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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The heat-stable inhibitor of cAMP-dependent protein kinase (PKI) was shown previously to export the kinase catalytic subunit (C) from the nucleus (Fantozzi, D. A., Harootunian, A. T., Wen, W., Taylor, S. S., Feramisco, J.R., Tsien, R. Y., and Meinkoth, J. L. (1994) J. Biol. Chem. 269, 2676-2686), in addition to its ability to inhibit kinase activity. In this study, the mechanism of PKI export is investigated. The injection of a C-PKI complex containing both labeled PKI and C-subunit revealed that both proteins exit the nucleus in unison. A fusion protein of C-subunit with glutathione S-transferase (GST) (140 kDa) cannot transverse the nuclear membrane in either direction, but can be exported from the nucleus when complexed with PKI, supporting the presence of a nuclear export signal (NES) in the C-PKI complex. Fusions of PKI alpha with GST (70 kDa) or PKI beta 1 with maltose-binding protein (MBP) (50 kDa) remain effective at exporting complexes with C-subunit. The export of C-PKI is also sensitive to temperature and energy depletion. Taken together, these results demonstrate that export is both energy- and temperature-dependent, but size-independent, consistent with an active signal-mediated export process. GST-PKI exits from the nucleus even in the absence of C-subunit, indicating that the NES resides entirely on PKI, but suggesting that fusion of PKI to GST leads to a conformational change that mimics the exposure of the NES caused by the binding of C. Since both PKI alpha and PKI beta 1 can export C-subunit, the predicted export signal is likely to reside on the residues conserved between PKI alpha and PKI beta 1.
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PMID:Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal. 779 21

We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, for "related to cAMP-dependent protein kinases and protein kinase Cs". To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehrac1 gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli. The thrombin-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro. The preferred substrate for the enzyme was histone H1 with a Km of approx. 14 microM. Histone H3 and kemptide were phosphorylated at about half the rate of histone H1. Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme. The protein kinase activity was higher in the presence of Mn2+ than Mg2+. Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity. The pH optimum of the enzyme was 7.5. The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM. Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents. The expression and purification of enzymatically active Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E. histolytica.
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PMID:Expression and characterization of a rac family protein kinase of Entamoeba histolytica. 798 73

A specific antiserum against the human m3-muscarinic receptor subtype was made by subcloning a variant region of the third intracellular loop of the m3-receptor (Ser345-Leu463) into a bacterial expression plasmid that produced a fusion protein with glutathione S-transferase. In immunoblot studies this anti-serum identified the human m3-receptor expressed in transfected Chinese hamster ovary (CHO) cells (CHO-m3 cells, 1343 fmol/mg protein) as a diffuse band at approximately 97-110 kDa. In vivo labeling of the ATP pool in CHO-m3 cells with [32P]orthophosphate followed by immunoprecipitation of solubilized m3-receptors revealed that the unstimulated receptor existed in a phosphorylated form. Incubation of CHO-m3 cells with the cholinergic agonist carbachol (1 mM) increased the phosphorylated state of the receptor dramatically, primarily at serine. The time course for agonist-dependent phosphorylation was very rapid occurring within seconds of agonist addition and was maintained for at least 30 min. The muscarinic antagonist atropine (10 microM) inhibited agonist-stimulated phosphorylation. Neither forskolin (10 microM) nor the calcium ionophore, ionomycin (1 microM), had any effect on the state of phosphorylation of the m3-receptor, eliminating a role for cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in the agonist-dependent phosphorylation of m3-receptors. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (100 nM) did increase m3-receptor phosphorylation, an effect that was inhibited by the selective protein kinase C inhibitor RO-318220 (10 microM). However, agonist-stimulated m3-receptor phosphorylation was not inhibited by RO-318220 indicating that protein kinase C was not involved in agonist-induced m3-receptor phosphorylation. In conclusion the phosphorylation of m3-receptors, in vivo, was increased following the application of muscarinic agonist or PMA. The response to agonist was mediated via a kinase distinct from protein kinase C, protein kinase A and Ca2+/calmodulin dependent protein kinase, whereas the effect of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate was mediated by protein kinase C.
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PMID:Rapid agonist-mediated phosphorylation of m3-muscarinic receptors revealed by immunoprecipitation. 848 62

