Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Epithelial chloride (Cl-) transport is achieved by the coordinated action of symporters such as the Na+-K+-2Cl- cotransporter (NKCC1) and chloride channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). As a secretory tissue, mammary epithelial cells are obvious candidates for such mechanisms, but Cl- transport and its hormonal regulation have been poorly delineated in mammary epithelial cells. We determined whether the mammary epithelial cell line, HC11, transports chloride and whether this was regulated by PRL, a hormone known to stimulate ion transport. HC11 cells express both CFTR and NKCC1. Exposure to PRL or PGE1 increased Cl- transport in HC11 cells. This was inhibited by the NKCC1 blocker, furosemide, and by the Cl- channel inhibitor, diphenylamine 2-carboxylate. Dose and time course of PRL action indicate that PRL had maximal effect on Cl- transport at 1 microg/ml and at 10 min of stimulation. Examination of the signaling pathways suggests that the PRL effect on Cl- transport does not involve an increase in [Ca2+]i or MAP kinase activity. RT-PCR analyses indicate that HC11 cells express mRNA for Janus kinase 1 (JAK1), JAK2, and signal transducer and activator of transcription 5 (STAT5) but not for JAK3. PRL treatment of HC11 cells increased phosphorylation of STAT5. The JAK2 inhibitor AG490 blocked phosphorylation of STAT5 and PRL-induced, but not PGE1-induced, Cl- transport. NKCC1, but not CFTR, is tyrosine phosphorylated in HC11 cells. PRL enhanced tyrosine phosphorylation of NKCC1, and this effect was attenuated by the JAK2 inhibitor AG490. These results are the first demonstrations of a role for tyrosine phosphorylation of NKCC1 and of the PRL-JAK2 cascade in the regulation of Cl- transport.
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PMID:Janus kinase 2 (JAK2) regulates prolactin-mediated chloride transport in mouse mammary epithelial cells through tyrosine phosphorylation of Na+-K+-2Cl- cotransporter. 1111 34

We investigated in this study the effects of ovine PRL on endocrine functions of cultured murine Leydig tumor cells (mLTC-1). The parameters studied were the activation of signal transduction systems involving cAMP and intracellular free Ca(2+), the expression of Janus kinase 2 (JAK2), expression and function of LH and PRL receptors (R), expression of the steroidogenic acute regulatory (StAR) protein, and stimulation of steroidogenesis. Very similar biphasic dose- and time-dependent responses of all the parameters studied were found upon PRL stimulation, comprising a fast inhibition within 24 h in response to high PRL doses (>/=30 microgram/liter), and a slow stimulation, between 48-72 h, in response to lower PRL doses (1-10 microgram/liter). In addition, extracellular Ca(2+) (1.5 mmol/liter) increased the effect of PRL on human CG (hCG)-stimulated StAR messenger RNA expression and progesterone (P) production. Importantly, the biphasic effects of PRL on LHR gene expression and hCG-mediated P production were abolished in the presence of anti-PRL antiserum, demonstrating specificity of PRL action. The PRL effects on StAR expression, and steroid and cAMP production, apparently reflect its effects on LHR function. The relevance of the PRL effects observed in mLTC-1 cells was supported by demonstration of similar PRL responses in hCG-stimulated testosterone production of isolated mouse Leydig cells. Collectively, these findings clearly demonstrate the biphasic regulatory actions of PRL, and clarify some facets of the controversial role of this hormone in Leydig cell function.
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PMID:Biphasic action of prolactin in the regulation of murine Leydig tumor cell functions. 1114 94

The human GH (hGH) antagonist B2036 combines a single amino acid substitution impairing receptor binding site 2 (G120K) with eight additional amino acid substitutions that improve binding site 1 affinity. B2036 does not bind, activate, or antagonize the human PRL receptor and therefore is suitable to determine cellular effects mediated specifically through the hGH receptor. We have used this hGH receptor specific antagonist in MCF-7 cells stably transfected with either the hGH gene (MCF-hGH) or a translation deficient hGH gene (MCF-MUT) to determine whether the effects of autocrine hGH on mammary carcinoma cell behavior are mediated via the hGH receptor. Enhanced JAK2 tyrosine phosphorylation observed in MCF-hGH cells compared with MCF-MUT cells is abrogated by B2036 as is the autocrine hGH stimulated increase in total cell number and DNA synthesis. Interestingly, autocrine hGH functions as a potent inhibitor of apoptosis induced by serum withdrawal compared with exogenously added hGH, and the protection against apoptosis afforded by autocrine hGH is abrogated by B2036. B2036 also inhibited autocrine hGH stimulated transcriptional activation mediated by either STAT5, CHOP (p38 MAP kinase specific) or Elk-1 (p44/42 MAP kinase specific). Finally, B2036 inhibited the autocrine hGH-dependent enhancement of the rate of mammary carcinoma cell spreading on a collagen matrix. Thus, the effects of autocrine hGH on human mammary carcinoma cell behavior are mediated via the hGH receptor.
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PMID:The effects of autocrine human growth hormone (hGH) on human mammary carcinoma cell behavior are mediated via the hGH receptor. 1115 49

