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Human apolipoprotein A-I. Post-translational modification by covalent phosphorylation.
Z H Beg1, J A Stonik, J M Hoeg
1Molecular Disease Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
The Journal of Biological Chemistry
|April 25, 1989
Summary
Human apolipoprotein A-I (apoA-I) undergoes reversible phosphorylation on a serine residue, affecting its charge and potentially its role in lipoprotein metabolism. This modification occurs both intracellularly and extracellularly in hepatocytes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Apolipoprotein A-I (apoA-I) is a key component of high-density lipoproteins (HDLs).
- Post-translational modifications can significantly alter protein function.
- The role of apoA-I phosphorylation in cellular processes was previously uncharacterized.
Purpose of the Study:
- To investigate the phosphorylation of human apolipoprotein A-I (apoA-I).
- To identify the specific site and characteristics of apoA-I phosphorylation.
- To determine the cellular localization and functional implications of apoA-I phosphorylation.
Main Methods:
- In vitro phosphorylation assays using purified human apoA-I and a Ca2+/calmodulin-dependent kinase.
- Isoelectric focusing and phosphoamino acid analysis to characterize phosphorylation.
- Automated Edman degradation to identify the phosphorylation site.
- Studies using HepG-2 cells and primary human hepatocytes to assess intracellular and secreted apoA-I phosphorylation.
Main Results:
- Human apoA-I is phosphorylated in vitro by a Ca2+/calmodulin-dependent kinase in a time-, Ca2+-, and calmodulin-dependent manner.
- Phosphorylation occurs on a single serine residue at position 201, adding approximately one phosphate per apoA-I molecule.
- Phosphorylation increases apoA-I's negative charge, shifting its isoelectric point.
- ApoA-I is secreted as a phosphoapolipoprotein by HepG-2 cells and primary human hepatocytes, and intracellular apoA-I is also phosphorylated.
- Dephosphorylation is reversible, with loss of phosphate correlating with loss of radioactivity.
Conclusions:
- This study describes the first evidence of reversible phosphorylation of apoA-I, occurring on serine 201.
- Phosphorylation affects apoA-I's charge and occurs both intracellularly and extracellularly in hepatocytes.
- The synthesis and secretion of apoA-I as a phosphoapolipoprotein may be crucial for lipoprotein assembly, transport, and secretion.