Pseudokinases

1st Edition - May 5, 2022

Editors: Natalia Jura, James Murphy

Hardback ISBN:

9 7 8 - 0 - 3 2 3 - 9 1 5 4 1 - 0

eBook ISBN:

9 7 8 - 0 - 3 2 3 - 9 1 5 4 2 - 7

Pseudokinases, Volume 667, the latest release in the Methods in Enzymology serial, highlights new advances in the field with this new volume presenting interesting chapters,… Read more

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Pseudokinases, Volume 667, the latest release in the Methods in Enzymology serial, highlights new advances in the field with this new volume presenting interesting chapters, including the Production and Purification of the PEAK pseudokinases for structural and functional studies, Structural biology and biophysical characterization of Tribbles pseudokinases, Detecting endogenous TRIB protein expression and its downstream signaling, Analysis of human Tribbles 2 pseudokinase, Expression, purification and examination of ligand-binding to IRAK pseudokinases, Characterization of pseudokinase ILK-mediated actin assembly, Biochemical examination of Titin pseudokinase, Approaches to study pseudokinase conformations, CRISPR editing cell lines for reconstitution studies of pseudokinase function, and much more.

Cover image
Title Page
Table of Contents
Copyright
Contributors
Preface
Chapter One: Production and purification of the PEAK pseudokinases for structural and functional studies
Abstract
1: Introduction
2: Recombinant protein expression
3: Recombinant protein purification
4: Overview and conclusion
Acknowledgments
References
Chapter Two: Structural and biophysical characterization of Tribbles homolog 1
Abstract
1: Introduction
2: Key resources table
3: Materials and equipment
4: Expression and purification of TRIB1
5: Crystallization of TRIB1 pseudokinase domain
6: Biophysical measurement of TRIB1-binding
7: Conclusion
Acknowledgments
References
Chapter Three: Detecting endogenous TRIB2 protein expression by flow cytometry and Western blotting
Abstract
1: Introduction
2: Flow cytometry-based method for staining of the intracellular TRIB2
3: TRIB2 identification by Western blotting method
4: Discussion
Acknowledgments
References
Chapter Four: Analysis of human Tribbles 2 (TRIB2) pseudokinase
Abstract
1: Introduction
2: Protocols
3: Summary
Acknowledgments
References
Chapter Five: Purification, crystallization and drug screening of the IRAK pseudokinases
Abstract
1: Introduction
2: Before you begin
3: Key resources table
4: Materials and equipment
5: Step-by-step method details
6: Expected outcomes
7: Discussion
Acknowledgments
References
Chapter Six: Characterization of pseudokinase ILK-mediated actin assembly
Abstract
1: Introduction
2: Preparation of ILK-PINCH1-α-parvin (IPP) complex
3: F-actin co-sedimentation assay to detect IPP binding to F-actin
4: Co-sedimentation assay to measure binding affinity of a protein to F-actin
5: Pyrene-actin polymerization: Direct observation of initial polymerization rates
6: Detection and visualization of IPP formed bundles
7: Summary
Acknowledgment
References
Chapter Seven: Production and analysis of titin kinase: Exploiting active/inactive kinase homologs in pseudokinase validation
Abstract
1: Introduction
2: Challenges in the mechanistic analysis of the titin pseudokinase
3: Impact of domain composition on sample production
4: Prediction of mutants for pseudokinase validation
5: Recombinant protein production
6: Experimental assessment of sample stability
7: Evaluating phosphotransfer activity
8: Summary
Acknowledgments
References
Chapter Eight: Co-expression of recombinant RIPK3:MLKL complexes using the baculovirus-insect cell system
Abstract
1: Introduction
2: Molecular cloning of baculovirus co-expression constructs
3: Baculovirus production, amplification, and recombinant protein expression
4: Conclusions
Acknowledgments
Competing interests
References
Chapter Nine: CRISPR deletions in cell lines for reconstitution studies of pseudokinase function
Abstract
1: Introduction
2: Selection and preparation of CRISPR and lentiviral delivery vectors
3: Preparation of virus and introduction into target cells
4: Conclusion
Acknowledgments
References
Chapter Ten: Protein engineering strategies to stimulate the functions of bacterial pseudokinases
Abstract
1: Introduction
2: Equipment and materials
3: Protocol
4: Data analysis
5: Summary
Acknowledgment
Reference
Chapter Eleven: Dynamics of protein kinases and pseudokinases by HDX-MS
Abstract
1: Introduction
2: Overview of HDX-MS
3: HDX-MS to probe dynamics of the kinase fold
4: HDX-MS as a tool to study pseudokinase dynamics
5: Protocol: Bottom-up HDX-MS to probe ligand-induced conformational dynamics of a pseudokinase domain
6: Data analysis and presentation
7: Summary
Acknowledgments
References
Chapter Twelve: Cellular thermal shift assay (CETSA) for determining the drug binding affinity using Ba/F3 clones stably expressing receptor pseudokinases
Abstract
1: Introduction
2: Generation of stable Ba/F3 clones expressing receptor pseudokinases using a nucleofection protocol
3: CETSA with stable Ba/F3 cell lines
4: Summary
5: Key resource table
References
Chapter Thirteen: Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions
Abstract
1: KSR identification
2: KSR domain structure
