Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein that exhibits greater substrate specificity for cGMP-dependent protein kinase than for cAMP-dependent protein kinase has been purified 8,000-fold from cytosol of rabbit cerebellum to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein, termed G-substrate, is a monomer of 23,000 daltons. It is heterogeneous on isoelectric focusing, exhibiting three isoelectric forms over the pH range of 5.2-5.6 cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of G-substrate, both into threonine residues. The protein has a high content of aspartate, glutamate, and proline. The hydrodynamic properties, heat stability, and acid solubility of this protein are consistent with an unfolded, nonglobular structure. G-substrate is localized primarily in the cytosol of cerebellum, although low concentrations of a phosphorylated protein with a similar molecular weight are detected in other brain regions.
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PMID:A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. I. Purification and characterization. 625 70

Kinetic studies on the activity of purified cGMP-dependent protein kinase and catalytic subunit of cAMP-dependent protein kinase have been carried out using a protein termed G-substrate (see preceding paper) as the phosphate acceptor. Each enzyme catalyzed the phosphorylation of 2.0-2.1 mol of 32P/mol of G-substrate, with phosphorylation occurring primarily at threonine residues. When phosphorylation was carried out in the simultaneous presence of the two enzymes, the stoichiometry increased only slightly, to a value of 2.4, suggesting that both enzymes phosphorylated the same two sites. Initial rate studies on the phosphorylation of G-substrate by cGMP-dependent protein kinase yielded a Km of 0.21 microM and a Vmax of 2.2 mumol/min/mg. Similar studies with the cAMP-dependent protein kinase yielded a Km of 5.8 microM and a Vmax of 2.3 mumol/min/mg. cGMP-dependent protein kinase thus exhibited a high degree of specificity towards this substrate which was apparently based on selective substrate binding rather than catalytic efficacy. The activity of cGMP-dependent protein kinase towards G-substrate was maximal at pH 7.5-8.0 and a Mg2+ concentration of 1-3 mM. Activity declined sharply at high ionic strength (greater than 20 mM KCl).
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PMID:A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. II. Kinetic studies on its phosphorylation by guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases. 625 71

Cyclic GMP (cGMP)-dependent protein kinase (PKG) has a limited substrate specificity, and only cerebellar G-substrate has been demonstrated in brain. In view of the physiological importance of cGMP and PKG in the nervous system, it is important to identify endogenous PKG substrates in rat brain. We devised a combination of ion-exchange and hydrophobic chromatographies to identify potential PKG substrates. Extracts from cytosol, peripheral membrane proteins, or a fraction enriched in Ca(2+)-sensitive lipid-binding proteins were partly purified and phosphorylated with purified PKG. Using whole extracts only a single specific PKG substrate-P34-was found. However, after chromatography we detected > 40 distinct proteins that were phosphorylated by PKG to a much greater extent than by cyclic AMP-dependent protein kinase or protein kinase C. Four PKG substrates--P140, P65, P32, and P18--were detected in the cytosol. Six PKG substrates--P130, P85 (doublet), P58, P54, and P38--were enriched from the Ca(2+)-sensitive lipid-binding protein fraction. In peripheral membrane fractions > 30 relatively specific PKG substrates were enriched after chromatography, especially P130, P94, P58, P52, P45, P40, P36, P34, P28, P26, P24, and P20. These results indicate that brain is not lacking in PKG substrates and show that many are apparently quite specific substrates for this enzyme. The identification of some of these novel PKG substrates will facilitate understanding the role of cGMP signaling in the brain.
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PMID:Cyclic GMP-dependent protein kinase substrates in rat brain. 761 14

