Substrate specificities of tyrosine-specific protein kinases toward cytoskeletal proteins in vitro.

Sepsis is a major cause of mortality in critically ill patients and associated with cardiac dysfunction, a complication linked to immunological and metabolic aberrations. Cardiac neutrophil infiltration and subsequent release of myeloperoxidase (MPO) leads to the formation of the oxidant hypochlorous acid (HOCl) that is able to chemically modify plasmalogens (ether-phospholipids) abundantly present in the heart. This reaction gives rise to the formation of reactive lipid species including aldehydes and chlorinated fatty acids. During the present study, we tested whether endotoxemia increases MPO-dependent lipid oxidation/modification in the mouse heart. In hearts of lipopolysaccharide-injected mice, we observed significantly higher infiltration of MPO-positive cells, increased fatty acid content, and formation of 2-chlorohexadecanal (2-ClHDA), an MPO-derived plasmalogen modification product. Using murine HL-1 cardiomyocytes as in vitro model, we show that exogenously added HOCl attacks the cellular plasmalogen pool and gives rise to the formation of 2-ClHDA. Addition of 2-ClHDA to HL-1 cardiomyocytes resulted in conversion to 2-chlorohexadecanoic acid and 2-chlorohexadecanol, indicating fatty aldehyde dehydrogenase-mediated redox metabolism. However, a recovery of only 40% indicated the formation of non-extractable (protein) adducts. To identify protein targets, we used a clickable alkynyl analog, 2-chlorohexadec-15-yn-1-al (2-ClHDyA). After Huisgen 1,3-dipolar cycloaddition of 5-tetramethylrhodamine azide (N3-TAMRA) and two dimensional-gel electrophoresis (2D-GE), we were able to identify 51 proteins that form adducts with 2-ClHDyA. Gene ontology enrichment analyses revealed an overrepresentation of heat shock and chaperone, energy metabolism, and cytoskeletal proteins as major targets. Our observations in a murine endotoxemia model demonstrate formation of HOCl-modified lipids in the heart, while pathway analysis in vitro revealed that the chlorinated aldehyde targets specific protein subsets, which are central to cardiac function.

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A new tool to dissect the function of p70 S6 kinase

Biochemical Journal ◽

10.1042/bj20101445 ◽

2010 ◽

Vol 431 (2) ◽

pp. e1-e3 ◽

Cited By ~ 13

Author(s):

Benoit Bilanges ◽

Bart Vanhaesebroeck

Keyword(s):

Metabolic Disease ◽

Insulin Resistance ◽

Mammalian Target Of Rapamycin ◽

Specific Protein ◽

Selective Inhibitor ◽

S6 Kinase ◽

Substrate Specificities ◽

P70 S6 Kinase ◽

Biochemical Journal

Developing small-molecule inhibitors that are highly selective for specific protein kinases has been and remains a serious challenge. This especially applies to members of families of related kinases with overlapping substrate specificities, such as the serine/threonine kinases of the AGC family. In this issue of the Biochemical Journal, Dario Alessi's group, in a collaboration with Pfizer, report on PF-4708671, a potent and highly selective inhibitor of S6K1 (p70 S6 kinase 1) in vitro and in cells. S6K1 is an AGC family member and a crucial effector of the mTORC1 (mammalian target of rapamycin complex 1) kinase. This is the first reported inhibitor that is highly selective for S6K1. This compound will help to understand the signalling and physiological roles of S6K1, and to dissect signalling downstream of mTORC1. S6K1 inhibitors may ultimately be useful in the treatment of diseases such as cancer where S6K1 is overexpressed, but most importantly in metabolic disease such as insulin resistance and obesity.

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Regulation and properties of extracellular signal-regulated protein kinases 1, 2, and 3.

Journal of the American Society of Nephrology ◽

10.1681/asn.v451104 ◽

1993 ◽

Vol 4 (5) ◽

pp. 1104-1110

Author(s):

D J Robbins ◽

E Zhen ◽

M Cheng ◽

S Xu ◽

C A Vanderbilt ◽

...

