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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The growth hormone (GH)-releasing action of GH-releasing factor (GRF) is known to be cAMP-dependent. However, definitive proof for the involvement of the cAMP-dependent enzyme protein kinase A (PKA) is still lacking. In this study, we characterized the PKA system in purified rat somatotrophs and examined its role in mediating GRF-stimulated GH release under static incubation conditions. PKA enzyme activity was detected only in the cytosolic, but not the particulate fraction of rat somatotrophs. This cytosolic PKA activity exhibited the characteristic cAMP dependence (with ED50 of 0.1 microM), ability to phosphorylate kemptide (a synthetic peptide with a PKA phosphorylation site), and susceptibility to inhibition by the bovine heat-stable PKA inhibitor. GRF treatment (1 pM-1 nM) stimulated the cytosolic PKA activity and GH release from rat somatotrophs in a dose-dependent manner. Time-course studies also demonstrated that activation of cAMP synthesis and PKA activity preceded the GH response to GRF. Stimulation of cytosolic PKA activity in rat somatotrophs by the adenylate cyclase activator forskolin (10 nM-1 microM) and membrane permeant cAMP analog db.cAMP (5 microM-0.5 mM) mimicked the GH-releasing effect of GRF. In contrast, Rp.cAMP, a cAMP antagonist for PKA regulatory subunits, blocked both the cytosolic PKA activity as well as GRF-induced GH release. Similar inhibitions were also observed when an inhibitor for PKA catalytic subunits, H89, was used. Somatostatin (SRIF) (1 nM), the physiological GH-release inhibitor, suppressed the GH response to GRF without affecting the basal or GRF-stimulated PKA activity. SRIF at a higher dose (10 nM) abolished the GH-releasing effect of GRF. In this case, SRIF also induced a small but significant inhibition of GRF-stimulated PKA activity. Taken together, the present study provides direct evidence that PKA enzyme activity is localized only in the cytosol of rat somatotrophs and constitutes an essential component of the signal transduction mechanism for GRF-stimulated GH release. This cytosolic PKA system, however, does not appear to be a major target for the GH-release inhibiting action of SRIF.
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PMID:Cytosolic protein kinase A mediates the growth hormone (GH)-releasing action of GH-releasing factor in purified rat somatotrophs. 761 38

We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine (CHO-GHR1-638 Y333F, Y338F) were generated by cDNA transfection. Compared with the wild type receptor the Y333F,Y338F mutant possessed normal high affinity ligand binding, hormone internalization, and ligand-induced receptor down-regulation. GH activation of mitogen-associated protein kinase was also similar in CHO clones expressing similar wild type and Y333F,Y338F receptor number. However, two GH-regulated cellular events (lipogenesis, and protein synthesis) were deficient in the tyrosine substituted receptor. In contrast, transcriptional regulation by GH (as evidenced by chloramphenicol acetyltransferase cDNA expression driven by the GH-responsive region of the SPI 2.1 gene) was not affected by Y333F,Y338F substitution. Thus we provide the first experimental evidence that specific tyrosine residues of the GH receptor are required for selected cellular responses to GH.
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PMID:Requirement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor for selected GH-stimulated function. 766 93