Nitric oxide and cGMP influence plasticity of nociceptive processing in spinal cord. However, effectors for cGMP have not been identified in sensory pathways. We now demonstrate that cGMP-dependent protein kinase I (cGKl) occurs in the DRGs at levels comparable to that in cerebellum, the richest source of cGKl in the body. Immunohistochemical studies reveal that cGKl is concentrated in a subpopulation of small- and medium-diameter DRG neurons that partially overlap with substance P and calcitonin gene-related polypeptide containing cells. During development, cGKl expression throughout the embryo is essentially restricted to sensory neurons and to the spinal floor and roof plates. Neuronal nitric oxide synthase (nNOS) is coexpressed with cGKl in sensory neurons during embryonic development and after peripheral nerve axotomy. The primary target for cGKl in cerebellum, G-substrate, is not present in developing, mature, or regenerating sensory neurons, indicating that other proteins serve as effectors for cGKl in sensory processing. These data establish sensory neurons as a primary locus for cGMP actions during development and suggest a role for cGKl in plasticity of nociception.
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PMID:cGMP-dependent protein kinase in dorsal root ganglion: relationship with nitric oxide synthase and nociceptive neurons. 862 52

G-substrate, a specific substrate of the cGMP-dependent protein kinase, has previously been localized to the Purkinje cells of the cerebellum. We report here the isolation from mouse brain of a cDNA encoding G-substrate. This cDNA was used to localize G-substrate mRNA expression, as well as to produce recombinant protein for the characterization of G-substrate phosphatase inhibitory activity. Brain and eye were the only tissues in which a G-substrate transcript was detected. Within the brain, G-substrate transcripts were restricted almost entirely to the Purkinje cells of the cerebellum, although transcripts were also detected at low levels in the paraventricular region of the hypothalamus and the pons/medulla. Like the native protein, the recombinant protein was preferentially phosphorylated by cGMP-dependent protein kinase (Km = 0.2 microM) over cAMP-dependent protein kinase (Km = 2.0 microM). Phospho-G-substrate inhibited the catalytic subunit of native protein phosphatase-1 with an IC50 of 131 +/- 27 nM. Dephospho-G-substrate was not found to be inhibitory. Both dephospho- and phospho-G-substrate were weak inhibitors of native protein phosphatase-2A1, which dephosphorylated G-substrate 20 times faster than the catalytic subunit of protein phosphatase-1. G-substrate potentiated the action of cAMP-dependent protein kinase on a cAMP-regulated luciferase reporter construct, consistent with an inhibition of cellular phosphatases in vivo. These results provide the first demonstration that G-substrate inhibits protein phosphatase-1 and suggest a novel mechanism by which cGMP-dependent protein kinase I can regulate the activity of the type 1 protein phosphatases.
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PMID:Phosphorylation-dependent inhibition of protein phosphatase-1 by G-substrate. A Purkinje cell substrate of the cyclic GMP-dependent protein kinase. 992 Aug 94

G-substrate, an endogenous substrate for cGMP-dependent protein kinase, exists almost exclusively in cerebellar Purkinje cells, where it is possibly involved in the induction of long-term depression. A G-substrate cDNA was identified by screening expressed sequence tag databases from a human brain library. The deduced amino acid sequence of human G-substrate contained two putative phosphorylation sites (Thr-68 and Thr-119) with amino acid sequences [KPRRKDT(p)PALH] that were identical to those reported for rabbit G-substrate. G-substrate mRNA was expressed almost exclusively in the cerebellum as a single transcript. The human G-substrate gene was mapped to human chromosome 7p15 by radiation hybrid panel analysis. In vitro translation products of the cDNA showed an apparent molecular mass of 24 kDa on SDS/PAGE which was close to that of purified rabbit G-substrate (23 kDa). Bacterially expressed human G-substrate is a heat-stable and acid-soluble protein that cross-reacts with antibodies raised against rabbit G-substrate. Recombinant human G-substrate was phosphorylated efficiently by cGMP-dependent protein kinase exclusively at Thr residues, and it was recognized by antibodies specific for rabbit phospho-G-substrate. The amino acid sequences surrounding the sites of phosphorylation in G-substrate are related to those around Thr-34 and Thr-35 of the dopamine- and cAMP-regulated phosphoprotein DARPP-32 and inhibitor-1, respectively, two potent inhibitors of protein phosphatase 1. However, purified G-substrate phosphorylated by cGMP-dependent protein kinase inhibited protein phosphatase 2A more effectively than protein phosphatase 1, suggesting a distinct role as a protein phosphatase inhibitor.
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PMID:Molecular identification of human G-substrate, a possible downstream component of the cGMP-dependent protein kinase cascade in cerebellar Purkinje cells. 1005 66

