Atrial natriuretic peptide is phosphorylated by intact cells through cAMP‐dependent ecto‐protein kinase

Abstract

. The question of the physiological role of externally directed kinase activity prompted a search for potential natural substrates present in the intercellular fluid. In the present study we have investigated the phosphorylation by ecto-PK A of the human atrial natriuretic peptide ANP99 -126, a hormone released by cardiac cells. This 28-amino-acid peptide carries the phosphorylation consensus sequence Arg-Arg-Ser-Ser for the PK A. Incubation of various cell lines (including epithelial, epidermal, myoblast and lymphoma cells) or freshly isolated blood cells (macrophages, erythrocytes and platelets) with ANP in the presence of low micromolar concentrations of ATP resulted in the phosphorylation of ANP at Ser residues. The ANP phosphorylation reaction proved strictly dependent on CAMP; CAMP could not be replaced by cCMP. The phosphorylation was inhibited by the PK A-specific inhibitory peptide and increased linearily for up to 15 min and with a K, value of 3 -5 pM for ANP. At higher ATP concentrations (> 100 pM) the incorporation rates amounted to about 0.3 mmol P (mol ANP)-' min-l. The rise of intracellular CAMP in HEL30 (an epidermal cell line) after application of the b-adrenergic receptor agonist isoproterenol led to an approximately three-fold stimulation of ANP phosphorylation which appears to be brought about by an efflux of intracellular CAMP. Employing cell supernatant fluids and cell sonicates, it could be shown that the phosphorylation of ANP results from the ecto-PK A. Comparison of ANP with ANP phosphorylated in vitro using purified catalytic subunit of PK A showed that phosphorylation is accompanied by certain changes in the average solution conformation of the peptide, consistent with the changes known to occur in its biological activity. Our results demonstrate CAMP-dependent phosphorylation of the peptide hormone analogue ANP99 -126 by intact cells through ecto-PK A, an intriguing mechanism for post-translational processing of ANP.The atrial natriuretic peptides (ANP) represent a family of molecules, 21 -33 amino acids in length, cleaved from the carboxy-terminal of inactive prohormones during their release from cardiac cell granules. On release from the cell, the circulating forms of ANP (e.g. ANP99 -126) in blood are highly efficient for the regulation of blood pressure through water and electrolyte balance, as reported previously 11, 21. The vasorelaxant and natriuretic property of ANP was found to be related to the secretion of renin and aldosteron 13, 41; it may also function as a neuropeptide [5, 61. For bioactivity ANP has to be in a loop form which is stabilized by a single disulfide bridge between Cysl05 and Cysl21. Sequence studies of the primary structure of many circulating forms of ANP peptides show the amino acid sequence Arg-Arg-Ser-Ser (101 -104) directly preceding the disulfide bridge. This represents a potential phosphorylation site for CAMP-dependent protein kinase (PK A) activity on

Evidence for cAMP-dependent Platelet Ectoprotein Kinase Activity That Phosphorylates Platelet Glycoprotein IV (CD36)

“…Using kemptide, a heptapeptide currently employed in assays for cell surface-located ecto-PKA activity (23,27), we clearly demonstrated the existence of an ecto-PKA activity on the outer surface of platelet membranes. Indeed, the addition of labeled ATP ([␥-32 P]ATP) to intact platelets in the presence of cAMP resulted in a rapid incorporation of radioactive phosphate into kemptide.…”

Proteins in unexpected locations.

“…The molecular cloning of a transmembrane kinase C isoform (Johannes et al, 1994) has corroborated earlier biochemical and cellular evidence supporting an ecto-location for kinases. Numerous substrates for ecto-kinases also have been identified (Vilgrain and Baird, 1991;Kubler et al, 1992;Jordan et al, 1994 Pepperbok et al (1994) showed further that casein kinase II is likely to exert at least some of its effects directly within the nucleus by demonstrating differential effects of antibodies against casein kinase II on the cell cycle when microinjected into the nucleus versus the cytoplasm of mitogenically stimulated primary fibroblasts. Luby-Phelps et al (1995) have directly visualized the translocation of calmodulin into the nuclei of living smooth muscle cells stimulated at micromolar levels of intracellular Ca2 .…”

Reinventing the Wheel of Cyclic AMP

“…The activity of ecto‐PKA was demonstrated further by phosphorylation of the PKA‐specific substrate Kemptide, occurring presumably at the surface of HeLa cells 36,37. Other studies also showed that the atrial natriuretic peptide is phosphorylated in a variety of intact cells by a cAMP‐dependent ectoprotein kinase 38. More recently, it has been definitively shown that purified membrane of human LS‐174T colon carcinoma cells contained PKA activity 39.…”

“…Since cAMP is generated in the cells by adenylyl cyclase in response to hormonal stimulation, activation of PKA that is localized in the extracellular milieu poses a logistical problem. However, both intracellular cAMP and ATP have been shown to be released into the extracellular space and are present in body fluids, thus providing the factors necessary to influence PKA activity extracellularly and therefore supporting a potential role for PKA in intercellular communication and regulation 35,36,38. Various ecto‐ and exoenzymes are implicated in developmental processes, intercellular communication, feedback regulation, and events such as cell‐cell interaction or receptor transduction of external stimuli.…”