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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using Chinese hamster ovary cell lysate, an in vitro assay has been developed to study the interaction of fibronectin with the alpha 5 beta 1 integrin in a cytosolic environment. In our solid phase assay, 96-well microtiter plates were coated with fibronectin in which cell lysate was incubated. A dose-dependent binding of the fibronectin receptor onto the coated plastic was immunodetected by specific polyclonal antibodies raised against the alpha 5 beta 1 integrin. Both soluble fibronectin and PB1, a monoclonal antibody raised against the fibronectin receptor, competed with the alpha 5 beta 1 integrin for binding to the fibronectin-coated plastic. General phosphatase inhibitors used during cell lysis completely abolished the fibronectin/integrin interaction in the assay, indicating that the affinity of the fibronectin receptor might be modulated by a protein phosphatase activity. Furthermore, in this assay, the interaction between the fibronectin receptor and its substrate in a cytosolic environment required intracellular calcium. Additionally, the action of more specific phosphatase inhibitors and the inhibition of the integrin/fibronectin interaction by a monoclonal antibody raised against the calcium/calmodulin-dependent protein phosphatase calcineurin suggested that calcineurin allowed the interaction between the alpha 5 beta 1 integrin and fibronectin. Metabolical labeling experiments showed that alpha 5 beta 1 itself was not the target of phosphorylation/dephosphorylation cascades involving calcineurin and leading to the modulation of integrin affinity. Taken together, these results showed that in vitro one substrate of the serine/threonine protein phosphatase calcineurin regulates the alpha 5 beta 1 integrin affinity by interacting with a yet unidentified effector.
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PMID:Control of the alpha 5 beta 1 integrin/fibronectin interaction in vitro by the serine/threonine protein phosphatase calcineurin. 753 36

Chemoattractants stimulate neutrophil migration by activating signalling pathways including repeated transient increases in intracellular free calcium, [Ca2+]i. A motile neutrophil sends out many pseudopods, some of which adhere to the substrate; to continue moving forward the cell must release these attachments. Adhesion can be actively regulated, and neutrophils in which [Ca2+]i transients are inhibited become stuck on fibronectin or vitronectin, extracellular matrix proteins that neutrophils encounter in vivo. Function-blocking antibodies to beta 3 integrins or the alpha v beta 3 heterodimer restore motility on vitronectin to [Ca2+]i-buffered cells (B. Hendey, M.A.L., E. Marcantonio and F.R.M., manuscript submitted), indicating that an alpha v beta 3-like integrin is responsible for the [Ca2+]i-sensitive adhesion. We show that the density of alpha v beta 3 integrins in the adherent membrane of neutrophils migrating on vitronectin is much higher at the leading edge than at the rear, but [Ca2+]i buffering or inhibition of Ca(2+)-calmodulin-activated protein phosphatase 2B (calcineurin) leads to accumulation of alpha v beta 3 on the adherent surface at the rear of the cell. We show that the polarized distribution of alpha v beta 3 integrins in migrating neutrophils is maintained by [Ca2+]i-dependent release of adhesion followed by endocytosis of these integrins and recycling to the leading edge.
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PMID:Ca(2+)- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils. 754 74

Neutrophils are guided to the sites of infection or inflammation by gradients of chemoattractants. Chemoattractants stimulate rapid and repeated changes in neutrophil intracellular calcium, [Ca2+]i, which correlate with cell spreading, pseudopod extension, motility, change of direction and phagocytosis. However, blocking the [Ca2+]i transients has little effect on cell spreading, polarization or pseudopod extension. Thus, either the [Ca2+]i transients are not required for cell spreading, polarization or pseudopod extension or other redundant mechanisms are present that allow the cells to perform these functions in vitro. In contrast, cell motility is [Ca2+]i dependent when the cells are examined on physiological substrates such as fibronectin or vitronectin. Calcium-buffered cells appear to make repeated attempts to move but are unable to detach from a fibronectin or vitronectin substrate. Motility can be restored to [Ca2+]i buffered cells by blocking substrate attachment with RGD peptides or by using a less adherent substrate such as albumin. A similar inhibition of motility on vitronectin could be induced by inhibitors of the calcium/calmodulin-dependent phosphatase, calcineurin. Thus, the periodic increases in [Ca2+]i apparently activate the phosphatase calcineurin to initiate a cycle of detachment from the vitronectin substratum. These data suggest that the [Ca2+]i transients regulate motility by coordinating a series of substrate-specific attachment/detachment events.
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PMID:Regulation of neutrophil motility and adhesion by intracellular calcium transients. 769 Dec 66

Analysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island.
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PMID:Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182. 861 77

The role of protein phosphatase-2A (PP-2A) in regulating the motility and adhesion of human head and neck squamous cell carcinomas (HNSCC) was investigated. Immunofluorescent staining of these HNSCC cells showed PP-2A can co-localize with microtubules. That the PP-2A influences motility was shown by the increase in HNSCC cell migration through laminin and vitronectin when PP-2A was selectively inhibited with low dose okadaic acid, and by the reduction in invasion through these same matrix components by elevators of PP-2A activity. Motility of HNSCC cells through collagen I or fibronectin was not modulated by PP-2A. The reduction in HNSCC migration through vitronectin or laminin that resulted from treatment with PP-2A elevators was associated with an increase in cellular adhesiveness to these same ECM components. These studies show the association of PP-2A with the cellular cytoskeleton and its role in restricting the invasiveness of tumor cells through select extracellular matrix components.
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PMID:Protein phosphatase-2A association with microtubules and its role in restricting the invasiveness of human head and neck squamous cell carcinoma cells. 902 32

We have shown that attachment to a fibronectin substrate stimulates two pathways of lipid biosynthesis in cultured human fibroblasts. Detachment of these cells (mechanically, with trypsin, or by RGDS peptides) caused a significant decrease in their 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and in their incorporation of [3H]acetate into fatty acids. This inhibition was substantially reversed by the reattachment of cells to fibronectin substrates, but not to poly-L-lysine substrates or to fibronectin in solution. Inhibiting phosphoprotein phosphatase activity with okadaic acid blocked the recovery of both biosynthetic activities. Both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid biosynthesis are known to be inhibited by the action of 5'-AMP-activated protein kinase, which is activated by an increase in the level of AMP relative to ATP. For example, in our system, sodium azide and 2-deoxy-D-glucose increased the ratio of cellular AMP to ATP and caused a decrease in lipid biosynthesis. We then verified the prediction that detachment of cells from substrates also caused an increase in the AMP/ATP ratio. We therefore conclude that the attachment of cells to fibronectin promotes lipid biosynthesis, presumably in coordination with the cellular growth response evoked by attachment to the extracellular matrix.
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PMID:Cell adhesion to fibronectin regulates membrane lipid biosynthesis through 5'-AMP-activated protein kinase. 923 31

Fibronectin binding on alpha5beta1 integrin is strictly dependent on intracellular calcium. Using an in vitro assay, we previously found that either calcineurin inhibitors or a blocking calcineurin monoclonal antibody added to cell lysates completely abolished the fibronectin/integrin interaction, which suggested that the activity of calcineurin, a calcium/calmodulin-dependent phosphatase, was required to counteract some kinase activity and maintain the high affinity state of alpha5beta1. In this paper, we show that blocking of the calcium/calmodulin kinase II (CaMKII) activity with the specific inhibitor KN-62 or with its pseudosubtrate Autocamtide-2 preserved the high affinity state of the integrin even under experimental conditions that inhibit calcineurin. Conversely, the addition of purified CaMKII to the cell lysate inhibited alpha5beta1 binding to fibronectin in vitro. Consistent with these results, cell adhesion on fibronectin was stimulated by KN-62. Moreover, Scatchard analysis of fibronectin binding on CHO cells revealed that KN-62 decreased the Kd value from 0.3 to 0.05 microM. Finally the expression of exogenous constitutively active CaMKII resulted in a dramatic defect in cell adhesion with no significant modification in alpha5beta1 cell surface expression. In summary our results demonstrate that CaMKII controls the affinity state of the integrin alpha5beta1 in vitro and in living cells.
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PMID:Calcium/calmodulin-dependent protein kinase II controls alpha5beta1 integrin-mediated inside-out signaling. 945 39

