Integrating bioinformatics tools to investigate protein phosphorylation

Directed Mutagenesis ◽

Phosphorylation Sites ◽

Cellular Processes ◽

Benchmark Study

Protein phosphorylation is one of the most important protein post-translational modifications and plays a role in numerous cellular processes including recognition, signaling and degradation. It can be studied experimentally by various methodologies, like employing western blot analysis, site-directed mutagenesis, 2 D gel electrophoresis, mass spectrometry etc. A number of in silico tools have also been developed in order to predict plausible phosphorylation sites in a given protein. In this review, we conducted a benchmark study including the leading protein phosphorylation prediction software, in an effort to determine which performs best. The first place was taken by GPS 2.2, having predicted all phosphorylation sites with a 83% fidelity while in second place came NetPhos 2.0 with 69%.

Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

10.1101/2020.07.21.213488 ◽

2020 ◽

Author(s):

Manish Bhattacharjee ◽

Navin Adhikari ◽

Renu Sudhakar ◽

Zeba Rizvi ◽

Divya Das ◽

...

Keyword(s):

Mass Spectrometry ◽

Plasmodium Falciparum ◽

Western Blot ◽

Wild Type ◽

Malaria Parasites ◽

Functional Conservation ◽

Cellular Processes ◽

Physiological Substrates

ABSTRACTA variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria parasites, we characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Gly76 mutated to Ala75Ala76) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (Δrub1) or NEDD8 conjugating E2 enzyme (ΔUbc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

Identification of protein phosphorylation sites within Ser/Thr-rich cluster domains using site-directed mutagenesis and hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry

Rapid Communications in Mass Spectrometry ◽

10.1002/rcm.3223 ◽

2007 ◽

Vol 21 (21) ◽

pp. 3443-3451 ◽

Cited By ~ 11

Author(s):

Julie B. King ◽

Julia Gross ◽

Christine M. Lovly ◽

Helen Piwnica-Worms ◽

R. Reid Townsend

Keyword(s):

Mass Spectrometry ◽

Cyclotron Resonance ◽

Ion Trap ◽

Quadrupole Ion Trap ◽

Directed Mutagenesis ◽

Ion Cyclotron Resonance ◽

Phosphorylation Sites ◽

Rich Cluster ◽

Linear Quadrupole Ion Trap

Methods in Enzymology - ABC Transporters: Biochemical, Cellular, and Molecular Aspects ◽

[25] Identification of in vivo phosphorylation sites for basic-directed kinases in murine mdr1b P-glycoprotein by combination of mass spectrometry and site-directed mutagenesis

10.1016/s0076-6879(98)92027-4 ◽

1998 ◽

pp. 342-358

Author(s):

J.S. Glavy ◽

M. Wolfson ◽

E. Nieves ◽

E.-K. Han ◽

C.-P.H. Yang ◽

...

Keyword(s):

Mass Spectrometry ◽

Directed Mutagenesis ◽

Phosphorylation Sites ◽

P Glycoprotein

Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

Cells ◽

10.3390/cells10071701 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1701

Author(s):

Lorenz Weidenauer ◽

Manfredo Quadroni

Keyword(s):

Mass Spectrometry ◽

K562 Cells ◽

Phosphorylation Sites ◽

Activity Regulation ◽

Wild Type ◽

Linker Region ◽

Cellular Processes ◽

Tryptic Cleavage ◽

Client Proteins

Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone.

Site-directed mutagenesis of the phosphorylation sites in the mouse glucocorticoid receptor.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)80568-7 ◽

1993 ◽

Vol 268 (29) ◽

pp. 21501-21504

Author(s):

S.A. Mason ◽

P.R. Housley

Keyword(s):

Glucocorticoid Receptor ◽

Directed Mutagenesis ◽

Phosphorylation Sites

The proteomic future: where mass spectrometry should be taking us

Biochemical Journal ◽

10.1042/bj20110363 ◽

2012 ◽

Vol 444 (2) ◽

pp. 169-181 ◽

Cited By ~ 52

Author(s):

Jay J. Thelen ◽

Ján A. Miernyk

Keyword(s):

Mass Spectrometry ◽

Gel Electrophoresis ◽

Protein Structures ◽

Research Area ◽

Mass Accuracy ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis ◽

Peptide Separation ◽

Research Areas

A newcomer to the -omics era, proteomics, is a broad instrument-intensive research area that has advanced rapidly since its inception less than 20 years ago. Although the ‘wet-bench’ aspects of proteomics have undergone a renaissance with the improvement in protein and peptide separation techniques, including various improvements in two-dimensional gel electrophoresis and gel-free or off-gel protein focusing, it has been the seminal advances in MS that have led to the ascension of this field. Recent improvements in sensitivity, mass accuracy and fragmentation have led to achievements previously only dreamed of, including whole-proteome identification, and quantification and extensive mapping of specific PTMs (post-translational modifications). With such capabilities at present, one might conclude that proteomics has already reached its zenith; however, ‘capability’ indicates that the envisioned goals have not yet been achieved. In the present review we focus on what we perceive as the areas requiring more attention to achieve the improvements in workflow and instrumentation that will bridge the gap between capability and achievement for at least most proteomes and PTMs. Additionally, it is essential that we extend our ability to understand protein structures, interactions and localizations. Towards these ends, we briefly focus on selected methods and research areas where we anticipate the next wave of proteomic advances.

Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015891 ◽

2020 ◽

Vol 295 (52) ◽

pp. 18105-18121

Author(s):

Andrew C. Hedman ◽

Dean E. McNulty ◽

Zhigang Li ◽

Laëtitia Gorisse ◽

Roland S. Annan ◽

...

Keyword(s):

Cell Function ◽

Scaffold Protein ◽

Human Lung Cancer ◽

Akt Activation ◽

Cellular Processes ◽

Src Signaling ◽

Chemical Inhibitors ◽

Hepatocyte Growth

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein β antibody in differentiating myoblasts

BMC Research Notes ◽

10.1186/s13104-019-4749-3 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Hamood AlSudais ◽

Nadine Wiper-Bergeron

Keyword(s):

Mass Spectrometry ◽

Monoclonal Antibody ◽

Transcription Factors ◽

Western Blot ◽

Light Chain ◽

Myosin Light Chain ◽

Intense Band ◽

Cellular Processes ◽

Enhancer Binding Protein ◽

Myoblast Cells

Abstract Objective CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPβ expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPβ, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPβ, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPβ, the band was present when C/EBPβ was knocked down, confirming specificity for a protein other than C/EBPβ. The objective of this work was to identify the contaminating reactivity. Results We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPβ is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPβ isoforms.