3: Higher-order KSR–RAF–MEK complexes: Heterodimers and heterotetramers
4: Is KSR an active kinase?
5: Purification of KSR1–MEK1 and KSR2–MEK1 for biochemical assays and cocrystallography
6: Assays to measure binding of ATP-competitive compounds within the orthosteric site on KSR1 and KSR2
7: Assays to measure binding of interfacial compounds of KSR-MEK complexes
8: Final conclusions
Acknowledgments
References
Chapter Fourteen: Computational tools and resources for pseudokinase research
Abstract
1: Introduction
2: Investigating pseudokinase functional specialization using comparative kinomics
3: Structural informatics and visualization tools for pseudokinase research
4: Protocols
5: Summary
Acknowledgments
References
Chapter Fifteen: Methods to assess small molecule allosteric modulators of the STRAD pseudokinase
Abstract
1: Introduction
2: Thermal stabilization assays
3: Nucleotide displacement from the STRAD/MO25 trimer
4: In vitro kinase assay
5: Summary and conclusions
References
Chapter Sixteen: Targeting the HER3 pseudokinase domain with small molecule inhibitors
Abstract
1: Introduction
2: Identifying small molecule binding to HER3 and HER2 by thermal shift assay
3: Validating the specificity and inhibitory capacity of HER3 binding compounds
4: Methods for assessing inhibitor mode of action
5: In silico modeling of inhibitor binding to HER3
6: Summary and perspectives
Acknowledgments
References
Chapter Seventeen: Considerations for studying phosphorylation of the mitotic checkpoint pseudokinase BUBR1
Abstract
1: Introduction
2: Experimental and theoretical considerations for studying BUBR1 during mitosis
3: Generation of stable BUBR1 cell lines using the Flp-In™ T-Rex™ system
4: Use of phosphospecific antibodies to infer function
5: Before you begin
6: Quantification and statistical analysis
7: Use of autophosphorylation and the characteristic mitotic upshift in SDS-PAGE as a readout
8: Before you begin
9: Summary
References
Chapter Eighteen: The pseudokinase domain in receptor guanylyl cyclases
Abstract
1: Introduction
2: Allosteric regulation of rGCs by the pseudokinase domain
3: Protocol
4: Summary
Acknowledgments
References
Chapter Nineteen: Methods for discovering catalytic activities for pseudokinases
Abstract
1: Introduction
2: Bioinformatics-based search for novel atypical kinases/pseudokinases. Sequence-based approach
3: Bioinformatics-based search for novel atypical kinases/pseudokinases. Approach using predicted three-dimensional protein structures
4: Cloning a candidate novel kinase for bacterial expression
5: Preparation of an active kinase complex from bacteria
6: Assigning novel catalytic activities
7: Conclusion
Acknowledgments
References
Chapter Twenty: Efficient expression, purification, and visualization by cryo-EM of unliganded near full-length HER3
Abstract
1: Introduction: The human epidermal growth factor receptor 3 (HER3)
2: Construct design and expression of near full-length HER3
3: Purification and initial visualization of homogeneous near full-length HER3 sample
4: The detergent exchange strategy
5: Reconstructions of the fully-glycosylated HER3 extracellular domain
6: Conclusion
References
Chapter Twenty-One: An effective strategy for ligand-mediated pulldown of the HER2/HER3/NRG1β heterocomplex and cryo-EM structure determination at low sample concentrations
Abstract
1: Introduction
2: Expression and purification of NRG1β
3: Purification, crosslinking and preparation of the HER2/HER3/NRG1β sample for single particle electron microscopy imaging
4: Sample preparation for freezing and imaging by cryo-EM
Acknowledgments
References
Chapter Twenty-Two: Enabling pseudokinases as potential drug targets
Abstract
1: Introduction
2: Construct design, cloning and test purification
3: Protein expression and purification
4: DSF fingerprint of pseudokinases
5: Crystallization and structure elucidation
6: Concluding remarks
Acknowledgments
References
Chapter Twenty-Three: Identification and characterization of TYK2 pseudokinase domain stabilizers that allosterically inhibit TYK2 signaling
Abstract
1: Introduction
2: Identification of TYK2-JH2 pseudokinase domain binders
3: Characterization of TYK2-JH2 pseudokinase domain binders
4: Probe displacement assays for binding energetics and lead identification
5: Summary
Acknowledgments
References
Chapter Twenty-Four: A suite of in vitro and in vivo assays for monitoring the activity of the pseudokinase Bud32
Abstract
1: Introduction
2: Purification of GST- and His-tagged KEOPS proteins and of in vitro transcribed tRNA
3: Monitoring the ATPase activity of Bud32 using the ADP Glo assay
4: Monitoring the tRNA binding activity of Bud32-containing complexes using a 647-CCA probe displacement fluorescence polarization assay
5: Monitoring the role of Bud32 in the t6A modification of tRNA using an HPLC analysis of nucleoside composition
6: Drop assay for monitoring the role of Bud32 in yeast cell growth in vivo
7: Summary and conclusions
Acknowledgments
References
Chapter Twenty-Five: Dissecting protein function in vivo: Engineering allelic series in mice using CRISPR-Cas9 technology
Abstract
1: Introduction
2: Before you begin
3: Allele design
4: Step-by-step method details
References

