We previously reported that long term treatment with insulin led to
sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells
overexpressing insulin receptors. Here we investigated the ...signaling
molecules involved in insulin inhibition of JNKs, focusing on
phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein
kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of
JNK inhibition for insulin-mediated proliferation and survival. Insulin
inhibition of JNKs was mediated by PI 3-K, as it was blocked by
wortmannin and LY294002 and required the de novo
synthesis of a phosphatase(s), as it was abolished by orthovanadate and
actinomycin D. MKP-1 was a good candidate because 1) insulin
stimulation of MKP-1 expression correlated with insulin inhibition of
JNKs; 2) insulin stimulation of MKP-1 expression, like insulin
inhibition of JNKs, was mediated by PI 3-K; and 3) the transient
expression of an antisense MKP-1 RNA reduced the insulin inhibitory
effect on JNKs. The overexpression of a dominant negative JNK1 mutant
increased insulin stimulation of DNA synthesis and mimicked the
protective effect of insulin against serum withdrawal-induced
apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA
reduced the proliferative and/or antiapoptotic responses to insulin.
Altogether, these results demonstrate that insulin inhibits JNKs
through a PI 3-K- and MKP-1-dependent pathway and provide evidence for
a key role for JNK inhibition in insulin regulation of proliferation
and survival.
Transforming growth factor β (TGF-β) is a multifunctional factor that induces a wide variety of cellular processes which affect
growth and differentiation. TGF-β exerts its effects through a ...heteromeric complex between two transmembrane serine/threonine
kinase receptors, the type I and type II receptors. However, the intracellular signaling pathways through which TGF-β receptors
act to generate cellular responses remain largely undefined. Here, we report that TGF-β initiates a signaling cascade leading
to stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation. Expression of dominant-interfering forms
of various components of the SAPK/JNK signaling pathways including Rho-like GTPases, mitogen-activated protein kinase (MAPK)
kinase kinase 1 (MEKK1), MAPK kinase 4 (MKK4), SAPK/JNK, and c-Jun abolishes TGF-β-mediated signaling. Therefore, the SAPK/JNK
activation contributes to TGF-β signaling.
Protein tyrosine kinases (PTKs) are key enzymes implicated in signal transduction pathways regulated by growth factors (GFs). We have previously shown by immunohistochemistry that the level of ...phosphotyrosine (pY) proteins is increased in prostatic basal epithelial cells following estrogen treatment in castrated dogs. In this study, we investigated if this treatment increases the level and distribution of prostatic PTK activity, and more specifically, if it alters the expression and/or activity of the Src family members p60
src and p53/56
lyn. Prostates from normal and hyperplastic dog prostates, as well as those from castrated dogs treated with androgens, were also examined. Only the glands obtained from estrogen-treated dogs had a significantly increased total and specific PTK activity, observed uniquely in the particulate extract, as compared to the other types of prostates studied. In addition, this increased activity was correlated upon gel filtration chromatography with the presence of an additional peak of activity with an apparent molecular weight of 130 kDa, which was absent in other prostate fractions presenting only 50 kDa peaks. Using antibodies, we demonstrate that active p60
src and p53/56
lyn kinases accounted for 81% of the activity in this 130 kDa peak. On the other hand, in situ renaturation also revealed the presence of still uncharacterized 50/55 kDa PTKs in the 130 kDa peak. Altogether, these findings raise the possibility that these PTKs contribute to the transmission of mitogenic signals originating directly or indirectly from estrogen stimulation of the basal cell layer of the prostate.
We recently showed that the antiapoptotic function of insulin requires nuclear factor κB (NF-κB) activation (Bertrand, F.,
Atfi, A., Cadoret, A., L'Allemain, G., Robin, H., Lascols, O., Capeau, J., ...and Cherqui, G. (1998) J. Biol. Chem. 273, 2931â2938). Here we sought to identify the NF-κB-dependent survival genes that are activated by insulin to mediate this
function. Insulin increased the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2) mRNA and protein
in Chinese hamster ovary cells overexpressing insulin receptors (IRs). This effect required (i) IR activation since it was
abrogated by IR mutation at tyrosines 1162 and 1163 and (ii) NF-κB activation since it was abolished by overexpression of
dominant-negative IκB-α(A32/36) and mimicked by overexpression of the NF-κB c-Rel subunit. TRAF2 contributed to insulin protection
against serum withdrawal-induced apoptosis since TRAF2 overexpression mimicked insulin protection, whereas overexpression
of dominant-negative TRAF2-(87â501) reduced this process. Along with its protective effect, overexpressed TRAF2 increased
basal and insulin-stimulated NF-κB activities. All effects were inhibited by IκB-α(A32/36), suggesting that an amplification
loop involving TRAF2 activation of NF-κB is implicated in insulin antiapoptotic signaling. We also show that insulin increased
manganese-superoxide dismutase (Mn-SOD) mRNA expression through NF-κB activation and that Mn-SOD contributed to insulin antiapoptotic
signaling since expression of antisense Mn-SOD RNA decreased this process. This study provides the first evidence that insulin
activates the NF-κB-dependent survival genes encoding TRAF2 and Mn-SOD and thereby clarifies the role of NF-κB in the antiapoptotic
function of insulin.
