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)

Downstream regulation of the cAMP-dependent protein kinase (PKA) pathway is mediated by anchoring proteins (AKAPs) that sequester PKA to specific subcellular locations through binding to PKA regulatory subunits (RI or RII). The RII-binding domain of all AKAPs forms an amphipathic alpha-helix with similar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms that regulate AKAP-PKA affinity are not clearly defined. Using surface plasmon resonance (SPR) spectroscopy, we measured real-time kinetics of RII interaction with various AKAPs. Base-line equilibrium binding constants (K(d)) for RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nm, 119 nm, and 6.6 microm, respectively. PKA stimulation of intact Chinese hamster ovary cells increased RIIalpha binding to AKAP100/mAKAP and AKAP15/18 by approximately 7- and 82-fold, respectively. These results suggest that differences in primary sequence of the RII-binding domain may be responsible for the selective affinity of RII for different AKAPs. Furthermore, RII autophosphorylation may provide additional localized regulation of kinase anchoring. In cardiac myocytes, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be involved in adding additional specificity in intracellular signaling in diverse cell types and under conditions of cAMP/PKA activation.
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PMID:Selectivity and regulation of A-kinase anchoring proteins in the heart. The role of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase. 1099 82

Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. Wnt family genes encode secreted glycoproteins that act as ligands for membrane receptors belonging to the Frizzled family of proteins. Wnt-1 originally was found as a proto-oncogene that was upregulated in tumors caused by the mouse mammary tumor virus. The Drosophila homologue of Wnt-1, wingless, is a segment polarity gene that regulates body patterning of the fly embryo. In Xenopus, the Wnt pathway regulates formation of the ventral-dorsal axis. Although Wnt proteins are expressed widely in mammals, the function of the Wnt signaling pathway in normal adult mammalian tissues is not understood. Downstream components of the Wnt pathway, APC (adenomatous polyposis coli) and beta-catenin, clearly are involved in human cancer. There are also several reports that Wnt ligands are highly expressed in tumors. Wnt stabilizes cytoplasmic beta-catenin and activates beta-catenin/Lef-1 (lymphoid enhancer factor), Tcf (T-cell factor)-dependent gene transcription. This regulation of cytosolic beta-catenin is mediated by glycogen synthase kinase-3 (GSK-3) activity but in neither case is the mechanism known. The mechanism by which Wnt inhibits GSK-3 is unknown. Recent studies have shown that some of the intracellular signaling molecules that mediate the Wnt pathway are in complexes, including Dishevelled (Dsh or Dvl), GSK-3beta, and APC protein. However, little is known about how Wnt or other upstream stimuli regulate these complexes to stabilize beta-catenin. We took a variety of approaches to identify new components of the Wnt pathway. Using an expression-cloning technique, we isolated casein kinase I (CKI)epsilon as a positive regulator of beta-catenin in the Wnt pathway. Overexpression of CKIepsilon mimics Wnt by stabilizing beta-catenin, thereby increasing expression of beta-catenin-dependent genes. Inhibition of endogenous CKIepsilon attenuated gene transcription stimulated by Wnt or by Dsh. CKIepsilon forms a complex with Axin and the other downstream components of the Wnt pathway. CKIepsilon is a positive regulator of the Wnt pathway and a possible functional link between upstream signals and the intracellular Axin signaling complex that regulates beta-catenin. In separate experiments, we have identified a Dishevelled-associated kinase (DAK) that binds to Dsh and regulates its functions. Dsh is required for two different pathways, the Wnt pathway and planar polarity pathway in Drosophila. DAK dramatically enhances the function of Dsh in the Wnt pathway and inhibits its function in the planar polarity pathway. This chapter will discuss these newly identified components of the Wnt pathway.
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PMID:New steps in the Wnt/beta-catenin signal transduction pathway. 1103 39

Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca(2+) cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca(2+) but no significant difference between Ht31-expressing and control cells in the rate of Ca(2+) cycling or amplitude of the Ca(2+) transient. The increase in PKA-dependent phosphorylation of troponin I and myosin binding protein C on isoproterenol stimulation was significantly reduced in Ht31-expressing cells compared with controls. Our results demonstrate that, in response to beta-adrenergic stimulation, cardiomyocyte function and substrate phosphorylation by PKA is regulated by targeting of PKA by AKAPs.
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PMID:AKAP-mediated targeting of protein kinase a regulates contractility in cardiac myocytes. 1117 96