Natalia Jura

Dr. Natalia Jura is an Associate Professor and an Investigator in the Department of Cellular and Molecular Pharmacology at the Cardiovascular Research Institute within the School of Medicine at the University of California, San Francisco (UCSF). She is also an Associate Director of the Quantitative Biosciences Institute at UCSF. Dr. Jura's group at UCSF focuses on understanding how soluble protein kinases and membraneassociated receptor kinases assemble into functional complexes and regulate their signaling through molecular interactions with regulatory proteins. Her group also investigates alternative non-catalytic roles of protein kinases as scaffolds in cellular signaling pathways. They apply this knowledge for design of small molecule inhibitors that target these poorly understood kinase functions in human diseases. Dr. Jura received her M.S in biochemistry from Jagiellonian University in Krakow, Poland and her Ph.D. in molecular and cellular biology from Stony Brook University.

Affiliations and expertise

Assistant Professor, Cardiovascular Research Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, USA

James Murphy

James Murphy is Associate Professor and the head of the Inflammation Division at the WEHI (formerly known as The Walter and Eliza Hall Institute of Medical Research) in Melbourne and is closely associated with The University of Melbourne and The Royal Melbourne Hospital. James’ lab studies the protein-protein interactions that underpin signal transduction. Much of his work is focused on understanding the molecular mechanisms by which protein kinases and their relatives, pseudokinases, regulate cell signaling. Their work on the pseudokinase, MLKL, provided a template for developing a detailed understanding of how the remaining ~50 uncharacterized pseudokinases modulate cell signaling.

Affiliations and expertise

Associate Professor, Head, Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Victoria, Australia