4 The Kinase Client (KiC) assay. Pools of synthetic peptides (black lines) are incubated with a recombinant protein kinase (blue) in the presence of ATP. These peptides are subsequently separated on a liquid chromatography column and subjected to mass spectrometry. The mass spectrometer identifies peptides that are phosphorylated (red line) and subjects these peptides to electron transfer dissociation to map the associated phosphorylation site in the MS

2

spectrum.

Chapter 12: Developing Inhibitors of STAT3: Targeting Downstream of the Kinases for Treating Disease

Figure 12.1 Molecular surface model of the STAT3 homodimer. Domains are represented by the color-coded schematic below (Protein Data Bank (PDB): 1BG1).

Figure 12.2 Schematic representation of the canonical JAK/STAT3 signaling pathway showing IL-6-mediated activation of STAT3-mediated transcription.

Figure 12.3 Peptidomimetic

PM-66

synthesized by McMurray

et al.

to bind the STAT3 SH2 domain.

Figure 12.4 STAT3 SH2 domain inhibitors discovered by high-throughput screening.

Figure 12.5 Small-molecule salicylic acid-based inhibitors designed to bind the STAT3 SH2 domain.

Figure 12.6 Small-molecule JAK2/STAT3 pathway inhibitors.

Figure 12.7 Natural product JAK2/STAT3 pathway inhibitors.

Figure 12.8 Timeline for identification of notable STAT3 inhibitors.

Chapter 13: Metal Compounds as Kinase and Phosphatase Inhibitors in Drug Development: The Role of the Metal and Ligands

Figure 13.1 Schematic drawing of ATP (a), staurosporine (b), and a model ruthenium complex (c) bound to the ATP binding site of a cyclin-dependent kinase. All compounds are located in the hydrophobic pocket and form hydrogen bonds with glutamate-81 and leucine-83 of the hinge region.

Figure 13.2 Human kinase dendrogram (a) showing the results of an active site-directed affinity screening (KINOMEscan, DiscoveRx) for protein kinase selectivity against 442 human protein kinases of a Ru(1,4,7-trithiacyclononane) complex featuring a 3-(2-pyridyl)-1,8-naphthalimide chelate ligand (b) [35].

Figure 13.3 Ruthenium-based protein kinase inhibitors and their binding to the ATP-binding pocket of PAK1. Surface view illustrating the shape complementarity between (a)

Λ-FL172

and (b)

(

R

)-DW12

[42] as well as (c)

(

R

)-1

(PDB ID: 4DAW) within the ATP binding site of PAK1.

Figure 13.4 Structures of the protein kinase inhibitor

SU5416

, ferrocene-based oxindoles

S1

and

S2

, and

S2

docked into kinase DYRK2 [48].

Figure 13.5 Structures of the clinically approved protein kinase inhibitors erlotinib and gefitinib and their ferrocene-based analogs.

Figure 13.6 General structure of cyclometallated rhodium JAK2 inhibitors.

Figure 13.7 Structure of the non-ATP-mimetic inhibitor

M1

(a) of Pim1 which forms hydrogen bonds with lysine-67 and aspartic acid-186 at the opposite site of the hinge region (b) [57].

Figure 13.8 Structures of different POMs and their binding to protein kinase CK2 through interactions not involving the ATP-binding pocket (here occupied by an organic kinase inhibitor).

Figure 13.9 Paullone backbone (left) and Ga

III

, Cu

II

, Ru

II

, and Os

II

complexes with paullone-derived ligands.

Figure 13.10 Complexes of indoloquinoline-based ligands.

Figure 13.11 Concentration-dependent inhibition of CDK2/cyclin E (a) and CDK1/cyclin B (b) activity by indoloquinoline

A10

L

and indolobenzazepine

A11

L

ligands and the corresponding Os complexes

A10

Os

and

A11

Os

. Flavopiridol (FP) was used as a positive control [69].

Figure 13.12 Indirubin and Ru

II

– and Os

II

–arene complexes of quinoxalinone derivatives.

Figure 13.13 Flavopiridol and the general structure of flavonol organoruthenium compounds as well as a hydroxypyridone-based ruthenium(II) complex.

Figure 13.14 Schematic overview of the hypoxia-mediated activation concept and chemical structure of a Co

III

prodrug linked to an EGFR inhibitor [112].

Figure 13.15 From the left: dihydrogenvanadate(V) H

2

VO

4

−

(mainly at pH 7) and dihydrogenphosphate , monoperoxo and diperoxo forms of pervanadate.

Figure 13.16 Crystal structure of

Yersinia

protein tyrosine phosphatase complexed with vanadate as a transition state analog of PTPs (PDB code 2I42) (J. Vijayalakshmi and M.A. Saper, unpublished.)

Figure 13.17 From left: oxodiperoxo(1,10-phenanthroline)vanadate(V), oxodiperoxo(bipyridine)vanadate(V), and bis(maltolato)oxovanadium(IV).

List of Tables

Chapter 2: “Genuine” Casein Kinase (Fam20C): The Mother of the Phosphosecretome

Table 2.1 Early elucidation of phosphorylated sequences in phosphoproteins

Chapter 4: Protein Kinases and Caspases: Bidirectional Interactions in Apoptosis

Table 4.1 Selected examples of caspases positively or negatively regulated by protein kinase-mediated phosphorylation

Table 4.2 Selected examples of protein kinases activated or inactivated by caspase-mediated proteolysis

Chapter 6: ATP Analogs in Protein Kinase Research

Table 6.1 Summary of ATP analogs and their applications

Chapter 7: Electrochemical Detection of Protein Kinase-Catalyzed Phosphorylations

Table 7.1 Electrochemical parameters of peptide-based gold electrode followed by Fc-phosphorylation by a given kinase

Table 7.2 List of substrates and protein kinases that were detected by Fc–ATP electrochemical assay

Chapter 9: Development of Species- and Process-Specific Peptide Kinome Arrays with Priority Application to Investigations of Infectious Disease

Table 9.1 Example of a DAPPLE output table

Table 9.2 Example of raw data derived from a single kinome microarray

Chapter 11: Methods for Large-Scale Identification of Protein Kinase Substrate Networks

Table 11.1 Summary of available web-based resources for the prediction of protein kinase–substrate relationships

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Edited by Heinz–Bernhard Kraatz and Sanela Martic

Kinomics

Approaches and Applications

The Editors

Heinz-Bernhard Kraatz

University of Toronto

Phys. & Environmental Sciences

1265 Military Trail

Toronto, ON, M1C 1A4

Canada

Sanela Martic

Oakland University

Dept. of Chemistry

2200 North Squirrel Road

Rochester

MI 48309

United States

Cover Illustration

Structure of the human c-Src protein kinase (PDB 2SRC) based on data by W. Xu, A. Doshi, M. Lei, M. J. Eck, and S. C. Harrison. 258081820 / Science Photo Library RF / Media Manager Getty Images

178899864 / Thadthum / Getty Images

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List of Contributors

Soha Ahmadi

University of Toronto

Department of Chemistry

St. George Street

M5S 3H6 Toronto

Canada

and

University of Toronto Scarborough