Keyword(s):

Protein Kinases ◽

Cytoskeletal Proteins ◽

Regulatory Proteins ◽

Wild Type ◽

Catalytic Core ◽

Extracellular Signal ◽

Pleiotropic Action ◽

Kinase Cascade ◽

Protein Kinase Cascade

The extracellular signal-regulated kinases ERK1 and ERK2 are 43- and 41-kd enzymes activated by many extracellular cues. They lie within a protein kinase cascade that is used to achieve many cellular responses. In addition to the wide variety of regulatory contexts in which they are activated, they phosphorylate important regulatory proteins, including receptors, transcription factors, cytoskeletal proteins, and other protein kinases. Thus, the stimulation of this kinase cascade is thought to have a pleiotropic action. ERK1 and ERK2 are controlled by phosphorylation on threonine and tyrosine. To understand the regulatory mechanisms, wild-type and mutant ERKs were expressed in bacteria and phosphorylated with MEK, the enzyme that is upstream of ERKs. Wild-type proteins could be activated 500- to 1,000-fold in vitro by MEK. ERK3, an enzyme of 62 kd and only 50% identical to ERK1 and ERK2 in the catalytic core, was also phosphorylated by MEK in vitro. This suggests that all three of these enzymes are targets of common signaling pathways.

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Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example

Molecules ◽

10.3390/molecules26164898 ◽

2021 ◽

Vol 26 (16) ◽

pp. 4898

Author(s):

Aileen Roth ◽

Adrian Gihring ◽

Florian Göser ◽

Christian Peifer ◽

Uwe Knippschild ◽

...

Keyword(s):

Protein Kinases ◽

Posttranslational Modifications ◽

High Throughput Screening ◽

Kinase Inhibitors ◽

Specific Inhibitor ◽

Specific Protein ◽

Highly Sensitive ◽

Experimental Approaches ◽

Inhibitory Potential

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.

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Specific Labeling of Protein Domains with Antibody Fragments

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098587 ◽

1981 ◽

Vol 39 ◽

pp. 416-417

Author(s):

U. Aebi ◽

L.E. Buhle ◽

W.E. Fowler

Keyword(s):

Image Processing ◽

Specific Protein ◽

Antibody Fragments ◽

Supramolecular Organization ◽

Two Dimensional ◽

Ordered Structures ◽

Virus Capsids ◽

Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

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EVALUATION OF TISSUE STEROID BINDING IN VITRO

Acta Endocrinologica ◽

10.1530/acta.0.068s223 ◽

1971 ◽

Vol 68 (1_Suppl) ◽

pp. S223-S246 ◽

Cited By ~ 2

Author(s):

C. R. Wira ◽

H. Rochefort ◽

E. E. Baulieu

Keyword(s):

Protein Binding ◽

Binding Sites ◽

Experimental System ◽

Specific Protein ◽

Coupling Mechanism ◽

Target Tissue ◽

Protein Binding Sites ◽

Definition Of ◽

Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

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In vitro characterization of Streptomyces avermitilis SAV1748 and SAV3637 phosphopantetheinyl transferase substrate specificities

10.36934/t2020-037 ◽

2020 ◽

Author(s):

Jeromy DiGiacomo

Keyword(s):

Streptomyces Avermitilis ◽

Phosphopantetheinyl Transferase ◽

Substrate Specificities ◽

In Vitro Characterization

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Study of Inhibitors of Neu and Related Tyrosine-Specific Protein Kinases: Implications for the Treatment of Breast Cancer.

10.21236/ada360940 ◽

1998 ◽

Author(s):

Arthur Edelman

Keyword(s):

Breast Cancer ◽

Protein Kinases ◽

Specific Protein

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Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

Biochemical Journal ◽

10.1042/bj20080234 ◽

2008 ◽

Vol 415 (1) ◽

pp. 27-33 ◽

Cited By ~ 45

Author(s):

Meghna Thakur ◽

Pradip K. Chakraborti

Keyword(s):

Protein Kinases ◽

Solid Phase ◽

Morphological Changes ◽

Binding Assays ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Peptidoglycan Biosynthesis ◽

Vitro Binding ◽

Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

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DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.254 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii63-ii63

Author(s):

Lakshmi Bollu ◽

Derek Wainwright ◽

Lijie Zhai ◽

Erik Ladomersky ◽

Kristen Lauing ◽

...

Keyword(s):

Protein Degradation ◽

Time Course ◽

Immune Cell ◽

Enzyme Inhibitors ◽

Tumor Development ◽

Ubiquitin Proteasome System ◽

Degradation Pathway ◽

Specific Protein ◽

Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

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