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Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase.

ABSTRACT: The form of inhibitor protein of the cAMP-dependent protein kinase (PKI) that has been most thoroughly studied is a protein purified from rabbit skeletal muscle. Beale et al. previously isolated a species of PKI from rat testis that appeared from its amino acid composition to be quite distinct from the rabbit skeletal muscle protein [Beale, E. G., Dedman, J. R. & Means, A. R. (1977) J. Biol. Chem. 252, 6322-6327]. The amino acid sequence of a form of rat testis PKI has now been determined both by sequencing overlapping peptide fragments for 95% of the protein and by the isolation of a cDNA clone containing the coding region for the 70-amino acid protein. The sequence of the 70-amino acid testis PKI displays a maximum of only 41% sequence identity with the previously sequenced 75-amino acid rabbit skeletal muscle PKI. However, the two forms have identical potency as inhibitors and the key amino acids of the pseudosubstrate site, shown to be critical for maximal inhibition with the rabbit skeletal muscle PKI, have been conserved in the testis protein. The rabbit skeletal muscle and rat testis PKIs most likely represent distinct isoforms. The nucleotide sequence of the rat testis PKI cDNA suggests that a second form of testis PKI, longer by 8 additional amino-terminal amino acids, might also be produced.

SUBMITTER: Van Patten SM

PROVIDER: S-EPMC51877 | biostudies-other | 1991 Jun

REPOSITORIES: biostudies-other

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Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase.

Van Patten S M SM Ng D C DC Th'ng J P JP Angelos K L KL Smith A J AJ Walsh D A DA

Proceedings of the National Academy of Sciences of the United States of America 19910601 12

The form of inhibitor protein of the cAMP-dependent protein kinase (PKI) that has been most thoroughly studied is a protein purified from rabbit skeletal muscle. Beale et al. previously isolated a species of PKI from rat testis that appeared from its amino acid composition to be quite distinct from the rabbit skeletal muscle protein [Beale, E. G., Dedman, J. R. & Means, A. R. (1977) J. Biol. Chem. 252, 6322-6327]. The amino acid sequence of a form of rat testis PKI has now been determined both b ...[more]

PMID: 2052616

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Project description:BackgroundInvestigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA) increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells. These cell-based assays are often utilized to test the involvement of PKA-dependent processes by using H-89, a reversible competitive inhibitor of PKA.Principal findingsWe present here data to show that H-89, in addition to being a competitive PKA inhibitor, attenuates the bioluminescence signal produced by Renilla luciferase (RLuc) variants in a population of cells and also in single cells. Using 10 microM of luciferase substrate and 10 microM H-89, we observed that the signal from RLuc and RLuc8, an eight-point mutation variant of RLuc, in cells was reduced to 50% (+/-15%) and 54% (+/-14%) of controls exposed to the vehicle alone, respectively. In vitro, we showed that H-89 decreased the RLuc8 bioluminescence signal but did not compete with coelenterazine-h for the RLuc8 active site, and also did not affect the activity of Firefly luciferase. By contrast, another competitive inhibitor of PKA, KT5720, did not affect the activity of RLuc8.SignificanceThe identification and characterization of the adverse effect of H-89 on RLuc signal will help deconvolute data previously generated from RLuc-based assays looking at the functional effects of PKA signaling. In addition, for the current application and future development of bioluminscence assays, KT5720 is identified as a more suitable PKA inhibitor to be used in conjunction with RLuc-based assays. These principal findings also provide an important lesson to fully consider all of the potential effects of experimental conditions on a cell-based assay readout before drawing conclusions from the data.

Dataset Information

Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase.

Publications

Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase.

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