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Fetal Growth Factors*
Published in Emilio Herrera, Robert H. Knopp, Perinatal Biochemistry, 2020
Philip A. Gruppuso, Thomas R. Curran, Roderick I. Bahner
A discussion of regulation by protein phosphorylation should include a discussion of protein phosphatases. Study of protein phosphatases has lagged behind that of kinases, but the last several years have seen rapid advances. Protein phosphatases can be divided into two broad categories: serine/threonine vs. tyrosine specific.64,65 Within each category there is broad diversity which is comparable to that of the protein kinases. Protein phosphatase regulation has long been known to be centrally involved in metabolic regulation; the first demonstration of the interconversion of an enzyme between active and inactive forms was the inactivation (through dephosphorylation) of glycogen phosphorylase by phosphorylase phosphatase. The importance of the phosphatases in regulation of cellular processes is certainly comparable to that of kinases. Insulin effects are mediated in large part by the dephosphorylation of target enzymes.64 Recent work has demonstrated the involvement of protein phosphatases in cell cycle progression.63
Regulation of Human CYP2D6
Published in Shufeng Zhou, Cytochrome P450 2D6, 2018
Reversible protein phosphorylation, principally on serine, threonine, or tyrosine residues, is one of the most important and well-studied posttranslational modifications (Beltrao et al. 2012). Phosphorylation plays critical roles in the regulation of many cellular processes including cell cycle, growth, apoptosis, and differentiation. There are limited data on the regulation of CYP2D6 by posttranslational modifications. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, eight human CYP phosphorylation sites are identified. The data from surgical human liver samples establish that CYP1A2, 2A6, 2B6, 2E1, 2C8, 2D6, 3A4, 3A7, and 8B1 are phosphorylated in vivo (Redlich et al. 2008).
Nuclear Protein Kinases
Published in Lubomir S. Hnilica, Chromosomal Nonhistone Proteins, 2018
Samuel J. Mitchell, Lewis J. Kleinsmith
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.
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.
Phosphoproteomics: a valuable tool for uncovering molecular signaling in cancer cells
Published in Expert Review of Proteomics, 2021
Jacqueline S. Gerritsen, Forest M. White
The transformative potential of multiple constitutively activated kinases, as well as the role of protein phosphorylation in regulating other aspects of biology, has fueled a deep interest in protein phosphorylation, including studies at the single protein level, protein complexes, enzyme-substrate relationships, or at the level of the phosphoproteome, the compendium of protein phosphorylation sites in a given biological sample. Phosphoproteomics, the large-scale analysis of protein phosphorylation sites, was pioneered in 2002 by Ficarro et al., and has developed rapidly over the past few decades [10]. Although phosphoproteomics may be performed using a variety of instruments and can encompass both targeted and discovery analyses, phosphoproteomics-based mapping of phosphorylation events in a large-scale, relatively unbiased manner mainly relies on mass spectrometry (MS)-based approaches [11,12]. Alternative techniques to measure protein phosphorylation include immunofluorescence/immunohistochemistry, phospho-flow, reverse-phase protein microarrays, and multiple different forms of western blotting. Although these techniques are widely used, they are dependent on antibody availability and specificity, and can be limited in the number of phosphorylation sites monitored per analysis [13,14]. By comparison, MS-based methods require minimal a priori knowledge, can identify and quantify >10,000 phosphorylation sites in a given sample, and provide high specificity by directly sequencing the site of protein phosphorylation.