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)

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.
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PMID:Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts. 138 79

Like many other cell surface receptors for nutrients and polypeptide hormones, the insulin receptor undergoes a complex endocytotic itinerary. Upon insulin binding, the receptor is activated as a tyrosine-specific protein kinase and autophosphorylates. This autophosphorylation is necessary for the receptor to internalize. After endocytosis, the ligand (insulin) and its receptor are dissociated. Most of the insulin is degraded, whereas the receptors are largely recycled to the cell surface. The signals in the receptor that control and specify its endocytotic pathway are beginning to be understood. Through the techniques of in vitro mutagenesis, noninternalizing receptors have been engineered and their structural and functional properties have been analyzed. For example, the immediate submembranous domain of the insulin receptor has been found to contain sequences (Gly-Pro-Leu-Tyr and, to a lesser extent, Asn-Pro-Gln-Tyr) that are necessary for normal endocytosis. Receptors deleted or mutated in these sequences retain tyrosine kinase activity but fail to undergo endocytosis. Unlike the better understood low density lipoprotein and transferrin receptors, however, these sequences are not sufficient for endocytosis. An insulin receptor with only these sequences exposed in the cytoplasm does not internalize. Tyrosine kinase activity is thought to be needed to lead to autophosphorylation and a conformational change that exposes the otherwise buried endocytosis sequences in the normally dimerized insulin receptor. Non-internalizing mutants of the insulin receptor have been used to examine the role of endocytosis in insulin action. It was found that an endocytosis-defective receptor could induce a short-term metabolic action of insulin (glycogen synthetase stimulation) as well as longer-term mitogenic effects of insulin. Furthermore, insulin action deactivated after the hormone was removed from the noninternalizing receptors. Apparently, endocytosis is not necessary for insulin action, but probably is important for removing the insulin from the cell so the target cell for insulin responds in a time-limited fashion to the hormone.
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PMID:Mechanism and role of insulin receptor endocytosis. 147 59

Sertoli cells cultured in the presence of germ cells responded by increasing the level of transferrin mRNA 3-fold as determined by solution hybridization and Northern blot analysis. In contrast, the steady state levels of other mRNAs, including sulfated glycoprotein 1 (SGP-1), sulfated glycoprotein 2 (SGP-2), transferrin receptor, regulatory subunit of cAMP dependent protein kinase, and ferritin light chain, were not influenced by coculture with germ cells. The transferrin mRNA stimulatory activity was found in conditioned medium from germ cells but was not associated with germ cell membrane components. The activity was abolished by treatment of the medium with trypsin. Partial characterization and isolation of the protein(s) from conditioned medium indicated that it has an apparent mol wt between 10 and 30 K. Studies using inhibitors of protein and nucleic acid synthesis indicated that the stimulation of transferrin mRNA by germ cell conditioned medium required both transcription and translation. Sertoli cell enriched (germ cell depleted) testes were obtained from male offspring of pregnant females irradiated at the 19th day of gestation. Testicular transferrin mRNA levels from irradiated rats decreased in comparison to levels in the normal rat, whereas SGP-2 mRNA levels were unchanged. These studies demonstrate that germ cell secretions may interact with Sertoli cells to specifically increase the level of transferrin mRNA and that this interaction may be a mechanism by which germ cells regulate the flow of iron across the seminiferous epithelium.
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PMID:Germ cell regulation of Sertoli cell transferrin mRNA levels. 234 75

We studied the effects of porcine FSH, forskolin, and (Bu)2cAMP [agents that stimulate steroidogenesis via the adenylate cyclase-cAMP pathway (cAMP system)] either alone or with concomitant addition of phorbol 12-myristate 13-acetate (TPA; a phorbol ester that activates protein kinase-C) on steroidogenesis in porcine granulosa cells cultured from small (less than 3 mm) and medium-sized (3-6 mm) ovarian follicles. We attempted to determine if granulosa cells from different maturational states had different responses to these agonists and antagonists. Cells were cultured in serum-free medium 199 supplemented with insulin (10 micrograms/ml), transferrin ( 5 micrograms/ml), and androstenedione (2.5 X 10(-7) M) for 48 h. Levels of progesterone (P) and estradiol (E2) were determined in spent medium by RIA. We found that FSH, forskolin, and cAMP all stimulated secretion of E2 and P in a dose-dependent manner in both developmental groups. When TPA was added alone to cultures, P levels were stimulated at low doses of TPA but inhibited at higher doses in granulosa from both sized follicles, whereas cells from both small- and medium-sized follicles demonstrated reductions in E2. TPA was also found to inhibit FSH-, forskolin-, and cAMP-induced steroidogenesis in a dose-dependent manner in cells from the two groups of follicles. The stimulatory effects of any of the secretagogues on E2 secretion were inhibited by TPA to a significantly greater extent in granulosa cells from small follicles. Although inhibition of FSH- and forskolin-induced P secretion by TPA was also greater in granulosa cells from small follicles, cAMP-treated cells did not show this differential inhibition. Thus, it appears that modulators of the protein kinase-C system regulate steroidogenesis differently in granulosa cells from small and medium follicles. These differences may involve alterations in the interplay between the protein kinase-C and cAMP pathways.
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PMID:Steroidogenesis of porcine granulosa cells from small and medium-sized follicles: effects of follicle-stimulating hormone, forskolin, and adenosine 3,'5'-cyclic monophosphate versus phorbol ester. 253 76

