SILAKin: A novel high throughput SILAC-based quantitative mass spectrometry assay to identify the substratome of kinases secreted by pathogens

ABSTRACT: We developed SILAkin, a novel and easy method to identify substrates that is applicable to most kinases. It combines phosphatase treatment, pulse heating, in vitro kinase assay and SILAC (Stable Isotope Labeling with Amino acids in Cell culture)-based quantitative mass spectrometry (MS). SILAkin was developed using the Leishmania casein kinase 1 (L-CK1.2) as experimental model. Leishmania, an intracellular parasite causing Leishmaniasis, releases L-CK1.2 in its host cell. Applying this novel assay allowed us to gain unprecedented insight into host-pathogen interactions through the identification of 225 host substrates phosphorylated by L-CK1.2. The substratome was validated experimentally by L-CK1.2 and human CK1δ, demonstrating the efficiency of SILAkin to identify new substrates and revealing novel regulatory pathways. Finally, SILAkin was instrumental in highlighting host pathways potentially regulated by L-CK1.2 in Leishmania-infected host cells.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Macrophage

DISEASE(S): Leishmaniasis

SUBMITTER: Valentin SABATET  

LAB HEAD: Damarys Loew

PROVIDER: PXD026220 | Pride | 2022-02-17

REPOSITORIES: Pride