INDUCED RELEASE OF CELL-SURFACE PROTEIN-KINASE YIELDS CK1-AND CK2-LIKE ENZYMES IN TANDEM

Several types of cell exhibit cell surface protein kinase (ecto-PK) ac tivities with Ser/Thr-specificity. Ecto-PK sharing certain characteris tics of protein kinase CK2 can be detached from intact cells by intera ction with exogenous substrates (Kubler, D., Pyerin, W., Burow, E., an d Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). Howe ver, a detailed molecular analysis of this ecto-PK was hampered by the vanishingly small amounts of labile enzyme protein obtained by substr ate-inducible enzyme release, We now describe the stabilization and en richment of released ecto-PK by precipitation with polyethylene glycol followed by affinity chromatography on heparin-agarose. Ecto-PK is sh own to consist of two separate forms released in tandem, ecto-PK I and ecto-PK II. Comparison with cell homogenates as well as cell surface biotinylation experiments excluded contamination with intracellular PK . Purified ecto-PK I and ecto-PK II exhibit respectively selective pho sphorylation of CK1- and CK2-specific peptide substrates, a complement ary sensitivity to inhibitory agents and a differential use of the cos ubstrates ATP and GTP. Ecto PK I consists of a 40-kDa moiety; the ecto -PK II is an ensemble of three components of 43- and 40-kDa (catalytic subunits) and a noncatalytic 28-kDa subunit. In addition, components of the ecto-PK II react with CK2-specific antibodies. Further, compara tive peptide mapping and the results of mass spectrometry in combinati on with assignment of amino acid sequences confirmed that ecto-PK II i s closely related if not identical to the protein kinase CK2. Assays w ith intact cells that result in the phosphorylation of a variety of en dogenous membrane proteins showed that both ecto-PKs participate, and further, certain ecto-PK substrates be come preferentially labeled by one or another of the enzymes, whereas others are phosphorylated by bo th ecto-PK activities.