Evidence for CKI and CKII at the cell surface

Ser/Thr-protein kinases at the cell surface (ecto-PK) use physiological concentrations of extracellular ATP for phosphorylation of endogenous cell surface proteins, as well as of soluble protein substrates in the extracellular environment (Kübler et al., 1982, 1989). One abundant ecto-PK component is believed to be a protein kinase CKII since it phosphorylates phosvitin and casein, is sensitive to heparin at low concentrations, and can use both ATP and GTP as cosubstrate. This ecto-PK activity can be detached from the surface of intact cells through interaction with exogenous substrates, a process termed "shedding" (Kübler et al., 1983). This study reports a method for the purification and identification of shedded ecto-PK. Affinity chromatography of the concentrated ecto-PK through a heparin-matrix resolved two phosvitin/casein kinase activities upon elution with a NaCl gradient, termed as peak I and peak II. Relative to the total protein load of the cells employed 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 subunit, clearly identified one of the enzymes as a CKI-like entity and the other one as CKII-like. Although the spatial arrangement on the cell surface of the two related ecto-PKs is unknown, their tandem appearance together in the cell supernatant might suggest the possibility of a functional unit.