Growth factor-stimulated MAP kinase induces rapid retrophosphorylation and inhibition of MAP kinase kinase (MEK1)

It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)–dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3–dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control. © 1998 by The American Society of Hematology.

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Cytochrome c Induces Caspase-Dependent Apoptosis in Intact Hematopoietic Cells and Overrides Apoptosis Suppression Mediated by bcl-2, Growth Factor Signaling, MAP-Kinase-Kinase, and Malignant Change

Blood ◽

10.1182/blood.v92.4.1235 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1235-1246 ◽

Cited By ~ 43

Author(s):

John M. Garland ◽

Claudius Rudin

Keyword(s):

Growth Factor ◽

Cytochrome C ◽

Map Kinase ◽

Leukemic Cell ◽

Growth Factor Signaling ◽

Intact Cells ◽

Cell Extracts ◽

American Society ◽

Induced Apoptosis ◽

Map Kinase Kinase

Abstract It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)–dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3–dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control. © 1998 by The American Society of Hematology.

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MAP kinase kinase kinase 8 (MAP3K8; COT; TPL2); c-Met proto-oncogene (MET; HGFR); hepatocyte growth factor/scatter factor (HGF/SF)

Science-Business eXchange ◽

10.1038/scibx.2012.1107 ◽

2012 ◽

Vol 5 (42) ◽

pp. 1107-1107

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Map Kinase ◽

Scatter Factor ◽

Hepatocyte Growth ◽

Map Kinase Kinase Kinase ◽

Map Kinase Kinase

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The threonine residues in MAP kinase kinase 1 phosphorylated by MAP kinase in vitro are also phosphorylated in nerve growth factor-stimulated rat phaeochromocytoma (PC12) cells

FEBS Letters ◽

10.1016/0014-5793(94)80252-1 ◽

1994 ◽

Vol 341 (1) ◽

pp. 119-124 ◽

Cited By ~ 29

Author(s):

Yuji Saito ◽

Nestor Gomez ◽

David G. Campbell ◽

Alan Ashworth ◽

Chris J. Marshall ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Map Kinase ◽

Pc12 Cells ◽

Nerve Growth ◽

Map Kinase Kinase

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Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts

The Journal of Cell Biology ◽

10.1083/jcb.122.5.1079 ◽

1993 ◽

Vol 122 (5) ◽

pp. 1079-1088 ◽

Cited By ~ 456

Author(s):

P Lenormand ◽

C Sardet ◽

G Pagès ◽

G L'Allemain ◽

A Brunet ◽

...

Keyword(s):

Growth Factor ◽

Map Kinase ◽

Protein Kinases ◽

Map Kinases ◽

Phorbol Esters ◽

Nuclear Translocation ◽

Polyclonal Antibodies ◽

Resting Cells ◽

Restriction Point ◽

Map Kinase Kinase

Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.

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