This study identifies a 100-residue domain within the rabbit skeletal muscle regulatory subunit (PP1G) that binds both type-1 protein phosphatase (PP1C) and glycogen. An N-terminal portion of PP1G was cloned by RT-PCR, and different sized fragments were expressed in bacteria as glutathione S-transferase (GST) fusion proteins. A GST-PP1G fusion containing residues 51-240 bound both PPIC and glycogen, whereas GST alone or fusions containing residues 51-140 or 241-360 bound neither PP1C nor glycogen. The PPIC in whole cell lysates or partially purified PP1C from skeletal muscle, or a complex of PP1C-MCLR-biotin, all bound more effectively than Mn(2+)-activated, recombinant PP1C purified from bacteria. Binding was enhanced by increasing the ionic strength and was disrupted by ethylene glycol, consistent with hydrophobic interactions being critical for stable association. Phosphorylation of the GST-PP1G fusion by cAMP-dependent protein kinase prevented completely association of PP1C. This domain of PP1G, from residues 141-240, contains two sequence motifs of hydrophobic residues: Gx8FEKx10W and DxFxFxIxL, that are conserved among the known glycogen-binding PP1 regulatory subunits. These segments are predicted to form an alpha helix and a beta sheet, and we propose that they are the sites for association with PP1C and glycogen, respectively.
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PMID:Protein phosphatase type-1 and glycogen bind to a domain in the skeletal muscle regulatory subunit containing conserved hydrophobic sequence motif. 890 29

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases. alpha1b-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse alpha1a-, alpha1b-, and alpha1d-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the alpha1b sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.
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PMID:Phosphorylation of the third intracellular loop of the mouse alpha1b-adrenergic receptor by cAMP-dependent protein kinase. 912 16

Two fusion proteins in which the regulatory domains of human protein kinase Calpha (Ralpha; amino acids 1-270) or mouse protein kinase Cepsilon (Repsilon; amino acids 1-385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase Calpha (PKCalpha) and other protein kinases in vitro. Both GST-Ralpha and GST-Repsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKCalpha. In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase. GST-Ralpha and GST-Repsilon, on a molar basis, were 100-200-fold more potent inhibitors of PKCalpha activity than was the pseudosubstrate peptide PKC19-36. In addition, a GST-Ralpha fusion protein in which the first 32 amino acids of Ralpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKCalpha activity. The three GST-R fusion proteins also inhibited protamine-activated PKCalpha and proteolytically activated PKCalpha (PKM), two lipid-independent forms of PKCalpha; however, the IC50 values for inhibition were 1 order of magnitude greater than the IC50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipid-activated PKCalpha may have resulted from sequestration of lipid activators. Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKCalpha catalytic activity through direct interactions. These data indicate that the R domains of PKCalpha and PKCepsilon are specific inhibitors of protein kinase Calpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.
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PMID:Inhibitory properties of the regulatory domains of human protein kinase Calpha and mouse protein kinase Cepsilon. 953 77

The fibrous sheath is a unique cytoskeletal structure in the sperm flagellum believed to modulate sperm motility. FSC1 is the major structural protein of the fibrous sheath. The yeast two-hybrid system was used to identify other proteins that contribute to the structure of the fibrous sheath or participate in sperm motility. When FSC1 was used as the bait to screen a mouse testis cDNA library, two clones were isolated encoding the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase. Deletion analysis using the yeast two-hybrid system and in vitro binding assays with glutathione S-transferase-FSC1 fusion proteins identified two RIalpha tethering domains on FSC1. A domain located at residues 219-232 (termed domain A) corresponds to the reported tethering domain for a type II regulatory subunit (RII) of cAMP-dependent protein kinase, indicating that this binding domain has dual specificity to RI and RII. Another RIalpha tethering site (termed domain B) at residues 335-344 shows specific binding of RIalpha and had no significant sequence homology with known RII tethering domains. However, helical wheel projection analysis indicates that domain B is likely to form an amphipathic helix, the secondary structure of RII tethering domains of protein kinase A anchoring proteins. This was supported by the finding that site-directed mutagenesis to disrupt the amphipathic helix eliminated RIalpha binding. This is apparently the first report of an RIalpha-specific protein kinase A anchoring protein tethering domain.
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PMID:Identification of tethering domains for protein kinase A type Ialpha regulatory subunits on sperm fibrous sheath protein FSC1. 985 4

