Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Annexin I (lipocortin I) binds to secretory granule membranes and promotes their aggregation in a Ca(2+)-dependent manner [Creutz, C. E., et al. (1987) J. Biol. Chem. 262, 1860-1868; Drust, D. S., & Creutz, C. E. (1988) Nature 331, 88-91]. It is also phosphorylated on serine residues when bovine chromaffin cells are stimulated to secrete [Michener, M. L., et al. (1986) J. Biol. Chem. 261, 6548-6555], suggesting phosphorylation may be involved in modulating the function of annexin I. We report here that phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold. This inhibition was readily reversed when the protein was dephosphorylated by protein phosphatase 2A. The inhibition was not due to inability of phosphorylated annexin I to bind to chromaffin granules, since the phosphorylated form bound to the granule membrane at slightly lower levels of calcium than the native form. The phosphorylated annexin I also bound to 20% phosphatidylserine/80% phosphatidylcholine vesicles at lower Ca2+ levels than the native form. The inhibitory effect of phosphorylation on the granule aggregating activity of annexin I was found to be amplified by an unusual mechanism: The phosphorylated form inhibited the activity of the unphosphorylated form. The possible importance of the regulation of annexin I activity by phosphorylation in exocytosis is discussed.
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PMID:Regulation of the chromaffin granule aggregating activity of annexin I by phosphorylation. 139 Jul 76

We have identified two major proteins in human neutrophils that are phosphorylated in vitro by protein kinase C (PKC) as lipocortins III and a fragment of a lipocortin-like 68-kDa protein. In electroporated cells, the 68-kDa protein was phosphorylated during stimulation of the cells with either FMLP or PMA. Lipocortins are of interest because of their Ca2(+)- and phospholipid-dependent actin binding properties and ability to inhibit phospholipase A2. Two crude fractions of enzymes and proteins exposed to [gamma-32]PATP in the presence of Ca2+, Mg2+, phosphatidylserine and 1,2-oleoyl-acetyl-rac-glycerol were analyzed by gel electrophoresis and autoradiography. A number of proteins in a detergent-free fraction, including proteins at 36 and 32 kDa, were phosphorylated in the presence of these cofactors. In contrast, only two major proteins (35 and 32 kDa) were phosphorylated in a detergent-extracted fraction. Phosphorylation of the 36, 35, and 32 kDa proteins required the presence of Ca2+, Mg2+, and phosphatidylserine in our soluble fraction and detergent extract, indicating PKC-dependent phosphorylation. The 32-kDa protein phosphorylated in both the soluble fraction and detergent extract was identified as lipocortin III by immunoprecipitation with a cross-reactive antibody that recognized lipocortin I and comparison of cyanogen bromide (CNBr) cleavage patterns of this protein with a lipocortin III standard. The 68-kDa protein was identified as a lipocortin VI-like protein by immunoprecipitation with anti-calelectrin. Additionally, the CNBr cleavage pattern of the 68-kDa protein was similar to that of the 36-kDa protein phosphorylated in our soluble fraction. Autoradiograms of the 68- and 36-kDa fragments immunoprecipitated from our soluble fraction with anticalelectrin and cleaved with CNBr showed that both of these proteins were phosphorylated in this sample. Because phosphorylation is known to change the functional characteristics of the lipocortins, the potential exists to link PKC and lipocortins in neutrophils to regulation of granulemembrane interactions or mediation of inflammation.
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PMID:Lipocortins are major substrates for protein kinase C in extracts of human neutrophils. 169 66

Calpactins are members of the annexin family of structurally related Ca2(+)-dependent membrane binding proteins. Recent studies suggest a role for calpactins in the membrane fusion event of exocytosis. We show in this work that two members of the annexin family which are immunologically related to calpactin I (p36, annexin II) and calpactin II (p35, annexin I) are present in anterior pituitary cells. When sheep adenohypophyseal cells are disrupted in the absence of a Ca2+ chelator, immunoreactive calpactins associate with the crude vesicle fraction. Further purification of this subcellular fraction by sucrose density gradient centrifugation revealed a differential distribution: calpactin I was associated with secretory granule membranes and with plasma membranes, whereas calpactin II was found primarily with the plasma membrane fraction. Consistent with the Ca2+ and phospholipid binding properties of the calpactins, extraction of these proteins from the pituitary membranous fractions required sequential treatment with a detergent, octylglucoside, in the presence of 1 mM Ca2+ followed by solubilization with EGTA. Calpactins contain sites for phosphorylation by protein kinase C, and in this study we found phosphoprotein substrates for protein kinase C associated with secretory granule and plasma membranes which could be immunoprecipitated with calpactin antisera. In summary, the characteristics in anterior pituitary secretory cells of these two members of the annexin family lend support to the hypothesis that calpactins, potentially regulated by Ca2+ and by phosphorylation, may have a role in exocytosis.
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PMID:Membrane-specific association of annexin I and annexin II in anterior pituitary cells. 182 59

Activation of phospholipase A2 (PLA2) in response to external stimuli may play a pivotal role in signal-transduction pathways via the generation of important cellular intermediates, including prostaglandins. Epidermal growth factor (EGF) has been shown to modulate prostaglandin production, possibly via direct activation of PLA2 or indirectly via interaction with a PLA2-modifying protein such as lipocortin I. We have investigated these pathways with two CHO cell-lines, one (CHOwt) transfected with the full-length human EGF receptor and the second (CHO 11) with a deletion mutant, delta 990, that has lost the autophosphorylation sites and part of the internalization domain. CHOwt cells responded to EGF with a rapid rise in lysophosphatidylcholine and arachidonic acid release concomitant with an increase in prostaglandin production. However, in the non-internalizing CHO 11 cells no such activation of PLA2 was observed. This was not due to an intrinsic lack of PLA2 in these cells, as PLA2 activation was shown on melittin addition, nor was this difference due to a defect in intracellular pathways, as arachidonic acid was released from both cell types by Ca2+ and protein kinase C modulators. However, only in CHOwt cells were these responses potentiated by concomitant addition of EGF. Thus the cytoplasmic subdomain of the EGF receptor, containing the major sites of autophosphorylation and the internalization domain, seems to be involved in the activation of PLA2 by EGF. In addition, we have shown that phosphorylation of lipocortin I is unlikely to play a role in PLA2 activation. In CHOwt cells and a positive control cell line, A431, activation of PLA2 was complete by 10 min, at which time there was no evidence of lipocortin I phosphorylation.
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PMID:Modulation of phospholipase A2 activity by epidermal growth factor (EGF) in CHO cells transfected with human EGF receptor. Role of receptor cytoplasmic subdomain. 182 22

Incubation of plasma membranes isolated from bovine aorta with either 0.5 mM CaCl2 or with a phorbol ester (1 microM phorbol 12,13-dibutyrate) and phosphatidylserine in an EGTA-containing buffer resulted in the phosphorylation of 10 proteins (Mr of 158, 105, 75, 62, 44, 39, 33, 22, 15 and 9 kDa), presumably due to activation of endogenous protein kinase C (PKC). After heat treatment of the aortic plasma membranes at 80 degrees C for 5 min in order to inactivate all endogenous protein kinase, phosphatase and ATPase activities, membrane phosphorylation was absolutely-dependent upon the addition of an exogenous, partially-purified PKC preparation from bovine aorta. Under these conditions, a total of 17 phosphoproteins could be detected (Mr of 158, 105, 75, 44, 39, 33, 30, 29, 27, 25, 22, 17.5, 16, 15, 11, 10 and 9 kDa). The most prominent phosphoprotein band in native membranes had a molecular weight of 75 kDa (p75); several characteristics suggest that p75 might be autophosphorylated PKC. The phosphorylation of aortic plasma membranes by exogenous PKC required phosphatidylserine and was calcium-dependent (10(-5) to 10(-7) M Ca2+); the addition of diolein resulted in little or no enhancement of phosphorylation. Replacement of phosphatidylserine with oleic acid resulted in the same number of phosphoproteins, but the extent of phosphorylation was diminished. The phosphorylation pattern was altered slightly if the aortic plasma membranes were isolated in the presence of 1 mM Ca2+ instead of EGTA buffers as in the standard procedure. Experiments were performed to determine if the p39 substrate of PKC in aortic plasma membranes was calpactin II (lipocortin I). Immunoblotting established that calpactin II was present in aortic plasma membranes, but there was no corresponding phosphoprotein on the autoradiographs.
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PMID:Phosphorylation of aortic plasma membranes by protein kinase C. 183 27

