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

TY - JOUR

T1 - Regulation of the 14-3-3-binding protein p39 by growth factors and nutrients in rat PC12 pheochromocytoma cells

AU - Harthill, Jean E.

AU - Pozuelo-Rubio, Mercedes

AU - Milne, Fiona C.

AU - MacKintosh, Carol

PY - 2002/12/1

Y1 - 2002/12/1

N2 - 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 fact or-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 nutrients 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.

AB - 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 fact or-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 nutrients 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.

U2 - 10.1042/BJ20020838

DO - 10.1042/BJ20020838

M3 - Article

SN - 0264-6021

VL - 368

SP - 565

EP - 572

JO - Biochemical Journal

JF - Biochemical Journal

ER -