Nuclear Protein Kinases
Lubomir S. Hnilica in Chromosomal Nonhistone Proteins, 2018
Attempts to define the physiological significance of a given protein phosphorylation reaction are complicated by the fact that proteins which do not appear to be phosphorylated in vivo can in some cases be made to serve as substrates for protein kinase in vitro.10–13 Such phosphorylation induced under artificial in vitro conditions may cause changes in the conformation and functional activity of the protein in question, but the issue arises as to whether these changes are of any physiological relevance. In order to ward against the possibility of such erroneous conclusions concerning the regulatory role of protein phosphorylation, Krebs and Beavo8 have established a set of criteria which must be met before one ascribes physiological significance to any particular protein phosphorylation reaction. Briefly, one must show that: (1) phosphorylation of the protein in question will occur in vitro using the appropriate protein kinase, (2) this phosphorylation causes changes in the function of the protein consistent with the role of the protein in vivo, (3) the levels of protein kinase in vivo are sufficient to induce the required level of phosphorylation, and (4) phosphorylation and dephosphorylation of the protein in vivo produce the same changes as in vitro. The last requirement is, of course, the most difficult one to fulfill, and yet is clearly essential because of the above-mentioned tendency to produce artifactual protein phosphorylations in vitro.
Phosphorylation of Phosphofructokinase — The Possible Role of Covalent Modification in the Regulation of Glycolysis
Rivka Beitner in Regulation of Carbohydrate Metabolism, 1985
Insulin was reported to decrease the activity of cAMP-dependent protein kinase.130 This observation raised the question whether or not insulin deficiency, under diabetic conditions, would stimulate protein phosphorylation. Bazaes et al.129 studied the phosphate content of phosphofructokinase from genetically diabetic (db/db) mice in relation to normal (db/m) controls. Based on the incorporation of radioactive phosphate, the phosphate content of phosphofructokinase promoters in the normal mice muscle was determined as 0.14 ± 0.03 mol/mol. The phosphofructokinase promoters from the diabetic mice muscle contained 0.27 ± 0.11 mol phosphate per mole, indicating enhanced phosphorylation of the enzyme. The specific activity of phosphofructokinase in the diabetic muscle was about 30% lower than in normal controls. A partial separation of phospho- and dephospho- forms of phosphofructokinase was obtained by anion-exchange chromatography. However, the two forms exhibited only minor differences in their allosteric kinetics. Therefore, the enhanced phosphorylation of phosphofructokinase in the muscle of diabetic mice did not explain the reduced glycolytic rate in the organ.
Cyclic Nucleotide Metabolism and Action During Senescence
Richard C. Adelman, George S. Roth in Endocrine and Neuroendocrine Mechanisms of Aging, 2017
The most commonly used method of detecting specific protein phosphorylation involves separation of 32P-labeled proteins by Polyacrylamide gel electrophoresis and quantitation by slicing and counting or autoradiography and densitometry. Labeling may be done by incubation of tissue with phosphate-free media containing carrier-free 32Pi, followed by treatment with the appropriate stimulus, or by incubation of homogenates or subcellular fractions with [33Ρ]-γ-ΑΤΡ of high specific activity in the presence and absence of a cyclic nucleotide or Ca2+. In either case, incubations are terminated as quickly as possible, usually by heating with added sodium dodecyl sulfate (SDS).108 Some distinction between particulate and cytosolic proteins can be made by homogenizing labeled tissue with a buffered solution containing sucrose, NaF (50 mM), NaHP04 (10 mM), EDTA (10 mM), and protease inhibitors to preserve states of phosphorylation. A major problem incurred with intact cells exists in the 32P-RNA that is generated and appears as a high background in the gels, thus interfering with measurement of low level phosphorylation of minor proteins. Extraction with the above solution of inhibitors plus NP-40, a detergent which does not readily solubilize nuclear but does solubilize plasma membranes, and removal of nuclei by centrifugation may partially alleviate this problem. Alternatively, treatment of samples with an RNAse that remains active in 0.3% SDS and mercaptoethanol may also help to reduce this back-ground.109
Molecular insights into cancer drug resistance from a proteomics perspective
Published in Expert Review of Proteomics, 2019
Yao An, Li Zhou, Zhao Huang, Edouard C. Nice, Haiyuan Zhang, Canhua Huang
Site-specific protein phosphorylation is a post-translational modification that orchestrates a diverse array of cellular processes. Recent findings have demonstrated that the regulation of phosphorylation is a key mechanism in cancer drug resistance, as this modification serves as a rapid and reversible means to modulate protein activity and signal transduction. Large-scale studies of phosphoproteins, termed phosphoproteomics, may provide new insights for discovery and understanding of drug targets, mechanisms of carcinogenesis and cancer drug resistance, as well as biomarker discovery. Due to the high complexity of protein samples and the low abundance of phosphopeptides in whole cell lysates, effective enrichment methods are needed to capture phosphopeptides from extremely complex biological samples. Antibody-based affinity enrichment is widely used for the study of phosphorylation. Additionally, ionic interaction-based enrichment is another possible strategy for enriching phosphopeptides, by taking advantage of the phosphate group using approaches such as immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2) enrichment [125]. It should be noted that changes in total protein abundance itself can affect the abundance of phosphopeptides. Therefore, parallel quantification of proteins is essential [126].
