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Protocol to characterize extracellular c-Src tyrosine kinase function through substrate interaction and phosphorylation.

ABSTRACT: Cellular Src tyrosine kinase (c-Src) exists in the secretomes of several human cancers (extracellular, e-Src). Phosphoproteomics has demonstrated the existence of 114 potential extracellular e-Src substrates in addition to Tissue Inhibitor of Metalloproteinases 2. Here, we present a protocol to characterize secreted tyrosine-phosphorylated substrates as a result of c-Src expression and secretion. We describe steps for collecting cell secretomes and extracts, performing antibody treatment and Ni-NTA pull-down, and detecting protein-protein interaction and substrate Y-phosphorylation. This protocol is adaptable for studies examining the function of other extracellular kinases. For complete details on the use and execution of this protocol, please refer to Backe et al. (2023)¹ and Sánchez-Pozo et al. (2018).².

SUBMITTER: Omini MP

PROVIDER: S-EPMC10701435 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Protocol to characterize extracellular c-Src tyrosine kinase function through substrate interaction and phosphorylation.

Omini Michael P MP Alsalih Deema D Votra SarahBeth D SD Bourboulia Dimitra D

STAR protocols 20231202 4

Cellular Src tyrosine kinase (c-Src) exists in the secretomes of several human cancers (extracellular, e-Src). Phosphoproteomics has demonstrated the existence of 114 potential extracellular e-Src substrates in addition to Tissue Inhibitor of Metalloproteinases 2. Here, we present a protocol to characterize secreted tyrosine-phosphorylated substrates as a result of c-Src expression and secretion. We describe steps for collecting cell secretomes and extracts, performing antibody treatment and Ni- ...[more]

PMID: 38043058

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