Cellular functions of protein phosphatase-1 (PP1) are determined by regulatory subunits that contain the consensus PP1-binding motif, RVXF. This motif was first identified as the site of phosphorylation by cAMP-dependent protein kinase (PKA) in a skeletal muscle glycogen-targeting subunit (G(M)). We reported previously that a recombinant fusion protein of glutathione S-transferase (GST) and the N-terminal domain of G(M) [GST-G(M)-(1-240)] bound PP1 in a pull down assay, and phosphorylation by PKA prevented PP1 binding. Here we report that substitution of either Ala or Val for Ser-67 in the RVS(67)F motif in GST-G(M)-(1-240) essentially eliminated PP1 binding. This was unexpected because other glycogen-targeting subunits have a Val residue at the position corresponding to Ser-67. In contrast, a mutation of Ser-67 to Thr (S67T) in GST-G(M)(1-240) gave a protein that bound PP1 the same as wild type and was unaffected by PKA phosphorylation. Full length G(M) tagged with the epitope sequence DYKDDDDK (FLAG) expressed in COS7 cells bound PP1 that was recovered by co-immunoprecipitation, but this association was prevented by treatment of the cells with forskolin. By comparison, PP1 binding with FLAG-G(M)(S67T) was not disrupted by forskolin treatment. Neither FLAG-G(M)(S67A) nor FLAG-G(M)(S67V) formed stable complexes with PP1 in COS7 cells. These results emphasise the unique contribution of Ser-67 in PP1 binding to G(M). The constitutive PP1-binding activity shown by G(M)(S67T) opens the way for studying the role of G(M) multisite phosphorylation in hormonal control of glycogen metabolism.
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PMID:Mutations of the serine phosphorylated in the protein phosphatase-1-binding motif in the skeletal muscle glycogen-targeting subunit. 1065 42

Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase (A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1. Until now the kinase that phosphorylates Ser(67) had not been identified and the physiological role of Ser(67) phosphorylation was unknown. In this study we detected a high level of kinase activity in brain extract when a glutathione S-transferase (GST) fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein kinase (NCLK) in the brain extract could not be separated from each other by a series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated with anti-NCLK antibody from kinase-active column fractions. Purified NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed mutagenesis determined that NCLK phosphorylates Ser(67) of I-1. NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC(50) values approximately 9.5 and 13. 8 nM, respectively. When compared, A kinase-phosphorylated I-1 was only approximately 1.2 times more inhibitory than NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1 kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1.
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PMID:Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor. 1081 8

Glycogen-binding subunits for protein phosphatase-1 (PP1) target the PP1 catalytic subunit (PP1C) to glycogen particles, where the enzymes glycogen synthase and glycogen phosphorylase are concentrated. Here we identify sites within the striated muscle glycogen-binding subunit (G(M)) that mediate direct binding to glycogen synthase. Both PP1C and glycogen synthase were coimmunoprecipitated with a full-length FLAG-tagged G(M) transiently expressed in COS7 cells or C2C12 myotubes. Deletion and mutational analysis of a glutathione S-transferase (GST) fusion of the N-terminal domain of G(M) (residues 1-240) identified two putative sites for binding to glycogen synthase, one of which is the WXNXGXNYX(I/L) motif that is conserved among the family of PP1 glycogen-binding subunits. Either deletion of this motif or Ala substitution of Asn-228 in this motif disrupted the binding of glycogen synthase. Expression of full-length FLAG-G(M) in cells increased the activity of endogenous glycogen synthase, but protein disabled in either PP1 binding or glycogen synthase binding did not produce synthase activation. The results show that efficient activation of glycogen synthase requires a scaffold function of G(M) that involves simultaneous binding of both PP1C and glycogen synthase. Isoproterenol and forskolin treatment of cells decreased glycogen synthase binding to FLAG-G(M), thereby limiting synthase activation by PP1. This response was insensitive to inhibition by H-89, therefore probably not involving cAMP-dependent protein kinase, but did require inclusion of microcystin-LR during cell lysis, implying that phosphorylation was modulating binding of glycogen synthase. Phosphorylation control of binding to a scaffold site on the G(M) subunit of PP1 offers a new mechanism for regulation of muscle glycogen synthase in response to beta-adrenergic signals.
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PMID:Glycogen synthase association with the striated muscle glycogen-targeting subunit of protein phosphatase-1. Synthase activation involves scaffolding regulated by beta-adrenergic signaling. 1085 1


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