The intracellular concentrations of cyclic AMP, polyphosphoinosides and free Ca2+ were unaffected during receptor-mediated endocytosis of the neoglycoprotein beta-D-galactosyl-bovine serum albumin (D-Gal-BSA) by isolated hepatocytes. Elevation of either intracellular cyclic AMP by glucagon or inositol phosphates and Ca2+ by vasopressin were without effect on the binding and internalization of D-Gal-BSA. The normal response of this cell to glucagon- and vasopressin-mediated mobilization of these second messengers was not modified in the presence of saturating concentrations of D-Gal-BSA. Receptor-mediated endocytosis of diferric transferrin (Fe3+-TRF) by both hepatocytes and HL60 cells was also shown to be independent of second messengers, although the unequivocal expression of the transferrin receptor by hepatocytes could not be satisfactorily demonstrated. The results of the present study are at variance with a suggested regulatory role for second messengers in receptor-mediated endocytosis of serum-derived ligands such as asialoglycoproteins and Fe3+-TRF. Receptor phosphorylation by protein kinase C in particular has been proposed to regulate the distribution and recycling of these receptors in actively endocytosing cells. We would suggest that if receptor phosphorylation has a regulatory role during endocytosis, it is likely to be mediated by a second-messenger-independent protein kinase analogous to casein kinase II. An alternative interpretation is that phosphorylation has no physiological significance and receptor-mediated endocytosis is a constitutive event coupled to membrane turnover.
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PMID:Receptor-mediated endocytosis of asialoglycoproteins and diferric transferrin is independent of second messengers. 254 88

The proliferative responses of natural killer (NK) cells to 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase c(PKC), and to the Ca2+ ionophores A23817 and ionomycin, known to enhance the intracellular calcium, have been investigated. Highly purified large granular lymphocytes (LGL) were cultured for 12-30 hr in the presence of TPA, ionomycin, or A23817. TPA alone (1-20 ng/ml) triggered rapid LGL proliferation, whereas the calcium ionophores were ineffective. The addition of either calcium ionophore to suboptimal doses or TPA (0.1-0.5 ng/ml) resulted in a synergistic effect on LGL proliferation. Under these conditions high levels of IL-2 activity were released by the LGL. Phenotypic analysis revealed the rapid loss of the Fc gamma receptors (CD16) on LGL and the induction of the expression of IL-2 (CD25) and transferrin receptors and of HLA-DR, but not of CD3. Removal of extracellular Ca2+ by addition of EGTA at the beginning of the culture greatly depressed LGL proliferation and IL-2 production, and blocked phenotypic changes, such as the expression of Tac antigen. Finally, progression to the proliferative phase of LGL, activated by TPA alone or with ionomycin, was completely abrogated by a hyperimmune anti-IL-2 antiserum.
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PMID:Proliferative effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophores on human large granular lymphocytes (LGL). 283 70

Murine interleukin 1 (IL 1) inhibited concentration dependently the proliferation of murine T cell lymphomas and the human leukemic cell line K 562. The cytostatic action of IL 1 was not associated with cytotoxicity and appeared to be irreversible. Changes in the expression of surface antigens, like a rapid decrease of transferrin receptors or, more delayed, an increase in HLA-A, B, C antigen density suggested that a differentiation step was induced by IL 1. This effect of IL 1 was a direct one and most likely mediated by a specific receptor molecule. In order to characterize the receptor for IL 1, highly purified plasma membranes from K 562 were incubated with murine IL 1, and the phosphorylation pattern of plasma membrane proteins was investigated by the addition of radiolabeled ATP. At 0 degree C, IL 1 induced the specific phosphorylation of a 41 kDa membrane protein in a time- and concentration-dependent manner. Analysis of the phosphoamino acid composition revealed that IL 1 induced specifically the phosphorylation of tyrosine residues of the 41 kDa protein. Crosslinking experiments proved that the 41 kDa protein had an IL 1 binding site, strongly suggesting that the 41 kDa protein was the receptor for IL 1 itself. Affinity labeling with an ATP-analogue showed that this protein possessed an ATP binding and cleaving site. We conclude from this that the receptor for IL 1 in the plasma membranes of K 562 is a transmembranous protein of 41 kDa, which possesses a tyrosine specific protein kinase activity with an autophosphorylating capacity.
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PMID:The receptor for interleukin 1 in plasma membranes of the human leukemia cell K 562: biological and biochemical characterization. 294 3

Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly regulate surface transferrin receptors. Regulation of transferrin receptors by phorbol diesters involves receptor internalization in association with increased receptor phosphorylation (hyperphosphorylation). The intracellular mechanism of action of phorbol diester involves binding to and activation of the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Present studies comparing results obtained with whole cells and those from a cell-free system reconstituted from purified protein kinase C and transferrin receptor components have revealed that the transferrin receptor is phosphorylated by protein kinase C activated by phorbol esters. Following tryptic digestion and two-dimensional separation of phosphopeptides of phosphorylated transferrin receptors, two major and several minor phosphoserine-containing fragments are resolved. These fragments are identical whether transferrin receptor is phosphorylated in whole cells incubated with phorbol diesters or following phosphorylation of affinity immobilized transferrin receptor in the in vitro reconstitution system. Phosphoamino acid analysis of these fragments indicates that serine is the only amino acid phosphorylated in whole cells or in the cell-free system. In addition, colchicine is shown to inhibit in a dose-dependent manner phorbol diester-induced internalization but not hyperphosphorylation of the surface transferrin receptor in whole cells. This inhibition is specific for colchicine since inactive beta- and gamma-Lumicolchicine have no such effect, while taxol reverses the inhibition. These results indicate that the phorbol diester-mediated process of down-regulation of the surface transferrin receptor is associated with phosphorylation of the receptor by activated protein kinase C and requires an intact cytoskeleton to affect receptor internalization.
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PMID:Mechanism of phorbol diester-induced regulation of surface transferrin receptor involves the action of activated protein kinase C and an intact cytoskeleton. 299 Dec 44

Transferrin-specific cDNA clones were isolated from a rat liver cDNA library prepared from transferrin-enriched mRNA. Hybrid selection and sequence analysis confirmed that the selected clone contained the carboxy-terminal coding region of the transferrin mRNA. Northern blot analysis was used to demonstrate the presence of transferrin mRNA in liver and Sertoli cells. Transferrin mRNA levels were measured in total RNA isolated from cultured rat Sertoli cells after treatment with FSH, insulin, retinol, and testosterone. The results showed a 2- to 4-fold increase in the level of transferrin mRNA, which peaked on the fourth day of culture after initiation of treatment, with FSH, insulin, retinol, and testosterone. This induction is gene specific, since no change in the mRNA levels for either the catalytic or regulatory subunits of cAMP-dependent protein kinase was observed. The effects of hormones, vitamin A (retinol), and Bu2 cAMP on transferrin mRNA and transferrin secretion (measured by RIA) in cultured Sertoli cells were compared. In general, a direct relationship between the amount of transferrin mRNA present in the cells and the amount of transferrin secreted into the culture medium was observed. These results demonstrate the important role that vitamin A, testosterone, and peptide hormones play in modulating transferrin gene expression in Sertoli cells.
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PMID:Transferrin messenger ribonucleic acid: molecular cloning and hormonal regulation in rat Sertoli cells. 302 31

To assess the role of protein kinase-C (PK-C) in the growth and differentiation of small intestinal enterocytes, IEC-6 cells (a cell line derived from the crypts of rat small intestine) were incubated with factors known to induce growth (insulin, epidermal growth factor, gastrin, somatostatin and transferrin) or differentiation (transforming growth factor-beta, retinoic acid and phorbol 12-myristate 13-acetate (PMA)). Cell proliferation (3H-thymidine incorporation) and PK-C activity (Ca++/phospholipid dependent) were measured. Among growth promoting factors only epidermal growth factor, insulin and transferrin were associated with increased 3H-thymidine incorporation, and none of these agents induced PK-C activation as measured by its translocation from cytosol to membrane fraction. Of the differentiation inducing factors, only PMA translocated PK-C from cytosol to membrane. PMA also inhibited 3H-thymidine incorporation in a dose dependent manner. These results suggest that growth and proliferation of enterocytes occur independent of PK-C signal transduction.
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PMID:Effects of growth and differentiation inducing factors on protein kinase-C of cultured intestinal crypt cells. 339 31


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