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Biomolecules
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Recent Advances in Protein Phosphorylation
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Recent Advances in Protein Phosphorylation (Closed)

A topical collection in Biomolecules (ISSN 2218-273X). This collection belongs to the section "Molecular Biology".

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Editors

Dr. Eiji Kinoshita

E-Mail Website
Guest Editor
Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan
Interests: phosphoproteomics; posttranslational modification; protein kinase; Phos-tag
Dr. Emiko Kinoshita-Kikuta

E-Mail Website
Guest Editor
Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan
Interests: protein phosphorylation; two-component signal transduction system; histidine kinase; Phos-tag

Topical Collection Information

Dear Colleagues,

Protein phosphorylation is a common type of posttranslational modification (PTM) that is present in all biological species, and it affects key properties of proteins involved in numerous cellular events. In living cells, the continuous and dynamic phosphorylation and dephosphorylation of proteins at specific amino acid residues are controlled by complex signaling networks, resulting in the production of a variety of phosphoproteins with various states of phosphorylation. Protein phosphorylation occurs on several amino acid residues, including His, Asp, Glu, Lys, Arg, and Cys, on which it is very labile and difficult to detect, while more stable phosphorylation takes place on three specific residues, Ser, Thr, and Tyr. In bacterial cells, His- and Asp-phosphorylated proteins are well known for their leading roles in the two-component signal transduction system. In higher eukaryotic cells, on the other hand, Ser-, Thr-, and Tyr-phosphorylated proteins are predominant. Since the standard free energies for bonds of imidazole-phosphate on the His residue and carboxyl-phosphate on the Asp residue are large, the phosphorylated His and Asp have the potential to serve as intermediates in phosphotransfer reactions to other amino acids. Therefore, in addition to Ser-, Thr-, and Tyr-phosphorylated proteins, His- and Asp-phosphorylated proteins play crucial roles as a sensor apparatus and a response regulator, respectively, of the two-component system in quick response to intra- and extracellular signals in prokaryotes as well as in fungi and plants.

This reversible PTM is generally catalyzed by the opposing activities of large families of protein kinase and phosphatase enzymes. For example, the human genome encodes more than 500 protein kinases and about 300 protein phosphatases. Approximately 13000 human proteins have sites that are phosphorylated and dephosphorylated. These numbers reflect the importance and the complexity of protein phosphorylation. Abnormal phosphorylation resulting from an imbalance in the enzymatic reactions of kinases and phosphatases has been implicated in a wide range of human diseases, including cancer, diabetes mellitus, neurodegeneration, and immune/inflammatory and vascular disorders. Therefore, methods for quantitative and qualitative monitoring of alterations in the phosphorylation states of certain proteins are also very important for studies on the proteome, particularly in relation to the elucidation of the molecular origins of diseases and the rational molecular design of drugs.

This Special Issue will focus on the role of protein phosphorylation in all living cells. Original manuscripts and reviews dealing with any aspect of protein phosphorylation and related pathophysiology and methodology are very welcome.

Dr. Eiji Kinoshita
Dr. Emiko Kinoshita-Kikuta
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the collection website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • protein phosphorylation
  • protein kinase
  • protein phosphatase
  • phosphoproteomics

Published Papers (5 papers)

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2022

Jump to: 2021

16 pages, 4097 KiB  
Article
Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase
by Mutsuki Amano, Yoko Kanazawa, Kei Kozawa and Kozo Kaibuchi
Biomolecules 2022, 12(2), 159; https://doi.org/10.3390/biom12020159 - 18 Jan 2022
Cited by 4 | Viewed by 2080
Abstract
Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase–substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 [...] Read more.
Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase–substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase–MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors. Full article
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2021