The molecular characterization of GHRH and the GHRH receptor provides a framework for understanding the hypothalamic regulation of pituitary somatotroph function. The signaling events discerned from our investigation of GHRH receptor structure and function form the basis of a model for GHRH action, which is shown in Fig. 20. GHRH interaction with its seven transmembrane domain Gs-coupled receptor on the somatotroph (step 1) leads to the release of growth hormone from secretory granules (step 2), which is likely to involve a G protein-mediated interaction with ion channels, and to a stimulation of intracellular cAMP accumulation (step 3) (Mayo, 1992; Lin et al., 1992; Gaylinn et al., 1993). In several cell types tested, elevated cAMP leads to the phosphorylation and activation of the transcription factor CREB by protein kinase A (Gonzalez and Montminy, 1989; Sheng et al., 1991), and one target gene for CREB action is the pituitary-specific transcription factor Pit-1 or GHF-1 (step 4) (Bodner et al., 1988; Ingraham et al., 1988; McCormick et al., 1990). Pit-1 is a prototypic POU domain protein that is required for the appropriate regulation of the growth hormone gene in somatotroph cells, thus providing a pathway by which a GHRH signal can lead to increased growth hormone synthesis in the pituitary (step 5). In addition, Pit-1 is likely to directly regulate the synthesis of the GHRH receptor (step 6), in that the receptor is not expressed in the pituitary of dw/dw mice that lack functional Pit-1 (Lin et al., 1992), and a cotransfected Pit-1 expression construct can activate the GHRH receptor promoter in transiently transfected CV1 cells (Lin et al., 1993). It remains to be determined whether additional direct regulation of the GHRH receptor gene in response to the cAMP signaling pathway occurs (step 7). The inhibitory peptide somatostatin presumably interacts with this same signaling pathway through G protein-mediated suppression of the cAMP pathway (Tallent and Reisine, 1992; Bell and Reisine, 1993). In agreement with the importance of this signaling system for normal growth, a transgene encoding a nonphosphorylatable mutant CREB protein, which blocks the function of the endogenous CREB protein, is able to cause somatotroph hypoplasia and dwarfism in mice when its expression is targeted to pituitary somatotrophs (Struthers et al., 1991). Several steps in the signaling pathway leading to growth hormone secretion are subject to disruption, resulting in growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Growth hormone-releasing hormone: synthesis and signaling. 774 Jan 67

Lactogens [prolactin (Prl) and growth hormone] stimulate phosphorylation of the 40S ribosomal protein, S6, in Nb2 cells by mechanisms that do not involve participation of cAMP or protein kinase A, protein kinase C, or cGMP-dependent protein kinase. However, inhibition of tyrosine kinase (TK) abrogates Prl-mediated macromolecular biosynthesis. Inasmuch as lactogen signaling may involve sequential activation of protein kinases, the effect of Prl on the well-characterized mitogen-activated protein kinase (MAPK) and S6 kinase (S6K), the enzyme responsible for S6 phosphorylation in vivo, and their relationship to Nb2 macromolecular biosynthesis and mitogenesis were investigated. The results show that MAPK stimulation is transient (peak activity, 30 min) and precedes that of S6K, which reaches a maximum at 1.5-2 h, and slowly returns towards control levels at 6 h. Both staurosporine which inhibits GH receptor-associated kinase (JAK2) and genistein (GEN), an inhibitor of membrane-associated and cytoplasmic TKs, abrogate Prl-stimulated TK, MAPK, and S6K. Rapamycin (RAP), a specific inhibitor of p70S6K, completely blocks S6K but does not affect TK and MAPK. TK and MAPK activity correlates with Prl-stimulated anabolism, i.e., protein and DNA synthesis and mitogenesis. Thus, concentrations of STR and GEN which abrogate TK and MAPK inhibit anabolism virtually 100%. However, RAP, which inhibits S6K (ca. 100%) but not TK or MAPK, only delays Prl-mediated anabolism. These results indicate that Prl signaling in Nb2 cells involves a protein kinase cascade and that regulation of receptor-associated kinase, TK, and MAPK correlates with anabolism. The role of S6K (and S6 phosphorylation) appears to be ancillary.
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PMID:Stimulation of receptor-associated kinase, tyrosine kinase, and MAP kinase is required for prolactin-mediated macromolecular biosynthesis and mitogenesis in Nb2 lymphoma. 784 Jun 14

Winter flounder renal proximal tubule primary monolayer cultures mounted in Ussing chambers were used to determine the effect of salmon somatolactin (sSL) on transepithelial Pi and Ca2+ transport. sSL stimulated Pi reabsorption in a dose-dependent manner at physiological levels of the hormone (12.5 ng/ml). Net Pi transport was significantly altered by sSL (200 ng/ml) within 2 h after the initial exposure. Ca2+ fluxes were unchanged by the addition of 200 ng/ml sSL. The sSL-induced Pi reabsorption was abolished by 10 microM H-89, a highly specific protein kinase A inhibitor. Moreover the production and release of adenosine 3',5'-cyclic monophosphate were significantly increased after 1 and 2 h of exposure to sSL. The data indicate that sSL directly stimulates net renal Pi reabsorption by an adenosine 3',5'-cyclic monophosphate-dependent pathway. In addition to sSL, flounder SL and rat prolactin greatly, and salmon growth hormone (2.3 micrograms/ml) slightly, increased net Pi reabsorptive flux, whereas salmon prolactin had no effect.
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PMID:Effect of somatolactin and related hormones on phosphate transport by flounder renal tubule primary cultures. 790 Aug 98

