scholarly journals Biphasic regulation of extracellular-signal-regulated protein kinase by leptin in macrophages: role in regulating STAT3 Ser727 phosphorylation and DNA binding

2002 ◽  
Vol 364 (3) ◽  
pp. 875-879 ◽  
Author(s):  
Lisa O'ROURKE ◽  
Peter R. SHEPHERD
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Leptin Receptor ◽  
Binding Activity ◽  
Mitogen Activated Protein Kinase ◽  
Time Points ◽  
Dna Binding Activity ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Stat3 Phosphorylation

Activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) requires dimerization that is induced by phosphorylation of Tyr705, but its activity can be further modulated by phosphorylation at Ser727 in a manner that is dependent on cell context and the stimulus used. The role of STAT3 Ser727 phosphorylation in leptin signalling is currently not known. While cells transfected with the signalling-competent long form of the leptin receptor (ObRb) have been used to study leptin signalling, these are likely to be of limited use in studying STAT3 Ser727 phosphorylation due to the importance of cell background in determining the nature of the response. However, we have recently found that J774.2 macrophages endogenously express high levels of ObRb, and using these cells we find that leptin stimulates STAT3 phosphorylation on both Tyr705 and Ser727. The phosphorylation of Ser727 was not affected by rapamycin or the protein kinase C inhibitor H7 [1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride]. While the MEK-1 [mitogen-activated protein kinase (MAP kinase)/extracellular-signal-related kinase (ERK) kinase-1] inhibitor PD98059 [(2-amino-3′-methoxyphenyl)oxanaphthalen-4-one] had no effect on leptin-stimulated phosphorylation of STAT3 Tyr705, it greatly attenuated leptin's effects on STAT3 Ser727 phosphorylation. Further, Ob's effect on the DNA binding activity of STAT3 was also greatly reduced at all time points by PD98059. Leptin-induced ERK activation in J774.2 cells shows a biphasic pattern, with an initial reduction in ERK phosphorylation for up to 10min following leptin stimulation, while at later time points phosphorylation of ERK was increased above basal levels. The increase in ERK activity corresponded with an increase in both phosphorylation of Ser727 and STAT3 DNA binding activity. These data provide the first evidence that ERK-mediated phosphorylation of Ser727 is required for full stimulation of STAT3 by leptin.

scholarly journals Tobacco Transcription Factor WRKY1 Is Phosphorylated by the MAP Kinase SIPK and Mediates HR-Like Cell Death in Tobacco

2005 ◽  
Vol 18 (10) ◽  
pp. 1027-1034 ◽  
Author(s):  
Frank L. H. Menke ◽  
Hong-Gu Kang ◽  
Zhixiang Chen ◽  
Jeong Mee Park ◽  
Dhirendra Kumar ◽  
...  
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Dna Binding ◽  
Kinase Inhibitor ◽  
Hybrid Approach ◽  
Binding Activity ◽  
Mitogen Activated Protein Kinase ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

The salicylic acid-induced protein kinase (SIPK) of tobacco, which is a mitogen-activated protein kinase (MAPK), is activated by various biotic and abiotic treatments. Overexpression of SIPK has been shown to trigger cell death. In this study, a targeted yeast two-hybrid approach identified the tobacco transcription factor WRKY1 as a potential substrate. SIPK phosphorylated WRKY1, which resulted in enhanced DNA-binding activity of WRKY1 to its cognate binding site, a W box sequence from the tobacco chitinase gene CHN50. SIPK-mediated enhancement of WRKY1 DNA-binding activity was inhibited by staurosporine, a general kinase inhibitor. Co-expression of SIPK and WRKY1 in Nicotiana benthamiana led to more rapid cell death than expression of SIPK alone, suggesting that WRKY1 is involved in the formation of hypersensitive response-like cell death and may be a component of the signaling cascade downstream of SIPK.