Viral-mediated gene transfer of troponin I (TnI) isoforms and chimeras into adult rat cardiac myocytes was used to investigate the role TnI domains play in the myofilament tension response to protein kinase A (PKA). In myocytes expressing endogenous cardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein C and decreased the Ca2+ sensitivity of myofilament tension. In marked contrast, PKA did not influence Ca2+-activated tension in myocytes expressing the slow skeletal isoform of TnI or a chimera (N-slow/card-C TnI), which lack the unique phosphorylatable amino terminal extension found in cTnI. PKA-mediated phosphorylation of a second TnI chimera, N-card/slow-C TnI, which has the amino terminal region of cTnI, caused a decrease in the Ca2+ sensitivity of tension comparable in magnitude to control myocytes. Based on these results, we propose the amino terminal region shared by cTnI and N-card/slow-C TnI plays a central role in determining the magnitude of the PKA-mediated shift in myofilament Ca2+ sensitivity, independent of the isoform-specific functional domains previously defined within the carboxyl terminal backbone of TnI. Interestingly, exposure of permeabilized myocytes to acidic pH after PKA-mediated phosphorylation of cTnI resulted in an additive decrease in myofilament Ca2+ sensitivity. The isoform-specific, pH-sensitive region within TnI lies in the carboxyl terminus of TnI, and the additive response provides further evidence for the presence of a separate domain that directly transduces the PKA phosphorylation signal.
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PMID:Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A. 1120 28

Untransformed CD4(+) Th1 cells stimulated with Ag and APC demonstrated a dependence on B7- and CD28-mediated costimulatory signals for the expression and function of AP-1 proteins. The induction of transactivation by the c-fos gene regulator Elk-1 mirrored this requirement for TCR and CD28 signal integration. c-Jun N-terminal kinase (JNK) (but not extracellular signal-regulated kinase or p38) protein kinase activity was similarly inhibited by neutralizing anti-B7 mAbs. Blockade of JNK protein kinase activity with SB 202190 prevented both Elk-1 transactivation and c-Fos induction. These results identify a unique role for B7 costimulatory molecules and CD28 in the activation of JNK during Ag stimulation in Th1 cells, and suggest that JNK regulates Elk-1 transactivation at the c-fos gene to promote the formation of AP-1 complexes important to IL-2 gene expression.
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PMID:CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation. 1144 Oct 89

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).
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PMID:Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p. 1144 92

Based on two criteria, the tightness of packing of myosin rods within the backbone of the filament and the degree of order of the myosin heads, thick filaments isolated from a control group of rat hearts had three different structures. Two of the structures of thick filaments had ordered myosin heads and were distinguishable from each other by the difference in tightness of packing of the myosin rods. Depending on the packing, their structure has been called loose or tight. The third structure had narrow shafts and disordered myosin heads extending at different angles from the backbone. This structure has been called disordered. After phosphorylation of myosin-binding protein C (MyBP-C) with protein kinase A (PKA), almost all thick filaments exhibited the loose structure. Transitions from one structure to another in quiescent muscles were produced by changing the concentration of extracellular Ca. The probability of interaction between isolated thick and thin filaments in control, PKA-treated preparations, and preparations exposed to different Ca concentrations was estimated by electron microscopy. Interactions were more frequent with phosphorylated thick filaments having the loose structure than with either the tight or disordered structure. In view of the presence of MgATP and the absence of Ca, the interaction between the myosin heads and the thin filaments was most likely the weak attachment that precedes the force-generating steps in the cross-bridge cycle. These results suggest that phosphorylation of MyBP-C in cardiac thick filaments increases the probability of cross-bridges forming weak attachments to thin filaments in the absence of activation. This mechanism may modulate the number of cross-bridges generating force during activation.
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PMID:Multiple structures of thick filaments in resting cardiac muscle and their influence on cross-bridge interactions. 1146 48