Synthesis, biological evaluation, and in silico studies of new CDK2 inhibitors based on pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold with apoptotic activity
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Asmaa A. Mandour, Ibrahim F. Nassar, Mohammed T. Abdel Aal, Mahmoud A. E. Shahin, Wael A. El-Sayed, Maghawry Hegazy, Amr Mohamed Yehia, Ahmed Ismail, Mohamed Hagras, Eslam B. Elkaeed, Hanan M. Refaat, Nasser S. M. Ismail
Protein kinases represent a large group of structurally related enzymes that are essential and regulate cell cycle progression involved in cell division1–3. Cyclin-dependent kinases (CDKs) are serine-threonine kinases responsible for cell cycle regulation and cell differentiation2. Cyclin-dependent kinases (CDK) are mainly responsible for the phosphorylation process of proteins4–6. Cyclin is the regulatory protein bound by CDK leading to ATP binding region modification2. CDKs in absence of cyclin have less activity where the activation loop (known as T-loop) blocks the cleft, and the key amino acid residues are not optimally positioned for ATP binding2. CDK2 has a catalytic effect in cyclin-dependent protein kinase complex7,8. Protein phosphorylation has a critical role in cellular function regulation. This essential role during the cell cycle could be altered in tumour cells7–11. Alteration in kinases may lead to the development of many diseases, including cancer. Hence, the control of CDK dependent cell cycle is essential for tumour progression management. Where overexpression of CDK enzymes occurs in cancer2. As uncontrolled CDK2 activation in human cancer is associated with overexpression of cyclins A and E in many human cancers12. Thus, CDKs are considered critical targets for the development of novel anticancer drugs9,10.
Strategies for mass spectrometry-based phosphoproteomics using isobaric tagging
Published in Expert Review of Proteomics, 2021
Xinyue Liu, Rose Fields, Devin K. Schweppe, Joao A. Paulo
Protein phosphorylation of serine, threonine, and tyrosine amino acids is a highly studied post-translational modification (PTM) that coordinates a wide and diverse array of cellular and biochemical processes. Phosphorylation events in human cells are dynamically regulated by more than 500 kinases and approximately 200 phosphatases [1]. As phosphorylation is a readily and rapidly reversible process, its importance in signal transduction and protein activity modulation cannot be understated. For example, protein phosphorylation and associated cellular machinery (e.g. kinase, phosphatases, and phosphoprotein-binding proteins) are intricately involved in processes, such as apoptosis, cell division, response to extracellular signals and growth factor stimulation, among many other pathways [2]. Recent studies have mapped over 50,000 phosphorylation sites in a human cell line [3]. However, estimates have been made that approximately 1.85 million phosphorylatable residues are available across the entire human proteome [4]. Assuming ~11,000 proteins are expressed in a given human cell roughly suggests ~700,000 potential phosphorylation sites, each contributing to the millions of potential proteoforms that exist at any one time in human cells [5,6]. Resources, such as PhosphoSitePlus [7] and eukaryotic phosphorylation site database (EPSD) [8] are available that extensively collect, curate, and annotate phosphorylation sites in eukaryotic proteins.
Related Knowledge Centers
- Dephosphorylation
- Phosphatase
- Protein
- Threonine
- Amino Acid
- Phosphorylation
- Serine
- Post-Translational Modification
- Kinase
- Protein Structure