Constitutive activation of STAT-3 and downregulation of SOCS-3 expression induced by adrenalectomy

2001 ◽  
Vol 281 (6) ◽  
pp. R2048-R2058 ◽  
Author(s):  
Abram M. Madiehe ◽  
Ling Lin ◽  
Christy White ◽  
H. Doug Braymer ◽  
George A. Bray ◽  
...  
Keyword(s):  
Dna Binding ◽  
Signaling Pathway ◽  
Leptin Receptor ◽  
Binding Activity ◽  
Janus Kinase ◽  
Adrenal Steroids ◽  
Western Blots ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Stat Signaling

Removal of adrenal steroids by adrenalectomy (ADX) slows or reverses the development of many forms of obesity in rodents, including those that are leptin or leptin receptor deficient. Obesity is associated with hyperleptinemia and leptin resistance. We hypothesized that glucocorticoids impair leptin receptor signaling and that removal thereof would activate the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathway. The inhibitory effect of leptin (2.5 μg icv) on food intake was enhanced in ADX rats. A combination of ribonuclease protection assays, RT-PCR, Western blots, and mobility shift assays was used to evaluate the leptin signaling pathway in whole hypothalami from sham-operated, ADX and corticosterone-replaced ADX (ADX-R) Sprague-Dawley rats that were treated acutely with either saline vehicle or leptin intracerebroventricularly. ADX increased the expression of leptin receptor mRNA, increased STAT-3 mRNA and protein levels, induced constitutive STAT-3 phosphorylation and DNA binding activity, and also reduced suppressor of cytokine signaling-3 (SOCS-3) mRNA and protein levels. ADX and leptin treatment increased STAT-3 phosphorylation, but with no concomitant increase in DNA binding activity. Leptin and ADX decreased NPY mRNA expression, but their combination did not further decrease NPY mRNA. Corticosterone supplementation of ADX rats partially reversed many of these effects. In conclusion, ADX through activation of STAT-3 and inhibition of SOCS-3 activates the JAK-STAT signaling pathway. These effects most probably explain the ability to prevent the development of obesity by removal of adrenal steroids.

scholarly journals Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

1992 ◽  
Vol 12 (10) ◽  
pp. 4694-4705 ◽  
Author(s):  
S J Baker ◽  
T K Kerppola ◽  
D Luk ◽  
M T Vandenberg ◽  
D R Marshak ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Glycogen Synthase ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Serum Stimulation ◽  
Dependent Protein

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

scholarly journals Signaling mechanisms regulating bombesin-mediated AP-1 gene induction in the human gastric cancer SIIA

2000 ◽  
Vol 279 (2) ◽  
pp. C326-C334 ◽  
Author(s):  
Hong Jin Kim ◽  
B. Mark Evers ◽  
David A. Litvak ◽  
Mark R. Hellmich ◽  
Courtney M. Townsend
Keyword(s):  
Gene Expression ◽  
Gastric Cancer ◽  
Protein Kinase ◽  
Dna Binding ◽  
Signaling Pathways ◽  
Binding Activity ◽  
Human Gastric Cancer ◽  
Rapid Induction ◽  
Dna Binding Activity ◽  
Jun B

The hormone bombesin (BBS) and its mammalian equivalent gastrin-releasing peptide (GRP) act through specific GRP receptors (GRP-R) to affect multiple cellular functions in the gastrointestinal tract; the intracellular signaling pathways leading to these effects are not clearly defined. Previously, we demonstrated that the human gastric cancer SIIA possesses GRP-R and that BBS stimulates activator protein-1 (AP-1) gene expression. The purpose of our present study was to determine the signaling pathways leading to AP-1 induction in SIIA cells. A rapid induction of c- jun and jun-B gene expression was noted after BBS treatment; this effect was blocked by specific GRP-R antagonists, indicating that BBS is acting through the GRP-R. The signaling pathways leading to increased AP-1 gene expression were delineated using phorbol 12-myristate 13-acetate (PMA), which stimulates protein kinase C (PKC)-dependent pathways, by forskolin (FSK), which stimulates protein kinase A (PKA)-dependent pathways, and by the use of various protein kinase inhibitors. Treatment with PMA stimulated AP-1 gene expression and DNA binding activity similar to the effects noted with BBS; FSK stimulated jun-B expression but produced only minimal increases of c- jun mRNA and AP-1 binding activity. Pretreatment of SIIA cells with either H-7 or H-8 (primarily PKC inhibitors) inhibited the induction of c- jun and jun-B mRNAs in response to BBS, whereas H-89 (PKA inhibitor) exhibited only minimal effects. Pretreatment with tyrphostin-25, a protein tyrosine kinase (PTK) inhibitor, attenuated the BBS-mediated induction of c- jun and jun-B, but the effect was not as pronounced as with H-7. Collectively, our results demonstrate that BBS acts through its receptor to produce a rapid induction of both c- jun and jun-B mRNA and AP-1 DNA binding activity in the SIIA human gastric cancer. Moreover, this induction of AP-1, in response to BBS, is mediated through both PKC- and PTK-dependent signal transduction pathways with only minimal involvement of PKA.

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