Signal transduction mechanisms involved in mouse growth hormone-releasing hormone (GRH) and somatostatin (SRIH) release were investigated using an in vitro perifusion system. Hypothalamic fragments were exposed to depolarizing agents, protein kinase A and C activators, and a calcium ionophore. The depolarizing agents, KCl (60 mM) and veratridine (50 microM), induced similar patterns of GRH and SRIH release. Somatostatin release in response to both agents was twofold greater than that of GRH. Forskolin (10 microM and 100 microM), an adenylate cyclase activator, stimulated both GRH and SRIH release, though with different secretory profiles. The SRIH response was prolonged and persisted beyond removal of the drug from the system, while the GRH response was brief, ending even prior to forskolin removal. Neither GRH nor SRIH were stimulated by 1,9-dideoxy-forskolin (100 microM), a forskolin analog with cAMP-independent actions. A23187 (5 microM), a calcium ionophore, stimulated the release of SRIH to a much greater extent than that of GRH. The GRH and SRIH secretory responses to PMA (1 microM), a protein kinase C activator, were similar, though delayed. The results suggest that 1) GRH and SRIH secretion are regulated by both protein kinase A and C pathways, and 2) depolarizing agents are important for the release of both hormones.
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PMID:Mouse hypothalamic growth hormone-releasing hormone and somatostatin responses to probes of signal transduction systems. 790 44

To investigate whether the expression of the renal angiotensinogen (ANG) gene is regulated by beta-adrenoceptors and the cAMP-dependent protein kinase A pathway, we introduced stably the fusion gene containing the 5'-flanking regulatory sequence of the ANG gene with a human growth hormone (hGH) gene as a reporter, pOGH (ANG N-1498/+18), into opossum kidney (OK) cells. We successfully obtained several stable transformants with a high expression of the pOGH (ANG N-1498/+18) fusion gene. One stable transformant (OK 27) that is able to maintain the expression of pOGH (ANG N-1498/+18) in culture for more than a year was used in the present study. The level of expression of the pOGH (ANG N-1498/+18) in OK 27 was evaluated by the amount of immunoreactive-hGH (IR-hGH) secreted into the culture medium. The addition of isoproterenol (10(-11) M to 10(-9) M) stimulated the expression of pOGH (ANG N-1498/+18) and increased the accumulation of intracellular cAMP. Higher concentrations of isoproterenol (that is, greater than 10(-9) M) had low or minimal effect. In contrast, the addition of 8-bromo-cAMP (8-Br-cAMP) and forskolin stimulated the expression of pOGH (ANG N-1498/+18) in a dose-dependent manner. The stimulatory effect of isoproterenol was blocked by the presence of propranolol, atenolol and ICI 118,551. The addition of ICI 118,551, however, was less effective than atenolol. Furthermore, the stimulatory effect of isoproterenol and 8-Br-cAMP on the expression of the pOGH (ANG N-1498/+18) was inhibited by the presence of Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isoproterenol and 8-bromo-cyclic adenosine monophosphate stimulate the expression of the angiotensinogen gene in opossum kidney cells. 799 91

