Regulation of the 14-3-3-binding protein p39 by growth factors and nutrients in rat PC12 pheochromocytoma cells.
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.
Harthill
Jean E
JE
Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Pozuelo Rubio
Mercedes
M
Milne
Fiona C
FC
MacKintosh
Carol
C
D048948:14-3-3 Proteins; D000596:Amino Acids / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D002457:Cell Extracts; D002867:Chromones / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D020932:Nerve Growth Factor / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000187:drug effects / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000187:drug effects / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000187:drug effects / Q000378:metabolism*
D048948:14-3-3 Proteins; D000596:Amino Acids; D002352:Carrier Proteins; D002457:Cell Extracts; D002867:Chromones; C499526:Eif4ebp1 protein, rat; D004791:Enzyme Inhibitors; D006133:Growth Substances; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D009025:Morpholines; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004815:Epidermal Growth Factor; D020932:Nerve Growth Factor; D003513:Cycloheximide; D014446:Tyrosine 3-Monooxygenase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D005947:Glucose; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde