Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of a cAMP-dependent secretogogue (VIP) on the phosphorylation of an endogenous, membrane-bound protein (pp170) was assessed in an intact cell preparation from the avian salt gland. The addition of VIP, in the presence of 100 microM isobutylmethylxanthine, resulted in a concentration-dependent increase in phosphorylation of pp170. This effect was rapid and transient with a 3-5-fold increase in phosphorylation occurring 1 min after the addition of VIP. Under similar incubation conditions, VIP stimulated a 4.6-fold increase in cAMP accumulation that paralleled phosphorylation. Exposure of cells to either forskolin or 8-Br-cAMP resulted in a 5-8-fold increase in the phosphorylation of pp170. The effect of forskolin was dose dependent with an EC50 similar to that for stimulation of secretion (35 nM). These results implicate an involvement for a cAMP-dependent protein kinase in the phosphorylation of pp170. The identity of pp170 was assessed utilizing a monoclonal antibody (Q3) directed against pp170. Q3 recognized a single 170-kDa band on Western blots of salt gland membrane protein. Immunoprecipitation of pp170 from salt gland cells resulted in the selective extraction of a single protein whose phosphorylation state was increased approximately 5-fold in response to carbachol or VIP. The identity of pp170 was established using two criteria. First, Q3 recognized affinity-purified Na:K:Cl cotransporter preparations from shark rectal gland membranes. Second, pp170 was selectively immunoprecipitated by monoclonal antibodies (J3, J4, and J7) that recognize different epitopes of the shark transport protein. These results suggest that pp170 is homologous to the shark rectal gland Na-K-Cl cotransporter, and thus the proteins may be functionally similar.
 ...
PMID:The Na-K-Cl cotransporter of avian salt gland. Phosphorylation in response to cAMP-dependent and calcium-dependent secretogogues. 128 Nov 59

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.
 ...
PMID:Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells. 164 51

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.
 ...
PMID:Properties of several protein kinases that copurify with rat spinal cord neurofilaments. 275 40

Cl- secretion in T84 cells evoked by a stimulus that activates protein kinase C, carbachol, was associated with elevated levels of 32P-labeled phosphatidic acid (PA). PA's role in the regulation of Cl- secretion was explored by examining the effect of exogenous PA (10(-4) M) on Cl- secretion and intracellular Ca2+ levels ([Ca2+]i) in monolayers. PA potentiated the effect of carbachol on [Ca2+]i and Cl- secretion, although it did not stimulate Cl- secretion by itself. PA had divergent effects on cyclic nucleotide-dependent Cl- secretion. It delayed Cl- secretion induced by vasoactive intestinal polypeptide [VIP, adenosine 3',5'-cyclic monophosphate (cAMP) dependent] but potentiated that induced by the heat-stable enterotoxin of Escherichia coli (STa; guanosine 3',5'-cyclic monophosphate dependent). PA did not alter AMP or GMP levels, suggesting that PA acts at a site distal to the generation of these second messengers. PA caused a slight increase in phosphorylation of protein kinase C substrates but not of cAMP-dependent protein kinase substrates. However, PA is probably not acting through a classical protein kinase C pathway, because we have previously shown that phorbol esters inhibit carbachol's actions, and the protein kinase C inhibitor staurosporine failed to block the effect of PA on VIP- or STa-stimulated Cl- secretion. Thus PA differentially regulates stimulated Cl- secretion in T84 cells, depending on the nature of the agonist.
 ...
PMID:Phosphatidic acid modulates Cl- secretion in T84 cells: varying effects depending on mode of stimulation. 838 32

The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol Pi incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express >/=99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+ mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization.
 ...
PMID:Phosphorylation of inositol 1,4,5-trisphosphate receptors by cAMP-dependent protein kinase. Type I, II, and III receptors are differentially susceptible to phosphorylation and are phosphorylated in intact cells. 948 97

A cellular suspension from rat submandibular glands was prepared with collagenase. The intracellular pH (pHi) was estimated with 2',7'-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH4Cl, the pHi transiently increased (diffusion of NH3) and then dropped (influx of NH4+). Isoproterenol increased 2.5-fold the rate of NH4+ influx; bumetanide, an inhibitor of the Na+-K+-2Cl(-)-cotransporter blocked the response to isoproterenol, confirming that the beta-adrenergic agonist stimulated the cotransporter. Forskolin (1 micromol/L) mimicked the response to isoproterenol. VIP (1 nmol/L(-1) micromol/L) also increased the activity of the cotransporter. Cyclic AMP rather than calcium was the mediator of this activation since 1) carbachol which increased the [Ca2+]i fivefold increased the uptake of NH4+ by only 50%; 2) only high concentrations of VIP significantly increased the [Ca2+]i; 3) incubation in the presence of EGTA had no effect on the response to VIP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransporter by VIP, forskolin, and isoproterenol was inhibited by H8, an inhibitor of cAMP-dependent protein kinase. It is concluded that the Na+-K+-2Cl(-)-cotransporter of rat submandibular glands is activated by isoproterenol, forskolin, and neuropeptides of the VIP family by a mechanism involving cAMP-dependent processes. The activation of the cotransporter by VIP could partly explain the potentiating effect of VIP on the response to sialagogues like substance P or muscarinic agonists.
 ...
PMID:Activation of the Na+-K+(NH4+)-2Cl(-)- cotransporter from rat submandibular glands in response to VIP. 988 83

We investigated the effect of VIP on the liver metastases and angiogenesis by Colon 26-L5 carcinoma cells in mice. Daily systemic administration of VIP, beginning 3 days after tumor inoculation into a portal vein of mice, inhibited significantly the development of their liver metastases. Immunohistochemical staining for factor VIII-related antigen in the sections of liver metastases showed that the systemic administration of VIP caused significant prevention of angiogenesis within tumor masses. VIP (10-(10) to 10(-6) M) inhibited the invasion of reconstituted basement membrane (Matrigel) by hepatic sinusoidal endothelial (HSE) cells in a concentration-dependent manner in a Transwell chamber assay in vitro and achieved approximately 50% reduction of control at 10(-6) M. VIP (10(-6) M) also significantly suppressed the haptotactic migration of HSE cells to fibronectin, laminin or type I collagen substrates with a similar inhibition rate to the invasion assay. Exposure of VIP to HSE cells induced accumulation of intracellular cAMP in a concentration-dependent manner. The inhibitory effect of VIP (10(-6) M) on HSE cell migration was significantly abrogated in the presence of 3 x 10(-6) M H-89, a cAMP-dependent protein kinase inhibitor. VIP (10(-6) M) inhibited the morphogenesis of HSE cells into capillary-like structures on Matrigel-coated wells. VIP did not affect the proliferation of HSE cells and the production of gelatinases in HSE cells in vitro at the concentrations used in the invasion assay. These observations suggest that the anti-metastatic effect of VIP on liver metastases by Colon 26-L5 carcinoma cells in mice is partly due to the prevention of tumor angiogenesis probably through suppression of the motility of endothelial cells.
 ...
PMID:Inhibition by vasoactive intestinal polypeptide (VIP) of angiogenesis induced by murine Colon 26-L5 carcinoma cells metastasized in liver. 1054 14