EVIDENCE FOR CKI AND CKII AT THE CELL-SURFACE

Ser/Thr-protein kinases at the cell surface (ecto-PK) use physiologica l concentrations of extracellular ATP for phosphorylation of endogenou s cell surface proteins, as well as of soluble protein substrates in t he extracellular environment (Kubler et al., 1982, 1989). One abundant ecto-PK component is believed to be a protein kinase CKII since it ph osphorylates phosvitin and casein, is sensitive to heparin at low conc entrations, and can use both ATP and GTP as cosubstrate. This ecto-PK activity can be detached from the surface of intact cells through inte raction with exogenous substrates, a process termed ''shedding'' (Kubl er et al., 1983). This study reports a method for the purification and identification of shedded ecto-PK, Affinity chromatography of the con centrated ecto-PK through a heparin-matrix resolved two phosvitin/case in kinase activities upon elution with a NaCl gradient, termed as peak I and peak II. Relative to the total protein load of the cells employ ed for ecto-PK shedding, the specific activities increased by a factor of about 10(4) times. The use of peptide substrates specific for CKI and CKII, of ATP and GTP, as well as of antibodies specific for CKII s ubunit, clearly identified one of the enzymes as a CKI-like entity and the other one as CKII-like. Although the spatial arrangement on the c ell surface of the two related ecto-PKs is unknown, their tandem appea rance together in the cell supernatant might suggest the possibility o f a functional unit.