Abstract
The heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) is a synthetic construct of a substrate for cAMP-dependent protein kinase (PK). In this work we show that Kemptide has all the properties of a cytophilic substrate, i.e. it is a molecule preserving cell membrane intactness when added to cultured cells. Kemptide thus satisfies the prerequisites for employment in assays for cell surface-located ecto-PK activity. Different types of intact cells catalyze the phosphorylation of Kemptide in the presence of extracellular ATP and cAMP with Km values of 3-4 microM for Kemptide. Kemptide phosphorylation was influenced by PKI, the inhibitory protein specific for cAMP-PK. The results of comparative experiments with intact cells and with cell extracts demonstrate the ectoenzyme nature of this cAMP-PK. Further, the possibility was ruled out of a transfer of enzyme activity from damaged cells to the surface of intact cells. The anchorage of the surface cAMP-PK activity to the plasma membrane appears to be relatively stable since (i) cell supernatants, obtained after preincubation of intact cells with cAMP or Kemptide, did not show Kemptide phosphorylation, and (ii) the cAMP-dependent PK activity remained with cells even after five consecutive washes with cAMP or Kemptide. This is in contrast to the ecto-cAMP-independent phosvitin/casein type PK (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025) which is released from intact cells through the addition of substrate. Data are presented which show that both ectokinase activities are exhibited independently. In conjunction with published evidence for an active export of cAMP from cells as well as for the appearance of extracellular ATP the demonstration of an ecto-cAMP-PK further supports the potential of PK for intercellular regulation. The potential of ecto-cAMP-PK is demonstrated by its ability to phosphorylate biologically active forms of atrial natriuretic peptide, the atrial natriuretic peptide, which possesses the specific sequence for a cAMP-PK-catalyzed phosphorylation.
Highlights
Theheptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly nase (PK)’ activitiecsomprises a formof protein modification (Kemptide) is a synthetic construct of a substrate for that plays an important role in the regulation of numerous
KemptidePhosphorylation Is Mediated by Cell Surfacelocated Protein Kinase Activity- phosphorylation of Kemptide has been obtained in the presence of intact cells, the measured kinaseactivity could alsoreside inthe cell supernatant
This possibility was interesting in view of our observation that a substrate-effected release of the phosvitin/casein type ecto-protein kinase (PK) activiotcycurs from intact cells (23) and, in addition, that the catalytic subunbietcomes detached from its regulatory subunit(s)duetobinding of cAMP to the latter and theirsesbeyt free from thecell surface as shown recently with yeast plasma membranes (24)
Summary
% activity in cell lysate % stained cells autoradiography (seeFig. 2). Recovery studies for phospho-Kemptide were done with ”P-Kemptide. To confirm that the analysis of phosphorylation in the system with intact cells was monitoring Kemptide phosphorylation and not adulteratedby any other phosphorylated cellular proteins, we introducedtrichloroacetic acid precipitation of the postcellular supernatants prior to assaying samples on theP81 ion exchange paper. In the presence of the specific inhibitory protein no trichloroaceticacid treatment, i.e. total protein; R, after removal for CAMP-PK (PKI), Kemptide phosphorylation seen in the of trichloroaceticacid precipitate; andC, after removal of trichloropresence confirm t of he cAMP was abolished The latter CAMP-dependent type of kinase results reaction not but only acetic acid precipitate andacetic acid extraction of the paper-bound radioactive material. Threaction reached a plateau at cAMP levels above M, indicatingsaturation
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