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

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%.

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Site-directed mutagenesis of herpesvirus glycoprotein phosphorylation sites by recombination polymerase chain reaction.

Genome Research ◽

10.1101/gr.1.3.205 ◽

1992 ◽

Vol 1 (3) ◽

pp. 205-207 ◽

Cited By ~ 29

Author(s):

Z Yao ◽

D H Jones ◽

C Grose

Keyword(s):

Polymerase Chain Reaction ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Phosphorylation Sites ◽

Chain Reaction ◽

Polymerase Chain

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Phosphorylation Controls Autoinhibition of Cytoplasmic Linker Protein-170

Molecular Biology of the Cell ◽

10.1091/mbc.e09-12-1036 ◽

2010 ◽

Vol 21 (15) ◽

pp. 2661-2673 ◽

Cited By ~ 29

Author(s):

Ho-Sup Lee ◽

Yulia A. Komarova ◽

Elena S. Nadezhdina ◽

Rana Anjum ◽

John G. Peloquin ◽

...

Keyword(s):

Binding Affinity ◽

Conformational Changes ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Phosphorylation Sites ◽

Deficient Mutant ◽

Rich Region ◽

The Third ◽

Open Conformation ◽

Intramolecular Association

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an “open” conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the “folded back” conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.

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