Interleukin 3 (IL-3) and other hematopoietic cytokines transduce signals that stimulate DNA synthesis and cell survival. In certain chronic myelomonocytic leukemias, a TEL/platelet-derived growth ...factor receptor beta (PDGFRbeta) fusion protein is produced as a consequence of the t(5;12) translocation. It contains the amino terminus of the transcription factor TEL fused to the transmembranous and cytoplasmic domains of the PDGFRbeta. It is oncogenic as it substitutes for IL-3, thus promoting cell growth and preventing apoptotic cell death. The mechanism by which TEL/PDGFRbeta generates survival signals remains undefined. Here, we report that both IL-3 and TEL/PDGFRbeta initiate a signaling cascade that leads to the activation of the transcriptional factor NF-kappaB. In fact, either cytokine deprivation of IL-3-dependent Ba/F3 cells or exposure of TEL/PDGFRbeta-expressing cells to the specific inhibitor of the PDGFR tyrosine kinase, CGP53716, caused a strong decrease in NF-kappaB activity followed by extensive cell death. Further, treatment with the proteasome inhibitor Z-IE(O-t-Bu)A-leucinal suppressed IL-3 and TEL/PDGFRbeta-dependent survival. The same result was seen upon overexpression of an unphosphorylable form of IkappaBalpha. Because both conditions inactivate NF-kappaB by preventing its translocation into the nucleus, that process seems to be essential for cell survival in response to IL-3 and TEL/PDGFRbeta. Moreover, overexpression of a dominant-negative mutant of the protooncogene c-Myc, a downstream target of NF-kappaB, had a similar effect. We conclude that NF-kappaB plays an important role in maintaining cell survival in response to IL-3 and TEL/PDGFRbeta and that c-Myc may be a downstream effector mediating this effect.
Protein tyrosine kinases (PTKs) are key enzymes implicated in signal transduction pathways regulated by growth factors (GFs). We have previously shown by immunohistochemistry that the level of ...phosphotyrosine (pY) proteins is increased in prostatic basal epithelial cells following estrogen treatment in castrated dogs. In this study, we investigated if this treatment increases the level and distribution of prostatic PTK activity, and more specifically, if it alters the expression and/or activity of the Src family members p60src and p53/56lyn. Prostates from normal and hyperplastic dog prostates, as well as those from castrated dogs treated with androgens, were also examined. Only the glands obtained from estrogen-treated dogs had a significantly increased total and specific PTK activity, observed uniquely in the particulate extract, as compared to the other types of prostates studied. In addition, this increased activity was correlated upon gel filtration chromatography with the presence of an additional peak of activity with an apparent molecular weight of 130 kDa, which was absent in other prostate fractions presenting only 50 kDa peaks. Using antibodies, we demonstrate that active p60src and pp53/56lyn kinases accounted for 81% of the activity in this 130 kDa peak. On the other hand, in situ renaturation also revealed the presence of still uncharacterized 50/55 kDa PTKs in the 130 kDa peak. Altogether, these findings raise the possibility that these PTKs contribute to the transmission of mitogenic signals originating directly or indirectly from estrogen stimulation of the basal cell layer of the prostate.
We previously reported that insulin activates nuclear factor κB (NF-κB) in Chinese hamster ovary (CHO)-R cells overexpressing
wild-type insulin receptors (IRs) through a pathway requiring IR ...tyrosine kinase and Raf-1 kinase activities. We now investigated
whether the activation of NF-κB by insulin could serve an antiapoptotic function. Insulin (10 â9 -10 â7 m ) inhibited apoptosis induced by serum withdrawal in CHO-R cells in a concentration-dependent manner. Insulin antiapoptotic
signaling: (i) was dependent on IR number and IR tyrosine kinase activity since it was reduced in parental CHO cells and was
abolished in CHO-Y2 cells overexpressing IRs mutated at Tyr 1162/1163 ; (ii) was, like insulin activation of NF-κB, dependent on Raf-1 but not on mitogen-activated protein kinase activity since
both processes were decreased by the dominant-negative Raf-1 mutant Raf-C4 whereas they persisted in mitogen-activated protein
kinase-depleted cells; and (iii) required NF-κB activation since it was decreased by proteasome inhibitors and the dominant-negative
IκB-α (A32/36) mutant and was mimicked by overexpression of the NF-κB c-Rel subunit. We also show that insulin antiapoptotic
signaling but not insulin activation of NF-κB involved phosphatidylinositol 3-kinase (PI 3-kinase), as supported by the inhibition
of the former but not of the latter process by the PI 3-kinase inhibitor LY294002. Inhibition of both NF-κB and PI 3-kinase
totally abolished insulin antiapoptotic signaling. Thus insulin exerts a specific antiapoptotic function which is dependent
on IR tyrosine kinase activity and is mediated by both a Raf-1-dependent pathway that leads to NF-κB activation and a PI 3-kinase-dependent
pathway.
