Regulation of the 14-3-3-binding protein p39 by growth factors and nutrients in rat PC12 pheochromocytoma cells

• Published in: Biochemical journal 2002-12, Vol.368 (Pt 2), p.565-572
• Main Authors: Harthill, Jean E, Pozuelo Rubio, Mercedes, Milne, Fiona C, MacKintosh, Carol
• Format: Article
• Language: English
• Subjects: 14-3-3 Proteins; Amino Acids - pharmacology; Animals; Carrier Proteins - drug effects; Carrier Proteins - metabolism; Cell Extracts; Chromones - pharmacology; Cycloheximide - pharmacology; Enzyme Inhibitors - pharmacology; Epidermal Growth Factor - pharmacology; Glucose - pharmacology; Growth Substances - pharmacology; Leupeptins - pharmacology; Morpholines - pharmacology; Nerve Growth Factor - pharmacology; PC12 Cells - drug effects; PC12 Cells - metabolism; Phosphoproteins - drug effects; Phosphoproteins - metabolism; Phosphorylation; Protein-Serine-Threonine Kinases; Proteins - drug effects; Proteins - metabolism; Proto-Oncogene Proteins - antagonists & inhibitors; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; Rats; Ribosomal Protein S6 Kinases, 70-kDa - drug effects; Ribosomal Protein S6 Kinases, 70-kDa - metabolism; Tyrosine 3-Monooxygenase - drug effects; Tyrosine 3-Monooxygenase - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20020838

Unstimulated PC12 pheochromocytoma cells contain many proteins that bound to 14-3-3s in competition with a 14-3-3-binding peptide. Additional proteins, including one of 39 kDa (p39), became capable of binding to 14-3-3s in phosphatidylinositol 3-kinase-dependent responses to epidermal growth factor or nerve growth factor in vivo. The growth factor regulation was unaffected by inhibitors of the mitogen- or stress-activated protein kinase pathways, or by glucose starvation, but was blocked by amino acid starvation and only partially blocked by rapamycin. p39 in extracts of unstimulated, nutrient-fed cells, but not nutrient-starved cells, was able to bind to 14-3-3s after phosphorylation by protein kinase B (PKB) in vitro. Nutrient starvation did not affect the growth factor-stimulated activation of PKB in vivo. Either cycloheximide (CHX) or the cysteine protease inhibitor, MG132, restored the responsiveness of p39 to growth factors in nutrient-starved cells. In contrast, MG132 could not replace amino acids in supporting the growth factor-stimulated phosphorylation of two downstream targets of mTOR (mammalian target of rapamycin), namely eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and p70 S6 kinase. CHX permitted complete growth factor-stimulated phosphorylation of both 4E-BP1 and p70 S6 kinase in nutrient- starved cells; however, unlike p39, phosphorylation of these proteins was blocked by rapamycin. These findings implicate PKB (or an enzyme with similar specificity) in the growth factor-triggered phosphorylation of p39. In addition, amino acid starvation induces a CHX- and MG132-sensitive pathway that targets p39 and appears to be distinct from the mechanism of regulation of 4E-BP1 and p70 S6 kinase.