SPECIFIC DETECTION OF AN INTERLEUKIN 1- AND TUMOUR NECROSIS FACTOR- ACTIVATED β-CASEIN KINSASE IN HeLa AND KB CELLS

Abstract Interleukin 1 (IL-1) and tumour necrosis factor (TNF) activate a novel protein kinase, TIP kinase, which phosphorylates β-casein in vitro. We have identified and purified to homogeneity a tryptic fragment of β-casein, called T1, which was phosphorylated by TIP kinase with kinetics similar to those of the intact protein ( K m =27±6 μM). Phosphopeptide maps of in vitro phosphorylated T1 and β-casein were identical, confirming that T1 contained the main phosphorylation site of the protein. T1 corresponded to residues 114 to 169 of β-casein. It was phosphorylated by constitutively active protein kinases to a much lesser extent than β-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 β-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.