Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein

Xu, Jie; Sun, Luo; Ghosh, Inca; Xu, Ming‐Qun

doi:10.2144/04366st02

Cited by 11 publications

(67 citation statements)

References 13 publications

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“…4a, b). Similarly, recombinant Cdh1 protein inhibited the kinase activity of Src in promoting CDK1 33 and p85 cortactin (CTTN) phosphorylation 34 in vitro (Fig. 4b and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 82%

Interplay between c-Src and the APC/C co-activator Cdh1 regulates mammary tumorigenesis

et al. 2019

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The Anaphase Promoting Complex (APC) coactivator Cdh1 drives proper cell cycle progression and is implicated in the suppression of tumorigenesis. However, it remains elusive how Cdh1 restrains cancer progression and how tumor cells escape the inhibition of Cdh1. Here we report that Cdh1 suppresses the kinase activity of c-Src in an APC-independent manner. Depleting Cdh1 accelerates breast cancer cell proliferation and cooperates with PTEN loss to promote breast tumor progression in mice. Hyperactive c-Src, on the other hand, reciprocally inhibits the ubiquitin E3 ligase activity of APC Cdh1 through direct phosphorylation of Cdh1 at its N -terminus, which disrupts the interaction between Cdh1 and the APC core complex. Furthermore, pharmacological inhibition of c-Src restores APC Cdh1 tumor suppressor function to repress a panel of APC Cdh1 oncogenic substrates. Our findings reveal a reciprocal feedback circuit of Cdh1 and c-Src in the crosstalk between the cell cycle machinery and the c-Src signaling pathway.

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“…4a, b). Similarly, recombinant Cdh1 protein inhibited the kinase activity of Src in promoting CDK1 33 and p85 cortactin (CTTN) phosphorylation 34 in vitro (Fig. 4b and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 82%

Interplay between c-Src and the APC/C co-activator Cdh1 regulates mammary tumorigenesis

et al. 2019

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“…Phosphoserine containing peptide PB1-pSer was derived from mouse Bcl2-antagonist of cell death (BAD) (12). Phosphotyrosine-containing peptide Cdc2-pTyr15 was derived from a phosphorylation site of human cyclindependent kinase (13). …”

Section: Methodsmentioning

confidence: 99%

Use of Intein-Mediated Phosphoprotein Arrays to Study Substrate Specificity of Protein Phosphatases

et al. 2007

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Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.

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“…There are over 500 protein kinases and about 30% of the human proteins undergo phosphorylation to perform essential cellular activities [8]. Therefore, it is not surprising that abnormal phosphorylation should turn out to be a cause of human disease [9]. There are over 400 diseases that are linked with the phosphorylation of proteins, with mostof the diseases caused by mutations in the genes that regulate kinases that alter the phosphorylation sites in a protein.…”

Section: Role Of Protein Phosphorylationmentioning

confidence: 99%

Biosensors for Screening Kinase Inhibitors

Bhalla

Estrela

2017

CTMC

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For successful drug discovery it is important to understand the fundamentals of the underlying causes and consequences of the diseases for which the drug is being developed. One such physiological process in eukaryotic cells is protein phosphorylation, which is the main post-translational modification of proteins responsible for the onset or progression of Alzheimer's disease, diabetes and various cancers. Protein phosphorylation is facilitated by kinases and inhibitors of kinases act as drugs in controlling or curing these diseases by reducing protein phosphorylation. This review discusses the technologies capable of detecting kinase activity and screening candidate compounds to identify novel inhibitors of protein kinases.

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Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein

Cited by 11 publications

References 13 publications

Interplay between c-Src and the APC/C co-activator Cdh1 regulates mammary tumorigenesis

Interplay between c-Src and the APC/C co-activator Cdh1 regulates mammary tumorigenesis

Use of Intein-Mediated Phosphoprotein Arrays to Study Substrate Specificity of Protein Phosphatases

Biosensors for Screening Kinase Inhibitors

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