In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from our laboratory has established that PRL stimulates ERalpha expression at the level of transcription and that the transcription factor Stat5 (signal transducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase, Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ERalpha expression was investigated. In primary luteinized granulosa cells, the general tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, AG490, prevented PRL stimulation of ERalpha mRNA levels, suggesting that PRL signaling to the ERalpha gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced Stat5a phosphorylation only and had little or no effect on Stat5b phosphorylation. These effects of AG490 were confirmed in COS cells overexpressing Stat5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ERalpha promoter activity and Stat5b phosphorylation while a constitutively active Jak2 could stimulate both in the absence of PRL. Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocation and DNA binding. Therefore, it seems that in the presence of AG490, Stat5b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest that PRL may activate a second tyrosine kinase, other than Jak2, that is capable of phosphorylating Stat5b without inducing transcriptional activity. To investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ERalpha promoter activity or on PRL-induced Stat5b phosphorylation. However, the combination of AG490 and LY294002 largely prevented PRL-induced Stat5b phosphorylation. These findings indicate that PRL stimulation of ERalpha expression requires Jak2 and also that PRL can induce Stat5b phosphorylation through two tyrosine kinases, Jak2 and one downstream of PI3K. Furthermore, these results suggest that the role of Jak2 in activating Stat5b may be through a mechanism other than simply inducing Stat5b phosphorylation.
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PMID:PRL-induced ERalpha gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase. 1168 25

Inhibition of PRL hormone signaling by suppressor of cytokine signaling (SOCS)/cytokine-inducible SH2-containing protein (CIS) was investigated in transfected HEK 293 cells. We used the physiologically relevant wild-type beta-casein promoter as a target gene for PRL action. We demonstrate that CIS produces a 70% inhibition of PRL signaling by a mechanism distinct from, and downstream of, the effect of SOCS-1 on JAK2. This inhibition involves association with the PRL receptor (PRLR), resulting in the inhibition of signal transducer and activator of transcription 5 (STAT5) activation. Further, we show that SOCS-3 coimmunoprecipitates with the PRLR. These data suggest that SOCS-3 involves a second pathway for the inhibition of PRL signaling other than JAK2 inhibition. Additional results indicate that SOCS-2 can play a more important potentiator role on PRL signaling, resulting in a restoration of 50% of transcriptional inhibition induced by SOCS-3 and a restoration of 100% of transcriptional inhibition induced by CIS. SOCS-2 was able to block the inhibitory effect of SOCS-1. These results indicate that SOCS-2 seems to be an antagonist of the other SOCS. SOCS-1 binds JAK2 and inhibits its phosphorylation; SOCS-3 does not bind JAK2 but binds the PRLR that may mediate its inhibition of JAK2; and finally, CIS binds the PRLR but inhibits signal transducer and activator of transcription 5 rather than JAK2.
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PMID:Cytokine-inducible SH2-containing protein suppresses PRL signaling by binding the PRL receptor. 1171 28

Treatment of HC11 mammary epithelial cells with the lactogenic hormone PRL promotes differentiation and induction of milk protein gene expression via stimulation of the Janus kinase (JAK)/signal transducer and activator of transcription pathway. We have previously shown that autocrine activation of epidermal growth factor (EGF) receptor interferes with normal PRL-induced differentiation. Here we show that PRL activation of JAK2 was dramatically reduced in HC11 cells pretreated with EGF, demonstrating that the target of EGF receptor activation is JAK2 kinase. Using an in-gel protein tyrosine phosphatase (PTP) assay, we observed that the activity of a 125-kDa PTP was up-regulated in HC11 cells in response to EGF. A specific antiserum was used to demonstrate that the 125-kDa PTP was PTP-PEST and to show that EGF treatment of HC11 cells led to an increase in the level of PTP-PEST. In intact HC11 cells, PTP-PEST was constitutively associated with JAK2, and in response to EGF treatment there was an increased level of PTP-PEST in JAK2 complexes. An in vitro phosphatase assay, using PRL-activated JAK2 as the substrate and lysates from HC11 cells as the source of PTP-PEST, revealed that JAK2 could serve as a PTP-PEST substrate. However, in intact cells the regulation of JAK2 by PTP-PEST was complex, since transient overexpression of PTP-PEST had a negligible effect on PRL-induced JAK2 activation. EGF's negative influence on JAK2 activity was blocked by actinomycin D treatment of HC11 cells, suggesting that EGF induced a protein that mediated the effects of PTP-PEST on JAK2. In support of this model, PTP-PEST-containing lysates from EGF-treated HC11 cells dephosphorylated JAK2 to a greater extent than lysates prepared from control cells.
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PMID:The protein tyrosine phosphatase-PEST is implicated in the negative regulation of epidermal growth factor on PRL signaling in mammary epithelial cells. 1173 19