Lipocortin I (LPC-I, also called annexin I) is a 35-kD protein that binds phospholipids and actin in a Ca(++)-dependent manner. It is also a major substrate for EGF receptor/kinase and protein kinase C, and a putative inhibitor of phospholipase A2, which produces chemical mediators to cause inflammation. Psoriasis (PS) is an inflammatory skin disease characterized by a rapid turnover of keratinocytes and a defect in keratinization with increased activities of phospholipase C and A2, and EGF receptor. To understand the mechanism of the PS lesion formation and the function of LPC-I, its distribution was studied in the epidermis of PS, subacute eczema and normal skin, and in tumor cells of seborrheic keratosis and Bowen's disease. This study involved immunofluorescence and immunoblotting using affinity-purified polyclonal and monoclonal antibodies specific to LPC-I and to its Ca(++)-bound form. In normal, nonlesional PS and subacute eczema epidermis, LPC-I was detected mainly in the cytoplasm of the suprabasal cells, although it was on the inner aspects of the plasma membrane in some parts of the granular layer. In lesional epidermis of PS, it was localized mainly on the inner aspects of the plasma membrane, but not in the cytoplasm of the whole suprabasal cells as the Ca(++)-bound form, indicating a preferential localization on the plasma membrane. This membrane-binding of LPC-I was also observed in seborrheic keratosis, but not in Bowen's disease. These results suggest that the binding of LPC-I to the plasma membrane occurs actually in living cells, plays a role, not necessarily disease specific, in the PS lesion formation, and has some relevance to normal or abnormal differentiation of keratinocytes.
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PMID:Lipocortin I (annexin I) is preferentially localized on the plasma membrane in keratinocytes of psoriatic lesional epidermis as shown by immunofluorescence microscopy. 183 17

When cultured in the absence of thyreostimulin (TSH), thyroid cells lose some of their differentiated functions such as iodide transport and its incorporation into thyroglobulin. In the presence of TSH (0.1 mU/ml), these differentiated functions are preserved ("TSH cells"). The addition of tetradecanoyl phorbol 13 acetate (TPA) inhibits some differentiated functions of the cells and provokes important modifications of bio-signalling pathways. The protein kinase C (pKC) activity, unchanged in "control" and "TSH cells", was dramatically modified in TPA treated cells. After translocation, the pKC activity was down-regulated and the phosphorylation of its endogenous substrates (35-38 kDa) disappeared. Among these substrates, we identified the lipocortin I (LC I) (35 kDa), a phospholipase A2 inhibitory protein related to the Ca2+ binding protein family. By monodimensional electrophoresis (PAGE-SDS) and western-blot, we evidenced the presence of LCI in cytosols and particulate extracts. By 2 dimensional electrophoresis (PAGE-SDS and IEF) and western-blot we identified a phosphorylated and unphosphorylated LCI protein. The phosphorylation of LCI by pKC decreased its isoelectric point from 6.9-6.6. The modifications of pKC activity and LCI phosphorylation and the changes in the bio-signalling pathways can partly account for the loss of differentiation observed in control or TPA treated cells.
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PMID:[Modifications of protein kinase C activity and phosphorylation of lipocortin I in cultures of pig thyroid cells]. 214 21

Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.
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PMID:Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C. 245 90

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.
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PMID:A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages. 249 91

To examine in vivo phosphorylation of lipocortin I we made use of a polyclonal antibody to an amino terminal peptide of lipocortin I. This antibody does not recognize any other member of the annexin protein family, and can both immunoprecipitate lipocortin I and recognize this protein on western blots. Using cleaved forms of lipocortin I, we have been able to demonstrate that protein kinase C phosphorylates this protein in vitro within the first 29 amino terminal amino acids. However, the addition of phorbol esters to A431 cells over a wide range of concentrations and for varying periods of time did not stimulate the phosphorylation of this protein. Since in vitro lipocortin I is an excellent substrate for all three isoforms, alpha, beta, gamma, of protein kinase C, the discrepancy in these findings is not secondary to the presence of varying forms of this protein kinase within different cell types.
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PMID:Lack of phosphorylation of lipocortin I in A431 epidermoid carcinoma cells treated with phorbol esters. 252 94


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