The significant others: Global search for direct kinase substrates using chemical approaches

Shah, Kavita; Kim, Hyunjin

doi:10.1002/iub.2023

Cited by 13 publications

(18 citation statements)

References 62 publications

(83 reference statements)

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“…The pocket in the kinase is created by mutating the conserved gatekeeper residue to a glycine residue ( a nalog- s ensitive1 aka-as1, example CDK5-as1). While most of the kinases only require a mandatory gatekeeper mutation, many other kinases require additional mutations [ 20 ]. We identified one such residue L194 in the subdomain IV of Aurora kinase A (AURKA) active site, which needed to be mutated to valine (L194V) in addition to the gatekeeper mutation.…”

Section: Resultsmentioning

confidence: 99%

LIMK2-NKX3.1 Engagement Promotes Castration-Resistant Prostate Cancer

Sooreshjani

Nikhil

Kamra

et al. 2021

Cancers

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NKX3.1’s downregulation is strongly associated with prostate cancer (PCa) initiation, progression, and CRPC development. Nevertheless, a clear disagreement exists between NKX3.1 protein and mRNA levels in PCa tissues, indicating that its regulation at a post-translational level plays a vital role. This study identified a strong negative relationship between NKX3.1 and LIMK2, which is critical in CRPC pathogenesis. We identified that NKX3.1 degradation by direct phosphorylation by LIMK2 is crucial for promoting oncogenicity in CRPC cells and in vivo. LIMK2 also downregulates NKX3.1 mRNA levels. In return, NKX3.1 promotes LIMK2’s ubiquitylation. Thus, the negative crosstalk between LIMK2-NKX3.1 regulates AR, ARv7, and AKT signaling, promoting aggressive phenotypes. We also provide a new link between NKX3.1 and PTEN, both of which are downregulated by LIMK2. PTEN loss is strongly linked with NKX3.1 downregulation. As NKX3.1 is a prostate-specific tumor suppressor, preserving its levels by LIMK2 inhibition provides a tremendous opportunity for developing targeted therapy in CRPC. Further, as NKX3.1 downregulates AR transcription and inhibits AKT signaling, restoring its levels by inhibiting LIMK2 is expected to be especially beneficial by co-targeting two driver pathways in tandem, a highly desirable requisite for developing effective PCa therapeutics.

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Section: Resultsmentioning

confidence: 99%

LIMK2-NKX3.1 Engagement Promotes Castration-Resistant Prostate Cancer

Sooreshjani

Nikhil

Kamra

et al. 2021

Cancers

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“…It is even more important when studying host cell signalling pathways exploited by pathogens during infection through the release of their kinases. Many high throughput methods exist and most of them have important drawbacks or are not adapted to host-pathogen interaction studies 56 . Here, we propose SILAkin, a novel, easy, and validated method applicable to most kinases from any organism that overcomes most of the limitations existing in other mass spectrometry-based methods: First, it allows direct detection of phosphorylation, contrary to phospho-proteomics comparing WT and mutant kinase compartments 39,57 , and recognises specific consensus sites, the limitations inherent to IVKA do not apply.…”

Section: Discussionmentioning

confidence: 99%

SILAKin: A novel high throughput SILAC and mass spectrometry-based assay to identify the substratome of kinases secreted by pathogens

Smirlis

Dingli

Sabatet

et al. 2021

Preprint

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Protein phosphorylation is one of the most important reversible post-translational modifications. It affects every cellular process including differentiation, metabolism and cell cycle. Eukaryotic protein kinases (ePK) catalyse the transfer of a phosphate from ATP onto proteins, which regulates fast changes in protein activity, structure or subcellular localisation. The systematic identification of substrates is thus crucial to characterise the functions of kinases and determine the pathways they regulate, and even more so when studying the impact of pathogens-excreted kinases on the host cell signal transduction. Several strategies and approaches have been used to identify substrates, but all show important limitations thus calling for the development of new efficient and more convenient approaches for kinase substrate identification.Herein, we present SILAkin, a novel and easy method to identify substrates that is applicable to most kinases. It combines phosphatase treatment, pulse heating, in vitro kinase assay (IVKA) and SILAC (Stable Isotope Labeling with Amino acids in Cell culture)-based quantitative mass spectrometry (MS). We developed SILAkin using the Leishmania casein kinase 1 (L-CK1.2) as experimental model. Leishmania, an intracellular parasite causing Leishmaniasis, releases L-CK1.2 in its host cell. Applying this novel assay allowed us to gain unprecedented insight into host-pathogen interactions through the identification of host substrates phosphorylated by pathogen-excreted kinases. We identified 225 substrates, including 85% previously unknown that represent novel mammalian CK1 targets, and defined a novel CK1 phosphorylation motif. The substratome was validated experimentally by L-CK1.2 and human CK1δ, demonstrating the efficiency of SILAkin to identify new substrates and revealing novel regulatory pathways. Finally, SILAkin was instrumental in highlighting host pathways potentially regulated by L-CK1.2 in Leishmania-infected host cells, described by the GO terms ‘viral & symbiotic interaction’, ‘apoptosis’, ‘actin cytoskeleton organisation’, and ‘RNA processing and splicing’. SILAkin thus can generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogen adapted for intracellular survival.

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“…The most critical limitation is the genetic manipulation of ATP binding pocket, which might lead to slightly attenuated catalytic activities of the mutant protein kinases and potentially lower the affinity for ATP of the created as protein kinase mutants. Additionally, some difficulties are also associated with the intracellular delivery of bulky ATP analogues and the higher catalytic efficiency of as protein kinase mutants when the bulky ATP analogue is used [ 101 ]. However, the possibility of the rapid and reversible inactivation of as protein kinase mutants in a dose-dependent manner clearly surpasses these limits.…”

Section: Analog-sensitive Kinase Technologymentioning

confidence: 99%

“…Recently, several advanced high-throughput strategies for the direct identification of protein kinase substrates have been developed. These approaches, represented by protein microarrays, kinase-interacting substrate screening (KISS), heavy ATP kinase assay combined with quantitative mass spectrometry (HAKA-MS), or the analogue-sensitive kinase approach (ASKA), allowed us, despite many false positive identifications, to screen and track down some novel biologically relevant substrates of protein kinases [ 8 , 65 , 101 , 125 , 126 , 127 , 128 ].…”

Section: Strategies For the Identification Of The Protein Kinase Tmentioning

confidence: 99%

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

Jurcik

Siváková

Cipakova

et al. 2020

IJMS

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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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The significant others: Global search for direct kinase substrates using chemical approaches

Cited by 13 publications

References 62 publications

LIMK2-NKX3.1 Engagement Promotes Castration-Resistant Prostate Cancer

LIMK2-NKX3.1 Engagement Promotes Castration-Resistant Prostate Cancer

SILAKin: A novel high throughput SILAC and mass spectrometry-based assay to identify the substratome of kinases secreted by pathogens

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

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