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)

We previously demonstrated that, in rat pancreatic acinar cells, the high-affinity cholecystokinin (CCK) receptor agonist JMV-180 utilizes the phospholipase A2 (PLA2) cascade to mediate Ca2+ oscillations and amylase secretion. In contrast, the low-affinity CCK receptor utilizes the phospholipase C beta 1 (PLC beta 1) pathway. We have investigated structural requirements of CCK analogues to activate different intracellular pathways. CCK analogues such as CCK-8 [Met28,31; half-maximal effective concentration (EC50) = 0.4 pM], CCK-7 (Met28,31; EC50 = 0.7 pM), and NONA (Thr28/Nle31; EC50 = 5 pM) caused a biphasic amylase secretion. Reduction of secretion occurred with high doses of these peptides (> 100 pM). In contrast, CCK-5 (Met31; EC50 = 20,000pM), JMV-180 (Nle28,31; 1,500 pM), and OPE (Nle28,31; 200 pM) caused a monophasic secretion. CCK-8, but not JMV-180, increased protein kinase C (PKC) activities. The PKC activator phorbol ester inhibited an increase in myo-inositol 1,4,5-trisphosphate levels induced by CCK-8 and abolished monophasic amylase secretion induced by OPE. CCK-8, CCK-7, and NONA caused Ca2+ oscillations (< 100 pM) or large Ca2+ transients (> 100 pM). In contrast, JMV-180 and OPE evoked Ca2+ oscillations, even in high doses. Ca(2+)-signaling modes induced by CCK-5 were intermediate types between CCK-8 and JMV-180. CCK-8- and CCK-7-stimulated Ca2+ spikes were inhibited by the PLC inhibitor U-73122, but not by the PLA2 inhibitor ONO-RS-082. The action of CCK-5 was only partially sensitive to the PLC inhibitor. In contrast, JMV-180- and OPE-stimulated Ca2+ oscillations were inhibited by the PLA2, but not by the PLC, inhibitor. NONA was sensitive to PLC and PLA2 inhibitors. Although JMV-180 differs from CCK-8 by having an Asp-2 phenylethylester, rather than an Asp-phenylalanine amide, it is unlikely that these differences in the carboxyl terminus are important in determining which second-messenger systems will be activated. This is because CCK-5 (Phe33-CONH2) causes monophasic amylase secretion and Ca2+ oscillation in a manner similar to those induced by JMV-180 (2-phenylethylester). Meanwhile NONA (Phe33-CONH2) appeared to activate PLC and PLA2 pathways. The actions of all CCK analogues were abolished by L-364,718, indicating mediation by CCK-A receptors. Therefore, depending on the agonists used, CCK-A receptor activation in pancreatic acini may result in differential involvement of second-messenger systems, Ca2+ signal transduction, and amylase secretion. On the basis of the amino acid sequence of the carboxy terminus of CCK analogues, it appears that key amino acids for this differentiation are Met28 (or Thr28) for PLC pathways and Nle28 for PLA2 pathways.
 ...
PMID:Structural requirements of CCK analogues to differentiate second messengers and pancreatic secretion. 876 Jan 1

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a widely expressed, prominent substrate for protein kinase C. MARCKS is largely associated with membranes in cells, and hydrophobic interactions involving the amino-terminal myristoyl moiety are thought to play a role in anchoring MARCKS to cellular membranes. In addition, experiments in cell-free systems have suggested that electrostatic interactions between the positively charged phosphorylation site/calmodulin binding domain (PSD) of MARCKS and negatively charged membrane lipids are also involved in this association. Although it has been inferred from phosphorylation experiments, the electrostatic nature of the interaction between the PSD and membranes has not been demonstrated directly in intact cells. We expressed human MARCKS mutated in the myristoylation site and the PSD in REF52 cells; the cells were then fractionated by ultracentrifugation. Both nonmyristoylatable MARCKS and MARCKS in which the four serines in the PSD were mutated to aspartic acids, mimicking phosphorylation, exhibited decreased membrane affinity when compared to the fully myristoylated, wild-type, tetra-Ser protein or a myristoylated, tetra-Asn mutant. A double mutant, nonmyristoylatable protein in which the four serines in the PSD were mutated to aspartic acids exhibited negligible membrane association. Similar results were obtained in 293 cells that stably expressed chicken MARCKS mutated in the same domains. The double mutant, nonmyristoylatable tetra-Asp chicken protein exhibited little membrane association as determined by both subcellular fractionation and immunoelectron microscopy. These results indicate that myristoylation and electrostatic interactions involving the PSD exert independent, essentially additive effects on the membrane association of MARCKS in intact cells.
 ...
PMID:Myristoylation-dependent and electrostatic interactions exert independent effects on the membrane association of the myristoylated alanine-rich protein kinase C substrate protein in intact cells. 879 48

alpha-Calponin is a thin-filament-associated protein which has been implicated in the regulation of smooth muscle contraction. Quantification of the tissue content of rat tail arterial smooth muscle revealed approximately half the amount of alpha-calponin relative to actin compared with chicken gizzard and other smooth muscles, suggesting that this tissue would be particularly suitable for investigation of the effects of exogenous alpha-calponin on the contractile properties of permeabilized muscle strips. Rat tail arterial strips demembranated with Triton X-100 retained approximately 90% of their complement of alpha-calponin, and exogenous chicken gizzard alpha-calponin (which conveniently has a slightly lower molecular mass than the rat arterial protein) bound to the permeabilized muscle, presumably through its high affinity for actin. Exogenous alpha-calponin inhibited force in demembranated muscle strips in a concentration-dependent manner when added at the peak of a submaximal Ca(2+)-induced contraction, with a half-maximal effect at approximately 3 microM alpha-calponin. Pretreatment of demembranated muscle strips with alpha-calponin inhibited subsequent force development at all concentrations of Ca2+ examined over the activation range. The inhibitory effect of alpha-calponin was shown to be Ca(2+)-independent, since exogenous alpha-calponin also inhibited force in the absence of Ca2+ in demembranated muscle strips containing thiophosphorylated myosin. Phosphorylation of alpha-calponin on Ser-175 by protein kinase C has been suggested to alleviate the inhibitory effect of alpha-calponin on smooth muscle contraction. To test this hypothesis, the effects on Ca(2+)-induced and Ca(2+)-independent contractions of demembranated muscle strips of phosphorylated alpha-calponin and three site-specific mutants of alpha-calponin (in which Ser-175 was replaced by Ala, Asp or Thr) were compared with the effects of unphosphorylated tissue-purified and recombinant wild-type alpha-calponins. The recombinant wild-type protein behaved identically to the unphosphorylated tissue-purified protein, as did the S175T mutant, which is known to bind actin with high affinity and to inhibit the actin-activated myosin MgATPase in vitro. On the other hand, phosphorylated alpha-calponin and the S175A and S175D mutants, which bind weakly to actin and have little effect on the actin-activated myosin MgATPase in vitro, failed to cause significant inhibition of force induced by Ca2+ or myosin thiophosphorylation. These results support a role for alpha-calponin in the regulation of smooth muscle contraction and indicate the functional importance of Ser-175 of alpha-calponin as a regulatory site of phosphorylation.
 ...
PMID:Inhibition by calponin of isometric force in demembranated vascular smooth muscle strips: the critical role of serine-175. 891 94

Low temperature induces platelet aggregation, but this phenomenon is slight and poorly reproduced. However, heparin potentiated the reaction in a dose dependent manner. The degree of aggregation increased as the temperature at which the platelet-rich plasma was chilled was lowered, and as the time of chilling lengthened. Acetylsalicylic acid, a cyclooxygenase inhibitor, and staurosporin, an inhibitor of protein kinase C, partially inhibited cold-induced platelet aggregation (CIPA), suggesting that at least part of the reaction mechanism involves production of thromboxane A2 and activation of protein kinase C. Prostaglandin E1 (PGE1), which inhibits platelet responses through elevating platelet cyclic AMP, completely blocked CIPA, suggesting that PGE1 dependent pathway in platelets plays an important role for CIPA. The inhibition of CIPA by these inhibitors suggests that CIPA is aggregation with platelet activation but not platelet agglutination. Extracellular Ca2+ is essential for CIPA because ethylene glycol-bis (beta-aminoethylether) N, N, N', N'-tetraacetic acid (EGTA), extracellular Ca2+ chelating agent, completely inhibited CIPA. Monoclonal antibodies against glycoprotein (GP) IIb/IIIa (10E5, P2) and Ang-Gly-Asp-Ser-peptide (RGDS-peptide) which inhibit fibrinogen binding to GPIIb/IIIa completely blocked CIPA but monoclonal antibodies against GPIb (6D1, SZ2) partially. In addition, CIPA occurred only when fibrinogen was added to washed platelets suspension. These results indicate that CIPA is dependent on binding of fibrinogen to GPIIb/IIIa and GPIb is partly related with the reaction.
 ...
PMID:The mechanism of cold-induced platelet aggregation in the presence of heparin. 892 96

Single-site variants in the calmodulin-binding domain of RC3/neurogranin were heterologously expressed in Xenopus oocytes to examine their effects on serotonin-evoked currents. RC3 variants serine36 -->alanine (Ser36-->Ala), serine36-->glycine (Ser36-->Gly), and phenylalanine37-->tryptophan (Phe37-->Trp), which bind calmodulin but are deficient in protein kinase C (PKC) phosphorylation, display serotonin-evoked Ca(2+)-dependent Cl- currents in oocytes similar to control oocytes. A serine36-->aspartate (Ser36-->Asp) variant, which does not bind calmodulin and mimics the PKC-phosphorylated state of RC3, significantly enhances serotonin-evoked currents in a manner similar to wild-type. The results suggest that RC3 not only regulates the availability of free calmodulin in a dendritic spine but also, when phosphorylated, independently stimulates G-protein coupled second messenger pathways that generate inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG) and intracellular Ca2+.
 ...
PMID:Functional studies of single-site variants in the calmodulin-binding domain of RC3/neurogranin in Xenopus oocytes. 897 10

Integrins, among the various classes of cell adhesion receptors, are particularly associated with cell adhesion to extracellular matrices. They are heterodimeric transmembrane proteins with large ectodomains and short cytoplasmic tails. In many cases the sequence recognized by the integrins in the extracellular matrix proteins is the tripeptide Arg-Gly-Asp (RGD). Short synthetic peptides containing this sequence can inhibit tumor cell invasion in vitro and tumor dissemination in vivo. Because the alpha 5 beta 1 integrin appears to be the target of the peptides in many types of tumors, we have used phage display libraries to analyze the specificity of alpha 5 beta 1 and have isolated potent and specific inhibitors for this integrin. Increased expression of the alpha 5 beta 1 integrin, which is a fibronectin receptor, can also suppress cell migration and tumor cell invasion. We suggest this effect may be mediated through increased deposition of fibronectin matrix around the cells, because we found that the fibrillar matrix fibronectin suppresses tumor cell migration. There is increasing evidence that signals are elicited by the binding of integrins to their target proteins. This possibility has generated a great deal of interest in the cytoplasmic molecules that might mediate the integrin-associated signaling. At least two kinases, a novel tyrosine kinase, focal adhesion kinase (fak), and protein kinase C (PKC), are activated by integrin-mediated cell attachment. Moreover, a phosphorylated 190 kDa protein-associated with the alpha v beta 3 integrin has been found Anchorage dependence of cells and the migration-promoting activity of cell adhesion molecules are likely to depend on signal transduction through such molecules.
 ...
PMID:Cell adhesion and tumor metastasis. 898 67

Intracellular proteolysis by the calpains, a family of Ca2+ activated cysteine proteases, is a ubiquitous yet poorly understood process. Their action is implicated in an array of cellular and pathologic processes, including long-term potentiation, synaptic remodeling, protein kinase C and steroid receptor activation, ischemic cellular injury, and apoptosis. Unlike most proteases, the calpains display unusually strict substrate specificity, often cleaving only one or two bonds in proteins with hundreds of potential sites. Studies of synthetic peptides have defined sequences that modulate their specificity, but little data exist in the context of a bona fide protein. A prominent substrate for mu-calpain is alpha II spectrin (fodrin, brain spectrin), which is cleaved between Tyr1176 and Gly1177 within spectrin's 11th structural repeat unit. We have cloned and characterized human fetal brain alpha II spectrin (GenBank no. U26396) and identified a new Thr1300 to Ile polymorphism. From this clone, recombinant GST-fusion proteins representing repeat units 8-14 have been prepared and used to systematically explore the in vitro determinants of mu-calpain sensitivity. Twenty different amino acids were substituted by site-directed mutagenesis for wild-type Val1175, the penultimate (P2) residue flanking the major calpain cleavage site in alpha II spectrin. Gly, Pro, and Asp, and to a lesser extent Phe and Glu, substantively inhibited the susceptibility of this site to mu-calpain; other substitutions yielded lesser effects. Dynamic molecular modeling of the 11th structural repeat of human alpha II spectrin incorporating the various mutations suggests that the calpain cleavage site with its flanking calmodulin binding domain interrupts helix C of alpha II spectrin's 11th repetitive unit without significantly disrupting the repeat's triple-helical motif. This model predicts that the critical Tyr1176-Gly1177 bond occurs in a highly exposed loop juxtaposed between helix C and the calmodulin binding domain and that mutations at the P2 position subtly alter the conformation about this site. We conclude that secondary and tertiary conformational features surrounding the cleavage site, rather than the linear sequence itself, dominate the determinants that define alpha II spectrin's mu-calpain susceptibility.
 ...
PMID:Site-directed mutagenesis of alpha II spectrin at codon 1175 modulates its mu-calpain susceptibility. 899 18

Serine 657 in protein kinase C-alpha (PKCalpha) is a site of phosphorylation on expression of the recombinant protein in mammalian cells. To define the function of this phosphorylation, PKCalpha species with mutations of this site were investigated. The alanine mutant, S657A PKCalpha, displayed slow phosphate accumulation in pulse-chase experiments, indicating a rate-limiting role in the initial phase of phosphorylation. Consistent with this, the aspartic acid mutant, S657D PKCalpha, showed an increased rate of phosphate accumulation. Both the S657D and S657A PKCalpha mutants were slow to accumulate as fully phosphorylated forms during a second phase of phosphorylation. This latter property is shown to correlate with an increased phosphatase sensitivity and decreased protein kinase activity for these two PKCalpha mutants. It is further shown that once fully phosphorylated, the S657D PKCalpha mutant displays WT PKCalpha properties with respect to thermal stability and phosphatase sensitivity in vitro and in vivo; in contrast, the S657A PKCalpha mutant remains sensitive. The properties of the Ser-657 site PKCalpha mutants define functional roles for this phosphorylation in both the accumulation of phosphate on PKCalpha as well as in its agonist-induced dephosphorylation. These results are discussed in the context of a working model of PKCalpha behavior, providing insight into the workings of other kinases with equivalent sites of phosphorylation.
 ...
PMID:Phosphorylation of protein kinase C-alpha on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. 901 3

As one of the first steps to elucidate the relationship between the structure and function of CTP:phosphocholine cytidylytransferase (EC 2.7.7.15) in plants, the cytidylyltransferase cDNA of Arabidopsis thaliana was cloned and characterized. The A. thaliana cytidylyltransferase cDNA is 1447 bp long and contains an open reading frame of 993 bp coding for a protein of 331 amino acids. The deduced structure of the enzyme was composed of three main regions; the catalytic domain in the N-terminal half, the hydrophilic C-terminal region and the amphipathic domain in the middle. The catalytic domain region was relatively well conserved among different organisms, showing 76 and 72% homology with the rat and yeast protein sequences, respectively. The hydropathy profile revealed that the C-terminal non-catalytic portion of the protein was very hydrophilic, highly enriched in negatively charged aspartic acid and glutamic acid residues. In the region between the catalytic domain and the C-terminal region, there was an amphipathic alpha-helical domain, which was believed to bind the membrane surface in the active formation. Unlike animal counterparts, there was only one potential site of phosphorylation by protein kinase C and none by Ca2+/calmodulin protein kinase II in the C-terminal region. The identity of cytidylyltransferase cDNA was verified by successful transformation of a yeast mutant defective in the enzyme activity, using an expression vector inserted with the A. thaliana cytidylyltransferase cDNA. This was further confirmed by in vivo analysis of the enzyme reaction product after labeling the yeast transformants with radioactive phosphocholine. Southern analysis indicated the presence of a single copy of the citidylyltransferase gene in A. thaliana.
 ...
PMID:Cloning of CTP:phosphocholine cytidylyltransferase cDNA from Arabidopsis thaliana. 908 66

Both protein kinase C and the retinoblastoma tumor suppressor protein have been linked to the regulation of cell growth and cell death, suggesting the differential roles these factors play in mediating cell fate. In some cells, protein kinase C-induced activation of the retinoblastoma protein results in G1 arrest. However, inducible overexpression and activation of the protein kinase Calpha isozyme or the addition of 12-O-tetradecanoylphorbol-13-acetate in the prostate epithelial cell line, LNCaP, resulted in apoptosis preceded by induction of p21 and dephosphorylation of the retinoblastoma protein. Consistent with a role for the retinoblastoma growth suppressor protein in protein kinase C-induced apoptosis, DU145 cells, which do not express functional retinoblastoma protein or LNCaP cells, which have been transfected with the retinoblastoma inhibitor, E1a, were resistant to apoptosis. LNCaP apoptosis was initiated by a unique conflict between the growth-suppressive activity of the retinoblastoma protein and growth-promoting mitogenic signals. Thus, when this conflict was prevented by serum depletion, apoptosis was suppressed. The caspase family of cysteine proteases is believed to encompass the execution machinery of mammalian apoptosis, and addition of the cell-permeable caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, afforded nearly total protection from protein kinase C-signaled apoptosis. This protection correlated with the total loss of caspase activity as measured by the proteolytic cleavage of nuclear poly(ADP-ribose) polymerase. On the basis of these results, we propose that protein kinase C regulates a novel cell death pathway that is initiated by a cellular conflict between retinoblastoma growth-suppressive signals and serum mitogenic signals in proliferating prostate epithelial cells and that is executed by the caspase family of cysteine proteases.
 ...
PMID:Retinoblastoma protein-dependent growth signal conflict and caspase activity are required for protein kinase C-signaled apoptosis of prostate epithelial cells. 927 34


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>