Specific detection of an interleukin 1- and tumour necrosis factor-activated beta-casein kinase in HeLa and KB cells.
Interleukin 1 (IL-1) and tumour necrosis factor (TNF) activate a novel protein kinase, TIP kinase, which phosphorylates beta-casein in vitro. We have identified and purified to homogeneity a tryptic fragment of beta-casein, called T1, which was phosphorylated by TIP kinase with kinetics similar to those of the intact protein (K[m] = 27 +/- 6 microM). Phosphopeptide maps of in vitro phosphorylated T1 and beta-casein were identical, confirming that T1 contained the main phosphorylation site of the protein. T1 corresponded to residues 114 to 169 of beta-casein. It was phosphorylated by constitutively active protein kinases to a much lesser extent than beta-casein and thus constituted a specific substrate of the cytokine-activated enzyme. This made possible the detection of TIP kinase in extracts of IL-1-stimulated HeLa and KB cells, which had been hampered by high background phosphorylation when beta-casein was used as substrate. Our results show that the use of fragment T1 allows detection of low levels of TIP kinase in crude samples. They also suggest that its activation, which had previously been observed only in connective tissue cells, may be a general response of many cell types to IL-1 or TNF.
Guesdon
F
F
Cytokine Laboratory, The Babraham Institute, Cambridge, United Kingdom. F.A.Guesdon@Sheffield.ac.uk
Waller
R J
RJ
Saklatvala
J
J
D047388:Casein Kinases; D002364:Caseins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D007624:KB Cells; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D002364:Caseins; D007375:Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D011494:Protein Kinases; D047388:Casein Kinases; C109305:TNF-IL1-activated protein kinase; D010450:Endopeptidases