Phosphorylation of high‐ and low‐molecular‐mass atrial natriuretic peptide analogs by cyclic AMP‐dependent protein kinase

Olins, Gillian M.; Mehta, Pramod P.; Blehm, Delores J.; Patton, Dennis R.; Zupec, Mark E.; Whipple, Deborah E.; Tjoeng, Foe S.; Adams, Steven P.; Olins, Peter O.; Gierse, James K.

doi:10.1016/0014-5793(87)80478-7

Cited by 18 publications

(4 citation statements)

References 16 publications

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“…Using the purified catalytic subunit of PK A, it was shown that ANP exhibits a K,,, value of 0.5 pM, the lowest K , among PK A substrates known. Studies with different ANP analogues by Olins et al [8] determined that the amino acid sequence ANP99 -126 was required for optimal phosphorylation. They also showed that ANP labeling occurred at Serl04, the putative phosphorylation site in this molecule [7].…”

Section: Discussionmentioning

confidence: 99%

“…Different physiological effects have been observed with native and phosphorylated ANP, including altered binding to receptors [8], changes of the Na'i K+/Cl-cotransport in cultured vascular smooth muscle cells [7], decrease of vasorelaxant activity in norepinephrine precontracted rabbit aorta muscle strips [9] as well as significant inhibition of proteolysis of ANP99 -126 by endoprotease 24.1 1, a specific protease present in kidney cortical membrane thought to be responsible for the termination of ANP activity in the renal system [34].…”

Section: Discussionmentioning

confidence: 99%

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

Kübler

Reinhardt

Reed

et al. 1992

European Journal of Biochemistry

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. 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

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Kübler

Reinhardt

Reed

et al. 1992

European Journal of Biochemistry

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show abstract

“…Though pro-ANP is apparently a poor intracellular substrate for PKA, processed extracellular ANP (ANP96−126) is phosphorylated by an “ecto” PKA associated with HeLa cells and other cell lines when the cells are treated with isoproterenol . There are conflicting reports on the effect of phosphorylation on its bioactivity. , The physiological relevance of ANP phosphorylation remains obscure. Likewise, the physiological relevance of a mammalian ecto-PKA is unresolved, though recent descriptions of ecto-PKA activity on the surface of a variety of other cell types − suggests that cAMP-dependent phosphorylation of other extracellular proteins may be a general phenomenon.…”

Section: Extracellular Proteinsmentioning

confidence: 99%

“…333 There are conflicting reports on the effect of phosphorylation on its bioactivity. 334,335 The physiological relevance of ANP phosphorylation remains obscure. Likewise, the physiological relevance of a mammalian ecto-PKA is unresolved, though recent descriptions of ecto-PKA activity on the surface of a variety of other cell types [336][337][338] suggests that cAMP-dependent phosphorylation of other extracellular proteins may be a general phenomenon.…”

Section: Extracellular Proteinsmentioning

confidence: 99%