Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays

TY - JOUR

T1 - Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays

AU - Snelling, Tom

N1 - Copyright: © 2024 The Authors.

PY - 2024/11/20

Y1 - 2024/11/20

N2 - ALPK1 is an atypical protein kinase that is activated during bacterial infection by ADP-heptose and phosphorylates TIFA to activate a cell signaling pathway. In contrast, specific mutations in ALPK1 allow it to also be activated by endogenous human nucleotide sugars such as UDP-mannose, leading to the phosphorylation of TIFA in the absence of infection. This protocol describes a quantitative, cell-free phosphorylation assay that can directly measure the catalytic activity of wildtype and disease-causing ALPK1 in the presence of different nucleotide sugars. In this method, overexpressed ALPK1 is first immunoprecipitated from the extracts of ALPK1 knockout HEK-Blue cells transfected with plasmids encoding either FLAG-tagged wildtype or mutant ALPK1, and then subjected to a radioactive phosphorylation assay in which the phosphorylation of purified GST-tagged TIFA by ALPK1 is quantified by measuring the incorporation of radioactivity derived from radiolabeled ATP.

AB - ALPK1 is an atypical protein kinase that is activated during bacterial infection by ADP-heptose and phosphorylates TIFA to activate a cell signaling pathway. In contrast, specific mutations in ALPK1 allow it to also be activated by endogenous human nucleotide sugars such as UDP-mannose, leading to the phosphorylation of TIFA in the absence of infection. This protocol describes a quantitative, cell-free phosphorylation assay that can directly measure the catalytic activity of wildtype and disease-causing ALPK1 in the presence of different nucleotide sugars. In this method, overexpressed ALPK1 is first immunoprecipitated from the extracts of ALPK1 knockout HEK-Blue cells transfected with plasmids encoding either FLAG-tagged wildtype or mutant ALPK1, and then subjected to a radioactive phosphorylation assay in which the phosphorylation of purified GST-tagged TIFA by ALPK1 is quantified by measuring the incorporation of radioactivity derived from radiolabeled ATP.

KW - ALPK1

KW - Nucleotide sugar

KW - ADP-heptose

KW - ROSAH

KW - Spiradenoma

KW - Phosphorylation

KW - Protein kinase

UR - https://www.scopus.com/pages/publications/85211041948

U2 - 10.21769/BioProtoc.5124

DO - 10.21769/BioProtoc.5124

M3 - Protocol

C2 - 39600973

SN - 2331-8325

VL - 14

JO - Bio-Protocol

JF - Bio-Protocol

IS - 22

M1 - e5124

ER -