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Human Apolipoprotein A-I. Post-translational Modification by Covalent Phosphorylation

Human Apolipoprotein A-I. Post-translational Modification by Covalent Phosphorylation

Z H Beg, J A Stonik, J M Hoeg, S J Demosky Jr, T Fairwell, H B Brewer Jr

J Biol Chem. 1989 Apr 25;264(12):6913-21.

PMID: 2496123

Abstract:

In vitro phosphorylation of purified human plasma apolipoprotein A-I (apoA-I) by a recently characterized Ca2+/calmodulin-dependent kinase (Beg, Z. H., Stonik, J. A., and Brewer, H. B., Jr. (1987) J. Biol. Chem. 262, 13228-13240) was time-, Ca2+-, and calmodulin-dependent. Maximal phosphorylation of human apoA-I revealed a stoichiometry of approximately 1 mol of PO4/mol of apoA-I. Phosphorylation of apoA-I resulted in an increase of two negative charges and consequently a shift to a more acidic pI for each apoA-I isoform following isoelectrofocusing. Dephosphorylation of 32P-apoA-I with either phosphatase I or a Ca2+/calmodulin-dependent phosphatase was associated with a virtually complete loss of 1 mol of 32PO4/mol of apoA-I. Phosphoamino acid analysis of a purified 32P-peptide established that the phosphorylation occurred on a single serine residue. Automated Edman degradation of the purified 32P-peptide revealed a single amino acid sequence and indicated that phosphorylation occurred on the serine at residue 201 in the apoA-I sequence. ApoA-I was shown to be secreted as a phosphoapolipoprotein by HepG-2 cells as well as primary human hepatocytes. Analysis of HepG-2 cells established that intracellular apoA-I, like secreted apoA-I, is phosphorylated. Dephosphorylation of both secreted and intracellular 32P-apoA-I revealed the loss of radioactivity in the apoA-I protein bands. These data provide the initial description of a post-translational modification involving reversible phosphorylation of extracellular as well as intracellular apoA-I on a serine residue. These combined results suggest that synthesis and secretion of apoA-I as a phosphoapolipoprotein in HepG-2 cells as well as primary human hepatocytes may play an important role in lipoprotein assembly, intracellular transport as well as processing, and lipoprotein secretion.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4247069	Apolipoprotein A-I from human plasma			Price

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