Mitotic checkpoints delay cell cycle progression in response to alterations in the mitotic apparatus, thus ensuring correct chromosome segregation. While improper spindle orientation activates the Bub2/Bfa1-dependent checkpoint in budding yeast, delaying exit from mitosis, lack of bipolar kinetochore-microtubule attachment activates a signal transduction cascade that prevents both anaphase onset and exit from mitosis by inhibiting the Cdc20/APC (Anaphase Promoting Complex)-mediated proteolysis of securin and inactivation of mitotic cyclin-dependent kinases (CDKs), respectively. Proteolysis of the securin Pdsl is necessary to liberate the separase Esp1, which then triggers sister chromatid separation, whereas inactivation of mitotic CDKs is a prerequisite for exit from mitosis and for starting a new round of DNA replication in the next cell cycle. In budding yeast, this latter checkpoint response involves the proteins Mad1, 2, 3, Bub1 and Bub3, whose vertebrate counterparts localize to unattached kinetochores. Mutations that alter other kinetochore proteins result in mitotic checkpoint activation, while the ndc10-1 mutation not only impairs kinetochore function, but also disrupts the checkpoint response, indicating a role for Ndc10 in this process. Here we present evidence that Ndc10 is not part of the Bub2/Bfa1-dependent pathway, and its role in the checkpoint response might also be different from that of the other Mad and Bub proteins. Indeed, Ndc10, unlike other mitotic checkpoint proteins, is not required for the mitotic block induced by overexpression of the Mpsl protein kinase, which is implicated in mitotic checkpoint control. Furthermore, the delay in mitotic exit caused by non-degradable Pds1, which does not require Mad and Bub proteins, depends on Ndc10 function. We propose that a pathway involving Ndc10 might monitor defects in the mitotic apparatus independently of the Mad and Bub proteins. Since the Espl separase is required for exit from mitosis in both ndc10-1 and nocodazole-treated mad2delta cells, the two signal transduction cascades might ultimately converge on the inactivation of Esp1.
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PMID:Role of the kinetochore protein Ndc10 in mitotic checkpoint activation in Saccharomyces cerevisiae. 1158 68

beta-Adrenergic stimulation increases stroke volume in mammalian hearts as a result of protein kinase A (PKA)-induced phosphorylation of several myocyte proteins. This study investigated whether PKA-induced phosphorylation of myofibrillar proteins directly affects myocyte contractility. To test this possibility, we compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKA. Consistent with previous studies, PKA increased phosphorylation levels of myosin binding protein C and troponin I, and reduced Ca(2+) sensitivity of force. PKA also significantly increased both maximal force (25.4+/-8.3 versus 31.6+/-11.3 microN [P<0.001, n=12]) and peak absolute power output (2.48+/-1.33 versus 3.38+/-1.52 microW/mg [P<0.05, n=5]) during maximal Ca(2+) activations. Furthermore, PKA elevated power output at nearly all loads even after normalizing for the increase in force. After PKA treatment, peak normalized power output increased approximately 20% during maximal Ca(2+) activations (n=5) and approximately 33% during half-maximal Ca(2+) activations (n=9). These results indicate that PKA-induced phosphorylation of myofibrillar proteins increases the power output-generating capacity of skinned cardiac myocytes, in part, by speeding the step(s) in the crossbridge cycle that limit loaded shortening rates, and these changes likely contribute to greater contractility in hearts after beta-adrenergic stimulation.
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PMID:Power output is increased after phosphorylation of myofibrillar proteins in rat skinned cardiac myocytes. 1173 84

Mutations in the glycogen synthase kinase 3beta (GSK3beta) phosphorylation sites of the beta-catenin gene exon 3 are found in 20-30% of human primary hepatocellular carcinoma (HCC), whereas mutations in the APC or AXIN genes are found in other HCC populations. These data strongly suggest that the Wnt signaling pathway is involved in hepatocarcinogenesis. To determine the role of beta-catenin in intestinal tumorigenesis, we earlier constructed a mutant mouse strain Catnb(lox(ex3)), in which exon 3 of the beta-catenin gene was sandwiched by loxP sequences. By genetic crosses of these mice with the Fabpl-cre transgenic mice that express the cre gene controlled by the fatty acid binding protein gene promoter, we introduced the beta-catenin stabilizing mutation into the small intestine and liver. Although numerous polyps were formed in the small intestine, we did not find any neoplastic (i.e., dysplastic) foci in the liver, and the mice died in 5 weeks after birth because of acute liver damage accompanying mitochondrial swelling. When a recombinant adenovirus that expresses the cre gene from a human cytomegalovirus early gene promoter was constructed and inoculated at a high multiplicity (10(9) plaque-forming units/mouse), the Catnb(lox(ex3)) mice showed marked hepatomegaly, with similar mitochondrial swelling in the hepatocytes, and died within 3 weeks after infection. On the other hand, when inoculated at lower multiplicities of infection (10(7) and 10(8) plaque-forming units/mouse, respectively), the Catnb(lox(ex3)) mice survived >6 months without any neoplastic foci in the liver, although the nuclear localization of beta-catenin was found in some hepatocytes even after 6 months. These results suggest that, in contrast to intestinal polyposis, the Wnt pathway activation by stabilized beta-catenin is not sufficient for hepatocarcinogenesis, but additional mutations or epigenetic changes may be required.
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PMID:Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin. 1192 13


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