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.