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

2002 ◽  
Vol 368 (2) ◽  
pp. 565-572 ◽  
Author(s):  
Jean E. HARTHILL ◽  
Mercedes POZUELO RUBIO ◽  
Fiona C. MILNE ◽  
Carol MacKINTOSH
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Binding Protein ◽  
Initiation Factor ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Pheochromocytoma Cells ◽  
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 39kDa (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 acidstarvation 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.

scholarly journals p70 S6 Kinase Is Regulated by Protein Kinase Cζ and Participates in a Phosphoinositide 3-Kinase-Regulated Signalling Complex

1999 ◽  
Vol 19 (4) ◽  
pp. 2921-2928 ◽  
Author(s):  
Angela Romanelli ◽  
Kathleen A. Martin ◽  
Alex Toker ◽  
John Blenis
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Independent Manner ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Dependent Signal ◽  
Catalytic Loop ◽  
Phosphoinositide 3 Kinase ◽  
P70s6k Activation

ABSTRACT p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCζ as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCζ mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCζ mutant (myristoylated PKCζ [myr-PKCζ]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCζ. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCζ in vivo in a growth factor-independent manner, while PDK-1 and PKCζ can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.

scholarly journals Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin

2000 ◽  
Vol 348 (2) ◽  
pp. 351-358 ◽  
Author(s):  
Katia COULONVAL ◽  
Fabrice VANDEPUT ◽  
Rob C. STEIN ◽  
Sara C. KOZMA ◽  
Françoise LAMY ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Protein Kinase B ◽  
Phosphatidylinositol 3 Kinase ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

The proliferation of most normal cells depends on the co-operation of several growth factors and hormones, each with a specific role, but the key events involved in the action of each necessary stimulant remain largely uncharacterized. In the present study, the pathways involved in the mechanism(s) of co-operation have been investigated in primary cultures of dog thyroid epithelial cells. In this physiologically relevant system, thyroid stimulating hormone (TSH) acting through cAMP, epidermal growth factor (EGF) and phorbol esters (such as PMA) induce DNA synthesis. Their effect requires stimulation of the insulin-like growth factor-1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents. In contrast, hepatocyte growth factor (HGF) is itself fully mitogenic. The results of the study demonstrate that cAMP, EGF, HGF and PMA stimulate p70 ribosomal S6 kinase (p70 S6 kinase). However, insulin/IGF-1 also stimulate p70 S6 kinase. Thus stimulation of p70 S6 kinase might be necessary, but is certainly not sufficient, for the induction of DNA synthesis and is not specific for any stimulated pathway. In contrast, phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) activation by insulin and HGF is strong and sustained, whereas it is weak and transient with EGF and absent in the presence of TSH or PMA. These findings suggest that: (i) stimulation of PI 3-kinases and/or PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, or in the action of PMA, (ii) the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF-1 in the proliferation of these cells, and (iii) the stimulation of this pathway by HGF may explain why this agent does not require insulin or IGF-1 for its mitogenic action.

scholarly journals 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro

1998 ◽  
Vol 8 (2) ◽  
pp. 69-81 ◽  
Author(s):  
Dario R. Alessi ◽  
Mark T. Kozlowski ◽  
Qing-Ping Weng ◽  
Nick Morrice ◽  
Joseph Avruch
Keyword(s):  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Dependent Protein

scholarly journals p70 S6 kinase negatively regulates fibroblast growth factor 2-stimulated interleukin-6 synthesis in osteoblasts: function at a point downstream from protein kinase C

2008 ◽  
Vol 197 (1) ◽  
pp. 131-137 ◽  
Author(s):  
S Takai ◽  
Y Hanai ◽  
R Matsushima-Nishiwaki ◽  
C Minamitani ◽  
T Otsuka ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Fibroblast Growth Factor ◽  
Interleukin 6 ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Kinase C

We have previously reported that protein kinase C negatively regulates basic fibroblast growth factor (FGF-2)-stimulated synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. To further clarify the mechanism underlying the synthesis of IL-6 in osteoblasts, we investigated whether p70 S6 kinase is involved in the FGF-2-stimulated IL-6 synthesis in these cells. Rapamycin, an inhibitor of p70 S6 kinase, significantly enhanced the FGF-2-stimulated IL-6 synthesis in a dose-dependent manner. Downregulation of p70 S6 kinase by siRNA markedly amplified the FGF-2-stimulated IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a direct activator of protein kinase C, induced the phosphorylation of p70 S6 kinase. Go6976 and bisindolylmaleimide I, inhibitors of protein kinase C, suppressed the TPA-stimulated phosphorylation of p70 S6 kinase. Additionally, protein kinase C inhibitors markedly reduced the phosphorylation of p70 S6 kinase induced by FGF-2. These results strongly suggest that p70 S6 kinase functions at a point downstream of protein kinase C and limits the FGF-2-stimulated IL-6 synthesis in osteoblasts.

Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells

1998 ◽  
Vol 251 (1-2) ◽  
pp. 195-200 ◽  
Author(s):  
Mirjana Andjelkovic ◽  
Hana S. Suidan ◽  
Roger Meier ◽  
Matthias Frech ◽  
Dario R. Alessi ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Nerve Growth ◽  
Pheochromocytoma Cells ◽  
Alpha Beta ◽  
Pc12 Pheochromocytoma Cells

scholarly journals Protein Kinase B Activation and Lamellipodium Formation Are Independent Phosphoinositide 3-Kinase-Mediated Events Differentially Regulated by Endogenous Ras

1998 ◽  
Vol 18 (4) ◽  
pp. 1802-1811 ◽  
Author(s):  
David H. J. van Weering ◽  
Johan de Rooij ◽  
Barbara Marte ◽  
Julian Downward ◽  
Johannes L. Bos ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Egf Receptor ◽  
Protein Kinase B ◽  
Ectopic Expression ◽  
Differential Sensitivity ◽  
Phosphorylated Proteins ◽  
Phosphoinositide 3 Kinase

ABSTRACT Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.

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