Although C-type natriuretic peptide (CNP) has been shown to exist at the highest concentration in the anterior pituitary in rat tissues, its physiological role(s) there is (are) not clear. In this study, we report a novel function of CNP examined with anterior pituitary-derived cell lines, GH3 and AtT20/D16v-F2. Both CNP and atrial natriuretic peptide (ANP) increased cellular cGMP levels in both cell lines in dose-dependent manners. CNP, but not ANP, stimulated growth hormone (GH) release from GH3 cells. In contrast, neither ANP nor CNP had any significant effect on the corticotropin release from AtT20/D16v-F2 cells. An activator for cGMP-dependent protein kinase (cGK), dibutyryl cGMP, mimicked the stimulation of GH release from GH3 cells by CNP. Constitutive GH release from GH3 cells was greatly diminished in the presence of inhibitors for cAMP-dependent protein kinase, while stimulative GH release by CNP was not affected. However, inhibitors which can block cGK almost completely diminished the stimulative effect of CNP. An inhibitor for protein kinase C did not show any effect on either constitutive or CNP-stimulative GH release. Our observations indicate that the stimulation of GH release from GH3 cells by CNP is mediated mainly by the cGK signal-transduction pathway, not by cAMP-dependent protein kinase or protein kinase C, through a CNP-specific receptor (possibly ANP-B receptor). Thus, CNP may act as a local modulator in the anterior pituitary.
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PMID:C-type natriuretic peptide stimulates secretion of growth hormone from rat-pituitary-derived GH3 cells via a cyclic-GMP-mediated pathway. 802 May 2

Cortisol inhibits growth hormone (GH) release in short-term culture and is stimulatory in long-term cultures of rat and human pituitary cells. This study sought to determine the in vitro effects of cortisol on GH release and the signal transduction pathways mediating the effects of cortisol on GH release from cultured ovine somatotrophs. Pituitary cells were dispersed with collagenase and placed in culture medium for 4 days. The data indicate that cortisol inhibited growth hormone-releasing hormone (GHRH)-stimulated GH release by at least 2 h. In short-term culture GHRH-, forskolin- and dibutyryl cyclic AMP-stimulated GH release were inhibited by cortisol, suggesting an effect distal to the membrane and involving a protein kinase A (PKA)-dependent pathway. GH release initiated by KCl was inhibited by cortisol, but GH release caused by the calcium ionophore A23187 was unaffected. This suggests a possible action of cortisol on the calcium channels. The inhibition by cortisol of the calcium-dependent secretion of GH release appeared to play a smaller role in mediating cortisol inhibition of GH release than that seen with PKA. Attempts to overcome cortisol inhibition of GH release using puromycin, arachidonic acid or pertussis toxin were unsuccessful. Since cortisol inhibition of GH release does not occur via the mechanisms found in other cell types, cortisol inhibition of pituitary cell secretions appears to be cell-specific rather than utilizing a single inhibitory mechanism. The majority of cortisol actions on the somatotroph appear to act at a site distal to the production of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cortisol inhibition of growth hormone-releasing hormone-stimulated growth hormone release from cultured sheep pituitary cells. 807 50

Human myometrium contains receptors for hCG/human LH (hLH). This suggested the possibility that hCG and hLH might regulate human myometrium, which has not previously been considered a direct target of gonadotropin regulation. To investigate such a possibility, highly pure and viable smooth muscle cells were isolated from nonpregnant human myometrium and cultured as monolayers. The cells contained hCG/LH receptor mRNA transcripts and a 50-kDa immunoreactive protein that can bind 125I-hCG in a ligand-specific manner. The presence of hCG during culture resulted in a significant increase of myometrial smooth muscle cell density. The hCG effect was time- and concentration-dependent and was mimicked by hLH but not by human FSH or human FSH or human thyroid-stimulating hormone. Human CG also greatly increased the size of a subpopulation of myometrial smooth muscle cells without affecting their chromosomal ploidy. Antibodies to hCG/LH receptors and hCG blocked hCG effects. Human prolactin and growth hormone, which do not bind to hCG/LH receptors, also increased the myometrial smooth muscle cell density. A protein kinase A inhibitor (H-89) blocked hCG response whereas calphostin (a protein kinase C inhibitor) and lavendustin A (a tyrosine kinase inhibitor) had no effect on hCG response, suggesting that a cAMP/protein kinase A signaling mechanism is involved in hCG action. Eicosanoids from cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism are probably not involved, because the inhibitors of these enzymes had no effect on hCG response. While progesterone and estradiol could not mimic or modify hCG action, epidermal growth factor did mimic hCG in increasing myometrial smooth muscle cell density.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human myometrial smooth muscle cells are novel targets of direct regulation by human chorionic gonadotropin. 828 97


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