Because protein tyrosine kinases play a crucial role in the regulation of cell division and carcinogenesis, we have herein measured such enzyme activities (specific activity and subcellular ...distribution) and compared their characteristics with respect to hydrodynamic properties and radiation inactivation sizes as well as renaturation after electrophoresis in denaturing conditions in canine prostatic epithelial cells either in a resting (freshly isolated) or in a dividing (cultured cells) state. In quiescent cells, most protein tyrosine kinase activity was expressed by soluble proteins with a Stokes' radius (Rs) of 3.05 nm, a sedimentation coefficient (S20,w) of 4.0 S, and a molecular mass of 50 kDa. By contrast, in dividing cells (three days in primary culture), the specific activity was higher and the enzyme was mainly membrane bound. The use of a detergent (Triton X-100) allowed the extraction of most of that enzyme; its partial specific volume, S20,w and Rs were then 0.883 cm3/g, 4.0 S, and 5.6 nm, respectively, hence yielding a molecular mass of 215 kDa, which decreased to 125-145 kDa when corrected for detergent binding. Probing these chromatography-peak fractions, 50 kDa from cytosol of resting cells and 215 kDa from membrane extracts of dividing cells, with a phosphotyrosine antibody following their incubation with ATP and electrophoresis in denaturing conditions revealed the presence of a common 50-kDa phosphotyrosylated protein along with three other bands (130, 75, and 40 kDa) in the high-Mr peak of enzyme. However, the radiation inactivation size for protein tyrosine kinases expressed in both resting and dividing cells were similar, 47.2 +/- 8.7 and 44.5 +/- 6.1 kDa, respectively. Furthermore, by renaturation after electrophoresis in denaturing conditions, major protein tyrosine kinase polypeptides of 50 kDa were identified in both cell populations. Taken together, these results indicate that, in dividing prostatic epithelial cells, membrane-bound protein tyrosine kinases of low molecular weight with properties similar to those of monomeric soluble forms present in quiescent cells are part of high-molecular weight complexes. This activation process may be critical for hormone-independent proliferation of prostatic epithelial cells.
Transforming growth factor beta (TGF-β) is a multifunctional cytokine that controls a wide variety of cellular processes, such as cell migration, adhesion, differentiation, proliferation, and cell ...death. Disruption of components of the TGF-β signaling pathway is associated with human diseases, including cancer. TGF-β initiates signaling from the cell surface by contacting two distantly related transmembrane serine/threonine kinases called receptors I (TβRI) and II (TβRII). Upon ligand binding, the constitutively active TβRII phosphorylates and activates TβRI, which in turn phosphorylates Smad2 or Smad3 on two serines at the carboxyl terminus, within a highly conserved SSXS motif. Following phosphorylation, Smad2 and Smad3 associate with the shared partner Smad4 and translocate to the nucleus where Smad complexes, in cooperation with coactivators and corepressors, participate in transcriptional regulation of TGF-β-responsive genes. Smad phosphorylation, cellular distribution, and activation are tightly regulated via crosstalk with other signaling pathways or through functional interactions with Smad partners and modulators that determine the specific target genes. The present knowledge of the mechanisms controlling phosphorylation and activation of Smad proteins is reviewed here.
La casomorphine1-7 et la casomorphine1-4 (morphiceptine) stimulent in vivo l'activité de la thymidine kinase de la prostate ventrale du rat adulte. Ajoutées au milieu de culture de cellules de ...prostate ventrale de rat immature, ces deux casomorphines stimulent la multiplication des cellules prostatiques. Dans les deux types d'expériences, la morphiceptine est plus active que le casomorphine1-7. Ces résultats démontrent que les casomorphines du lait exercent des propriétés autres que les effets morphinomimétiques qui leur ont été attribués initialement.
Thymidine kinase activity of the ventral prostate from adult rats could be stimulated by administration to the animal of casomorphin1-7 or casomorphin1-4 (morphiceptin). On the other hand addition of both drugs to the culture medium of prostatic cells from immature rats enhanced the cells growth. In both cases, morphiceptin was more efficient than casomorphin)1-7. These results suggest than casomorphins have other properties than their morphinomimetic effects previously described.
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