We used a fluorescence differential display-PCR (FDD-PCR) technique to analyze the genes expressed in mouse brains collected at nine different developmental stages ranging from 3 days to 15 months after birth, and 5 age-dependently expressed genes were found. Age-dependent expression of each of these 5 genes was confirmed by quantitative real-time PCR analysis. Of the 5 genes, 4 (B1-B4) had high homology with the nucleotide sequences of cDNA clones of known mouse genes (myelin proteolipid protein, transferrin, embryo cDNA from the RIKEN full-length enriched library, and protein tyrosine phosphatase), and the rest (B5) with expressed sequence tags of an unknown gene. Sequencing analysis of the full-length cDNA constructed based on the B5 sequence demonstrated that the gene product of B5 was identical to G-substrate, a specific substrate for cGMP-dependent protein kinase. The expression patterns of known genes obtained in our study may provide a further opportunity to investigate the biological and physiological roles of the proteins they encode.
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PMID:Five age-dependently expressed genes in mouse brain revealed by the fluorescence differential display-PCR technique. 1221 62

Rat G-substrate cDNA was isolated from a cerebellar library and characterized. The deduced amino acid sequence of rat G-substrate contained two putative phosphorylation sites for PKG at Thr72 and Thr123; the amino acid sequences (KPRRKDT(p)PA) around these sites are conserved in human, mouse and rabbit. G-substrate phosphorylated by PKG inhibited the catalytic subunits of both protein phosphatase-1 (IC(50) 14.1 nM) and -2A (IC(50) 5.9 nM). Mutation of Thr123 (site 2) to Ala significantly reduced the inhibition of both PP-1 and PP-2A, while mutation of Thr72 (site 1) to Ala had little effect on inhibitory activity. In situ hybridization analysis revealed that G-substrate mRNA was localized exclusively in cerebellar Purkinje cells. Immunoperoxidase staining showed that in Purkinje cells, G-substrate was present in somata, dendrites and axons. In rat cerebellar slices, activation of PKG with a nitric oxide (NO) donor, NOR3, or 8-Br-cGMP, increased phosphorylation of G-substrate, as demonstrated with a phosphorylation-specific antibody. These results characterize further the inhibition of PP-1 and PP-2A by phospho-G-substrate, and demonstrate its physiological phosphorylation in rat Purkinje cells.
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PMID:Thr123 of rat G-substrate contributes to its action as a protein phosphatase inhibitor. 1250 27

The aim of this study was to determine the distribution and function of G-substrate, a specific substrate of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-cGMP-dependent protein kinase (PKG) signaling pathway, in normal rat retina and in G-substrate knockout mice. The retinas of adult wild-type rats and mice and G-substrate knockout mice were studied immunohistologically to characterize the upstream and downstream components of the NO-cGMP-PKG pathway. Immunoblot analysis showed that the molecular weight of retinal G-substrate was similar to that of cerebellar G-substrate. In adult rats and mice, retinal G-substrate was located in a subpopulation of amacrine cells and in C38-positive retinal ganglion cells (RGCs) but not in alpha RGCs. In addition, retinal G-substrate was co-expressed with other upstream and downstream signaling components of the NO-cGMP-PKG-G-substrate-phosphatase pathway in the adult retina. Electroretinographic (ERG) analysis demonstrated that there was no significant difference between the ERGs of wild-type and G-substrate knockout mice. These results suggest that retinal G-substrate plays a role as a downstream component of the NO-cGMP-PKG pathway. The co-localization of retinal G-substrate with protein Ser/Thr phosphatases suggests that it acts as an endogenous protein phosphatase inhibitor as in the cerebellum.
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PMID:Retinal G-substrate, potential downstream component of NO/cGMP/PKG pathway, is located in subtype of retinal ganglion cells and amacrine cells with protein phosphatases. 1585 69

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate-deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10-15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.
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PMID:Dual involvement of G-substrate in motor learning revealed by gene deletion. 1921 32


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