Functional PRL receptors are expressed in the human endometrium during the secretory phase of the menstrual cycle in which PRL stimulates tyrosine phosphorylation of Janus kinase 2 and STAT (signal transducer and activator of transcription) 1 and 5. In this study, we investigated the effect of PRL on the MAPK/ERK pathway in the human endometrium. Human endometrial tissue was collected during the mid to late secretory phase of the menstrual cycle. Western blot analysis performed on proteins, extracted after up to 30 min culture with PRL, demonstrated rapid tyrosine and threonine phosphorylation of ERK 1 and 2 MAPKs. The phosphorylation of ERK, in response to PRL, was localized by immunohistochemistry to glandular epithelial cells and a subset of stromal cells. Using immunofluorescence histochemistry, PRL-induced phosphorylation of ERK in the stromal compartment was localized to the uterine-specific CD56(+) natural killer (NK) cells. We have demonstrated that the PRL receptor is expressed in uterine CD56(+) NK cells in situ by immunofluorescence and in purified decidual CD56(+) NK cells by RT-PCR and Western blotting analysis. We have further demonstrated phosphorylation of ERK 1 and 2 in cultures of purified uterine CD56(+) NK cells, in response to PRL. Our data demonstrate that PRL stimulates the ERK pathway in multiple cellular compartments of the human endometrium and identify uterine CD56(+) NK cells as novel PRL target cells.
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PMID:Prolactin induces ERK phosphorylation in epithelial and CD56(+) natural killer cells of the human endometrium. 1199 84

To characterize the molecular mechanisms by which progesterone withdrawal initiates milk secretion, we examined global gene expression during pregnancy and lactation in mice, focusing on the period around parturition. Trajectory clustering was used to profile the expression of 1358 genes that changed significantly between pregnancy day 12 and lactation day 9. Predominantly downward trajectories included stromal and proteasomal genes and genes for the enzymes of fatty acid degradation. Milk protein gene expression increased throughout pregnancy, whereas the expression of genes for lipid synthesis increased sharply at the onset of lactation. Examination of regulatory genes with profiles similar or complementary to those of lipid synthesis genes led to a model in which progesterone stimulates synthesis of TGF-beta, Wnt 5b, and IGFBP-5 during pregnancy. These factors are suggested to repress secretion by interfering with PRL and IGF-1 signaling. With progesterone withdrawal, PRL and IGF-1 signaling are activated, in turn activating Akt/PKB and the SREBPs, leading to increased lipid synthesis.
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PMID:Functional development of the mammary gland: use of expression profiling and trajectory clustering to reveal changes in gene expression during pregnancy, lactation, and involution. 1497 74

The H19 gene is transcribed in an mRNA-like noncoding RNA. When tumors of various organs or cell types are considered, H19 oncogene or tumor-suppressor status remains controversial. To address the potential regulation of H19 gene expression by an androgen steroid hormone (DHT: dihydrotestosterone) or by a peptidic hormone (PRL: prolactin), we performed experiments in rats systemically treated with chemical mediators. This range of in vivo experiments demonstrated that chronic hyperprolactinemia upregulated the H19 expression in epithelial and stromal cells whereas DHT downregulated the gene. PRL and DHT appeared to be opposite mediators in the H19 RNA synthesis. We investigated these hormonal effects in three human prostate epithelial cell lines. In LNCaP cancer cells, the opposite effect of PRL and DHT was corroborated. However, in normal cells (PNT1A), H19 remained insensitive to the hormones in fetal calf serum (FCS) medium but became responsive in a serum-stripped medium. In the DU-145 cancer cell line, tested for its androgen-independence and aggressiveness, the hormones had no effect on H19 expression whatever the culture conditions. Finally, we demonstrated that PRL upregulated the H19 expression in LNCaP cells by the JAK2-STAT5 transduction pathway. We conclude that H19 expression is regulated by both a peptidic and a male steroid hormone.
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PMID:Hormonal regulation of H19 gene expression in prostate epithelial cells. 1552 75

The problem of pancreas donor shortage could be addressed through in vitro islet-cell proliferation prior to transplantation into diabetic patients. Therefore, we set out to evaluate the effects of prolactin (rhPRL) and laminin on primary cultures of human pancreatic islets. Our results showed that rhPRL induced an increase in islet-cell number and in cumulative insulin secretion (p<0.01). However, glucose-induced insulin secretion was enhanced only in the presence of both laminin and rhPRL. In addition, we describe, for the first time in human islets, the PRL-induced activation of JAK2, and signal transducer and activator of transcription (STAT) 1, 3 and 5. Our results demonstrate a significant beneficial effect of rhPRL and laminin on human islets and support widely held notion that the closer physiological stimuli and environment of beta cells are mimicked, the better are the results in cell proliferation and secretory function, both essential for successful islet transplantation.
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PMID:Beneficial effects of prolactin and laminin on human pancreatic islet-cell cultures. 1708 83


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