Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.

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Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6241 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6241-6252 ◽

Cited By ~ 214

Author(s):

M L Samuels ◽

M J Weber ◽

J M Bishop ◽

M McMahon

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Map Kinases ◽

Growth Media ◽

Hormone Binding ◽

Map Kinase Cascade ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Hormone Binding Domain ◽

Map Kinase Kinase

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

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The Mitogen-activated Protein (MAP) Kinase p38 and Its Upstream Activator MAP Kinase Kinase 6 Are Involved in the Activation of Signal Transducer and Activator of Transcription by Hyperosmolarity

Journal of Biological Chemistry ◽

10.1074/jbc.274.42.30222 ◽

1999 ◽

Vol 274 (42) ◽

pp. 30222-30227 ◽

Cited By ~ 79

Author(s):

Johannes G. Bode ◽

Petros Gatsios ◽

Stephan Ludwig ◽

Ulf R. Rapp ◽

Dieter Häussinger ◽

...

Keyword(s):

Map Kinase ◽

Signal Transducer ◽

Protein Map ◽

Mitogen Activated Protein ◽

Map Kinase Kinase

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Mitogen-Activated Protein (MAP) Kinase Phosphorylation of MAP Kinase Kinase: Determination of Phosphorylation Sites by Mass Spectrometry and Site-Directed Mutagenesis1

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a124524 ◽

1994 ◽

Vol 116 (2) ◽

pp. 304-314 ◽

Cited By ~ 68

Author(s):

Sam J. Mansour ◽

Katheryn A. Resing ◽

Julian M. Candi ◽

April S. Hermann ◽

Jason W. Gloor ◽

...

Keyword(s):

Mass Spectrometry ◽

Map Kinase ◽

Phosphorylation Sites ◽

Protein Map ◽

Mitogen Activated Protein ◽

Kinase Phosphorylation ◽

Map Kinase Kinase

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A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

The Journal of Cell Biology ◽

10.1083/jcb.137.2.433 ◽

1997 ◽

Vol 137 (2) ◽

pp. 433-443 ◽

Cited By ~ 73

Author(s):

Xiao Min Wang ◽

Ye Zhai ◽

James E. Ferrell

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Map Kinases ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitogen Activated Protein Kinase ◽

Mitotic Progression ◽

Map Kinase Phosphatase ◽

Mitogen Activated Protein ◽

Map Kinase Kinase

The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.

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Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6241-6252.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6241-6252

Author(s):

M L Samuels ◽

M J Weber ◽

J M Bishop ◽

M McMahon

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Map Kinases ◽

Growth Media ◽

Hormone Binding ◽

Map Kinase Cascade ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Hormone Binding Domain ◽

Map Kinase Kinase

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

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