We investigated the effects of signaling molecule inhibitors on the expression and function of beta1 integrins in Jurkat cells. Jurkat cells expressed alpha4beta1 and alpha5beta1, with significant levels of constitutively activated beta1 integrins as assessed by labeling with mAb 15/7 that distinguishes between activation states. Adhesion to fibronectin (Fn) was mediated equally through alpha4 and alpha5 subunits, and was potentiated by the beta1 integrin activating mAb 8A2. Fn adhesion was decreased by okadaic acid through effects on both alpha4beta1, and alpha5beta1. Tyrphostin A23 also decreased adhesion but was less potent. Neither inhibitor had any effect on the surface expression of total or activated beta1 integrins. The effect of tyrphostin was completely reversed by 8A2; the effect of okadaic acid was only partially reversed. Using Calyculin A, we determined that Jurkat adhesion to Fn was regulated via protein phosphatase 1, independent of the levels of integrins or integrin activation epitopes. Activation of Jurkat cells with a CD3-stimulating mAb enhanced adhesion to Fn and was partially blocked by okadaic acid. These data demonstrate different regulatory pathways for constitutive versus activation-dependent adhesion via beta1 integrins, and implicate both tyrosine kinases and serine-threonine phosphatases in integrin function.
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PMID:Beta 1 integrin-dependent binding of Jurkat cells to fibronectin is regulated by a serine-threonine phosphatase. 985 Jan 57

Cytostatin, which is isolated from a microbial cultured broth as a low molecular weight inhibitor of cell adhesion to extracellular matrix (ECM), has anti-metastatic activity against B16 melanoma cells in vivo. In this study, we examined a target of cytostatin inhibiting cell adhesion to ECM. Cytostatin inhibited tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin upon B16 cell adhesion to fibronectin. While the amount of FAK was not affected by cytostatin, electrophoretically slow-migrating paxillin appeared. Alkaline phosphatase treatment diminished cytostatin-induced slow-migrating paxillin. Furthermore, cytostatin increased intracellular serine/threonine-phosphorylated proteins and was found to be a selective inhibitor of protein phosphatase 2A (PP2A). Cytostatin inhibited PP2A with an IC(50) of 0.09 microgram/ml in a non-competitive manner against a substrate, p-nitrophenyl phosphate, but it had no apparent effect on other protein phosphatases including PP1, PP2B and alkaline phosphatase even at 100 microgram/ml. On the contrary, dephosphocytostatin, a cytostatin analogue, without inhibitory effect on PP2A did not affect B16 cell adhesion including FAK and paxillin. These results indicate that cytostatin inhibits cell adhesion through modification of focal contact proteins such as paxillin by inhibiting a PP2A type protein serine/threonine phosphatase. This is the first report that describes a drug with anti-metastatic ability that inhibits PP2A selectively.
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PMID:Cytostatin, an inhibitor of cell adhesion to extracellular matrix, selectively inhibits protein phosphatase 2A. 1055 74

Integrin-mediated substrate adhesion of endothelial cells leads to intracellular signaling, including the activation of ERK 1/2 (extracellular regulated kinases 1 and 2), members of the mitogen-activated protein kinase (MAPK) family. MKP-1 is a dual-specificity protein phosphatase that may play an important role in regulating MAPK activity through dephosphorylation of threonine and tyrosine. Adhesion of human umbilical vein endothelial cells to fibronectin increased MKP-1 protein and mRNA levels, which reached a maximum at 60 min, while MAPK activity was maximal at 30 min. The MEK inhibitor PD98059 blocked activation of MAPK as well as the induction of MKP-1 during adhesion. The transcription inhibitor actinomycin D blocked MKP-1 induction and produced prolonged MAPK activation during adhesion. In contrast, endothelial adhesion to poly-L-lysine did not alter MAPK activity or MKP-1 levels. These findings demonstrate that integrin-mediated adhesion of endothelial cells to fibronectin results in transcriptional activation of MKP-1 through a MAPK-dependent mechanism. Regulation of MKP-1 by MAPK likely represents an important negative-feedback mechanism.
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PMID:Adhesion to fibronectin enhances MKP-1 activation in human endothelial cells. 1087 41


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