Abstract
Several types of cell exhibit cell surface protein kinase (ecto-PK) activities with Ser/Thr-specificity. Ecto-PK sharing certain characteristics of protein kinase CK2 can be detached from intact cells by interaction with exogenous substrates (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). However, a detailed molecular analysis of this ecto-PK was hampered by the vanishingly small amounts of labile enzyme protein obtained by substrate-inducible enzyme release. We now describe the stabilization and enrichment of released ecto-PK by precipitation with polyethylene glycol followed by affinity chromatography on heparin-agarose. Ecto-PK is shown 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 phosphorylation of CK1- and CK2-specific peptide substrates, a complementary sensitivity to inhibitory agents and a differential use of the cosubstrates 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, comparative peptide mapping and the results of mass spectrometry in combination with assignment of amino acid sequences confirmed that ecto-PK II is closely related if not identical to the protein kinase CK2. Assays with intact cells that result in the phosphorylation of a variety of endogenous membrane proteins showed that both ecto-PKs participate, and further, certain ecto-PK substrates become preferentially labeled by one or another of the enzymes, whereas others are phosphorylated by both ecto-PK activities.
Highlights
The activity of protein kinases (PK)1 is well established as a major mediator by which cells relay important signals for cell growth, metabolism, and homeostasis
9080 22670 a Calculation was based on the proportion of total activities of ecto-PK I and ecto-PK II after their separation on heparin-agarose. b These values mainly represent phosvitin that has been added for the purpose of ecto-PK release. c Protein measurement was done from comparative protein staining on SDS-polyacrylamide gels (Coomassie) or after western blotting to polyvinylidene difluoride (PVDF)
The release of ecto-PK from intact cells by protein kinase substrates such as phosvitin or casein appears to be a common phenomenon [17, 33, 34]
Summary
Materials—Peptides specific for protein kinase CK1, (DDDDVASLPGLRRR) [22], RRKDLHDDEEDEAMSITA) [23], and protein kinase CK2, (RRRAADSDDDDD [24] and RRREEETEEE) [23]), were prepared by the peptide synthesis unit of the DKFZ. Phosphorylation of enzyme-specific peptide subunits (1 mg/1 ml) by purified ecto-PK samples was carried out for 10 min in a total of 100 l containing [␥-32P]ATP or [␥-32P]GTP (specific activity, 25 GBq/mmol) and was stopped by the addition of 100 l of ice-cold 10% trichloroacetic acid and 15 l of 0.63% bovine serum albumin for coprecipitation on ice (15 min) Under these conditions the peptides under investigation remained soluble, whereas larger proteins were precipitated and could be removed by centrifugation (14000 ϫ g for 10 min). Cell Surface Phosphorylation—Cell surface phosphorylation by ecto-PK activity on intact cells and analysis of radioactively phosphorylated proteins was carried out for 12 min as detailed earlier [11] in the presence of the PK inhibitors heparin or CK I-7 at concentrations given in the particular experiment.
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