Jump to: 2022

27 pages, 3534 KiB  
Review
Diversity in Sensing and Signaling of Bacterial Sensor Histidine Kinases
by Eiji Ishii and Yoko Eguchi
Biomolecules 2021, 11(10), 1524; https://doi.org/10.3390/biom11101524 - 15 Oct 2021
Cited by 21 | Viewed by 5587
Abstract
Two-component signal transduction systems (TCSs) are widely conserved in bacteria to respond to and adapt to the changing environment. Since TCSs are also involved in controlling the expression of virulence, biofilm formation, quorum sensing, and antimicrobial resistance in pathogens, they serve as candidates [...] Read more.
Two-component signal transduction systems (TCSs) are widely conserved in bacteria to respond to and adapt to the changing environment. Since TCSs are also involved in controlling the expression of virulence, biofilm formation, quorum sensing, and antimicrobial resistance in pathogens, they serve as candidates for novel drug targets. TCSs consist of a sensor histidine kinase (HK) and its cognate response regulator (RR). Upon perception of a signal, HKs autophosphorylate their conserved histidine residues, followed by phosphotransfer to their partner RRs. The phosphorylated RRs mostly function as transcriptional regulators and control the expression of genes necessary for stress response. HKs sense their specific signals not only in their extracytoplasmic sensor domain but also in their cytoplasmic and transmembrane domains. The signals are sensed either directly or indirectly via cofactors and accessory proteins. Accumulating evidence shows that a single HK can sense and respond to multiple signals in different domains. The underlying molecular mechanisms of how HK activity is controlled by these signals have been extensively studied both biochemically and structurally. In this article, we introduce the wide diversity of signal perception in different domains of HKs, together with their recently clarified structures and molecular mechanisms. Full article
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14 pages, 2322 KiB  
Article
Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems
by Emiko Kinoshita-Kikuta, Eiji Kinoshita, Misaki Suga, Mana Higashida, Yuka Yamane, Tomoka Nakamura and Tohru Koike
Biomolecules 2021, 11(10), 1448; https://doi.org/10.3390/biom11101448 - 02 Oct 2021
Cited by 2 | Viewed by 2222
Abstract
The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require [...] Read more.
The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation. Full article
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18 pages, 2554 KiB  
Review
The Regulation of Rab GTPases by Phosphorylation
by Lejia Xu, Yuki Nagai, Yotaro Kajihara, Genta Ito and Taisuke Tomita
Biomolecules 2021, 11(9), 1340; https://doi.org/10.3390/biom11091340 - 10 Sep 2021
Cited by 15 | Viewed by 4572
Abstract
Rab proteins are small GTPases that act as molecular switches for intracellular vesicle trafficking. Although their function is mainly regulated by regulatory proteins such as GTPase-activating proteins and guanine nucleotide exchange factors, recent studies have shown that some Rab proteins are physiologically phosphorylated [...] Read more.
Rab proteins are small GTPases that act as molecular switches for intracellular vesicle trafficking. Although their function is mainly regulated by regulatory proteins such as GTPase-activating proteins and guanine nucleotide exchange factors, recent studies have shown that some Rab proteins are physiologically phosphorylated in the switch II region by Rab kinases. As the switch II region of Rab proteins undergoes a conformational change depending on the bound nucleotide, it plays an essential role in their function as a ‘switch’. Initially, the phosphorylation of Rab proteins in the switch II region was shown to inhibit the association with regulatory proteins. However, recent studies suggest that it also regulates the binding of Rab proteins to effector proteins, determining which pathways to regulate. These findings suggest that the regulation of the Rab function may be more dynamically regulated by phosphorylation than just through the association with regulatory proteins. In this review, we summarize the recent findings and discuss the physiological and pathological roles of Rab phosphorylation. Full article
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12 pages, 838 KiB  
Article
Phosphorylated Osteopontin Secreted from Cancer Cells Induces Cancer Cell Motility
by Yoshinobu Kariya, Midori Oyama, Yukiko Kariya and Yasuhiro Hashimoto
Biomolecules 2021, 11(9), 1323; https://doi.org/10.3390/biom11091323 - 07 Sep 2021
Cited by 8 | Viewed by 2617
Abstract
Osteopontin (OPN) plays a pivotal role in cancer cell invasion and metastasis. Although OPN has a large number of phosphorylation sites, the functional significance of OPN phosphorylation in cancer cell motility remains unclear. In this study, we attempted to investigate whether phosphorylated OPN [...] Read more.
Osteopontin (OPN) plays a pivotal role in cancer cell invasion and metastasis. Although OPN has a large number of phosphorylation sites, the functional significance of OPN phosphorylation in cancer cell motility remains unclear. In this study, we attempted to investigate whether phosphorylated OPN secreted from cancer cells affect cancer cell migration. Quantitative PCR and Western blot analyses revealed that MDA-MB435S, A549, and H460 cells highly expressed OPN, whereas the OPN expression levels in H358, MIAPaca-2, and Panc-1 cells were quite low or were not detected. Compared with the cancer cell lines with a low OPN expression, the high OPN-expressing cancer cell lines displayed a higher cell migration, and the cell migration was suppressed by the anti-OPN antibody. This was confirmed by the OPN overexpression in H358 cancer cells with a low endogenous OPN. Phos-tag ELISA showed that phosphorylated OPN was abundant in the cell culture media of A549 and H460 cells, but not in those of MDA-MB435S cells. Moreover, the A549 and H460 cell culture media, as well as the MDA-MB435S cell culture media with a kinase treatment increased cancer cell motility, both of which were abrogated by phosphatase treatment or anti-OPN antibodies. These results suggest that phosphorylated OPN secreted from cancer cells regulates cancer cell motility. Full article
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