Amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase from rabbit skeletal muscle.

Scott, John D.; Fischer, Edmond H.; Takio, Koji; Demaille, Jacques; Krebs, Edwin G.

doi:10.1073/pnas.82.17.5732

Cited by 97 publications

(57 citation statements)

References 30 publications

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“…These included PKI, a highly specific inhibitor of the cyclic AMP (cAMP)-dependent protein kinase that targets its peptide substrate binding site (8,46), as well as several compounds that target the ATP-substrate binding sites of eukaryotic protein kinases with various degrees of selectivity: staurosporine (38), genistein (1), M9 (40), and H7 (40). While the aforementioned compounds produced no detectable inhibition at even the highest concentrations that we tested, tamoxifen, a compound that inhibits protein kinase C (39, 59) and a variety of calmodulin-dependent enzymes such as cardiac myosin light chain kinase (59) and a cAMP-phosphodiesterase (30), did inhibit the protein kinase activity of rSsoPK3 with an apparent 50% inhibitory concentration (IC 50 ) of 0.5 mM.…”

Section: Vol 186 2004mentioning

confidence: 99%

A Phosphoprotein from the Archaeon Sulfolobus solfataricus with Protein-Serine/Threonine Kinase Activity

2004

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Sulfolobus solfataricus contains a membrane-associated protein kinase activity that displays a strong preference for threonine as the phospho-acceptor amino acid residue. When a partially purified detergent extract of the membrane fraction from the archaeon S. solfataricus that had been enriched for this activity was incubated with [␥-32 P]ATP, radiolabeled phosphate was incorporated into roughly a dozen polypeptides, several of which contained phosphothreonine. One of the phosphothreonine-containing proteins was identified by mass peptide profiling as the product of open reading frame [ORF] sso0469. Inspection of the DNA-derived amino acid sequence of the predicted protein product of ORF sso0469 revealed the presence of sequence characteristics faintly reminiscent of the "eukaryotic" protein kinase superfamily. ORF sso0469 therefore was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein formed insoluble aggregates that could be dispersed using urea or detergents. The solubilized polypeptide phosphorylated several exogenous proteins in vitro, including casein, myelin basic protein, and bovine serum albumin. Mutagenic alteration of amino acids predicted to be essential for catalytic activity abolished or severely reduced catalytic activity. Phosphorylation of exogenous substrates took place on serine and, occasionally, threonine. This new archaeal protein kinase displayed no catalytic activity when GTP was substituted for ATP as the phospho-donor substrate, while Mn 2؉ was the preferred cofactor.The versatility of covalent phosphorylation-dephosphorylation as a mechanism for regulating protein function and transducing extracellular signals has been compellingly demonstrated in numerous studies encompassing a broad spectrum of eucaryal and bacterial organisms (reviewed in references 5, 15, 19, 22, 23, and 28). However, while protein phosphorylation has been detected in several members of the third phylogenetic domain, the Archaea (24,41,50,51,53,54,56,57), we know relatively little concerning the chemical nature, enzymatic catalysts, and physiological roles of archaeal protein phosphorylation-dephosphorylation events (reviewed in reference 27). Only a few archaeal proteins have been implicated as the targets of protein phosphorylation to date. They include a CheY homolog in Halobacterium salinarium (45), a methyltransferase-activating protein from Methanosarcina barkeri (11), an aminopeptidase from Sulfolobus solfataricus (9), and a glycogen synthase from Sulfolobus acidocaldarius (7). In addition, the N-terminal sequences of three phosphotyrosine-containing proteins from Thermococcus kodakaraensis KOD1 have been determined, although the full sequences of these phosphoproteins have yet to be identified (24 (26,27,32,42,49). However, in only a few instances have the inferences of these in silico analyses been translated into the direct, empirical identification and characterization of defined gene products displaying the predicted functional capabilities. Included among ...

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Section: Vol 186 2004mentioning

confidence: 99%

A Phosphoprotein from the Archaeon Sulfolobus solfataricus with Protein-Serine/Threonine Kinase Activity

2004

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“…Apart from the R subunits, another endogenous modulator of the C subunit is also present in most tissues: protein kinase inhibitor (PKI). The PKIs are heat-stable proteins, 70 to 75 amino acids in length, that are high-affinity, specific inhibitors of PKA (24). The N-terminal region of PKI contains the sequence RRNAI, which acts as a pseudosubstrate site for PKA and is required for PKI's inhibitory activity.…”

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Deficient Gene Expression in Protein Kinase Inhibitor α Null Mutant Mice

Gangolli

Belyamani

Muchinsky

et al. 2000

Molecular and Cellular Biology

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Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKI␣, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RI␣ subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

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“…The heat stable protein kinase inhibitors (PKIs) are a family of small proteins that inhibit PKA by binding to the substrate-binding site of the catalytic subunit with sub-nanomolar affinity. 32 The high affinity binding motif is found at residues 5 to 24, and the respective 20-mer peptide spanning this range (named PKI(5-24) or IP20) binds to the C-subunit with high affinity, with a K i of 2.3 nM. 33 Thus, IP20 is the ideal probe for this novel method.…”

Section: Assay Designmentioning

confidence: 99%

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

et al. 2006

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Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named Ligand-Regulated Competition (LiReC), in an effort to find non-ATP competitive small molecule regulators for type Iα cAMP-dependent Protein Kinase A (PKA-Iα), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Iα complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.

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Amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase from rabbit skeletal muscle.

Cited by 97 publications

References 30 publications

A Phosphoprotein from the Archaeon Sulfolobus solfataricus with Protein-Serine/Threonine Kinase Activity

A Phosphoprotein from the Archaeon Sulfolobus solfataricus with Protein-Serine/Threonine Kinase Activity

Deficient Gene Expression in Protein Kinase Inhibitor α Null Mutant Mice

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

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