Isolation and properties of the bovine brain protein inhibitor of adenosine 3′:5′‐monophosphate‐dependent protein kinases

Demaille, Jacques; Peters, Kristine A.; Strandjord, Thomas P.; Fischer, Edmond H.

doi:10.1016/0014-5793(78)80110-0

Cited by 34 publications

(18 citation statements)

References 19 publications

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“…6, a and c). Multiple charge forms of PKI␣ were evident in DEAE separation, as has been described previously for skeletal muscle and brain PKI␣ (29,30). The major forms of cerebellum PKI␣ comigrated with purified skeletal muscle PKI␣ (data not shown).…”

Section: Quantification Of Pki␤ Isoform Composition In Adult Ratsupporting

confidence: 70%

Multiplicity of the β Form of the cAMP-dependent Protein Kinase Inhibitor Protein Generated by Post-translational Modification and Alternate Translational Initiation

Kumar

Patten

Walsh

1997

Journal of Biological Chemistry

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Two distinct species of the thermostable inhibitor of the cAMP-dependent protein kinase, PKI␣ and PKI␤, exist that are the products of separate genes. The PKI␤ form, as first isolated from rat testis, is a 70-amino acid protein, but the genomic sequence suggested that an alternate form might exist, arising as a consequence of alternate translational initiation. This species, now termed PKI␤-78, has been synthesized by bacterial expression, demonstrated to be equipotent with PKI␤-70, and also now demonstrated to occur in vivo. By Western blot analyses, six additional species of PKI␤ are also evident in tissues. Two of these represent the phospho forms of PKI␤-78 and PKI␤-70. The other four represent phospho and dephospho forms of two higher molecular mass PKI␤ species. These latter forms are currently termed PKI␤-X and PKI␤-Y, awaiting the full elucidation of their molecular identity. In adult rat testis and cerebellum, PKI␤-70, PKI␤-X, and PKI␤-Y constitute 39, 23, and 32% and 15, 29, and 54% of the total tissue levels, respectively. In adult rat testis, 35-42% of each of these three species is present as a monophospho form, whereas no phosphorylation of them is evident in cerebellum. PKI␤-78 is present at much lower levels in both rat testis and cerebellum (ϳ6 and 2% of the total, respectively) and almost entirely as a monophospho species. PKI␤-78, like PKI␤-70, is a high affinity and specific inhibitor of the cAMP-dependent protein kinase. PKI␤-Y and PKI␤-X, in contrast, also significantly inhibit the cGMP-dependent protein kinase.The cAMP-dependent protein kinase (PKA) 1 plays a central role in the signal transduction of many hormones. The thermostable protein kinase inhibitor (PKI) is a highly specific competitive inhibitor of this enzyme that binds with high affinity to the protein substrate-binding site of the kinase (1). Its role as a regulator of PKA remains to be fully identified and appears to include not only the reduction of protein kinase activity, but also the trafficking of the protein kinase catalytic subunit between subcellular sites (2-4). Two genetically distinct forms of PKI (␣ and ␤) have been shown to be present in many tissues (5-9). Both forms act as specific pseudosubstrate inhibitors of PKA, have similar K i values in the subnanomolar range, and are likely both involved in intracellular trafficking (4). PKI␣ and PKI␤ share ϳ40% identity at the amino acid level (5), share very closely the same recognition determinants for the catalytic subunit of PKA (5, 10) and for nuclear export (9), but are notably distinct in the C-terminal half of the molecule. PKI␣ and PKI␤ each have a distinct tissue distribution (6).The PKI␤ form was first identified in rat testis (5) in a follow-up of observations made earlier by Means and co-workers (11, 12). The PKI␤ form purified was one of what appeared to be multiple forms of the protein present in testis, as was apparent from the DEAE chromatographic profile of the partially purified protein. The form purified was shown by protein sequencing and amino acid...

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Section: Quantification Of Pki␤ Isoform Composition In Adult Ratsupporting

confidence: 70%

Multiplicity of the β Form of the cAMP-dependent Protein Kinase Inhibitor Protein Generated by Post-translational Modification and Alternate Translational Initiation

Kumar

Patten

Walsh

1997

Journal of Biological Chemistry

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“…cAMP-dependent protein kinase type II and its separated catalytic subunit were prepared from pig heart according to Peters et aL (14). cAMP-dependent protein kinase inhibitor was isolated from rabbit skeletal muscle according to Demaille et al (15,16 turbidity measurement at 350 nm; 0.1 mM GTP was added to the warm solution at time 0 to initiate polymerization. After assembly, the solutions were chilled to 000 for 10 min and then rewarmed for another assembly cycle.…”

mentioning

confidence: 99%

Regulation of microtubule cold stability by calmodulin-dependent and -independent phosphorylation.

Job¹,

Rauch²,

Fischer³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Cold-labile microtubule protein can be rendered cold-stable by addition of a fraction containing a small number of polypeptides that are derived from cold-stable microtubules. These polypeptides can be obtained from purified cold-stable microtubules by passage through a DEAE-cellulose (DE-52) ion.exchange column from which they emerge in the first eluate fraction. The stabilizing. activity of these proteins is abolished by phosphoryla-.tion catalyzed by two types of protein kinases, one dependent on calmodulin and the other independent of that regulatory protein. The calmodulin-dependent reaction appears to phosphorylate mainly two polypeptides, 56 and 72 kilodaltons; the reaction is blocked by trifluoperazine. The calmodulin-independent reaction appears to phosphorylate different cold-stable microtubule-associated proteins. That reaction is observed only in purified material obtained from vigorously homogenized brain tissue. Gentle homogenization yields cold-stable microtubules that are responsive only to the calmodulin-dependent protein kinase. A distinguishing feature of the calmodulin-independent reaction is that it does not occur on polypeptides while they are bound to the microtubules.

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“…1) . Clearly then, F:PKI must bind to the substrate site of C (2,3,16,17), with an avidity like that of the unconjugated inhibitor . It follows that F:PKI behaves similarly when employed as a cytochemical probe .…”

Section: Discussionmentioning

confidence: 99%

“…To do so would require the ability to monitor an extremely minute amount of fluorescence, but at the moment this is technologically not feasible. We are, however, unaware of any published or unpublished evidence that the inhibitor binds to cellular proteins other than the free C from cAMP-dependent protein kinase (2)(3)(4)(15)(16)(17)(18)(19)(20) . Even so, these limitations made it necessary to establish that as a cytochemical probe the F:PKI binds predominantly, perhaps exclusively, to the free catalytic unit of cAMP-dependent protein kinase .…”

Section: Discussionmentioning

confidence: 99%

“…A number of laboratories have reported the presence of a heat-stable protein that inhibits free C phosphotransferase activity (4) . This inhibitor (PKI), found in a variety of tissues and characterized (14)(15)(16)(17)(18)(19)(20) has a high affmity and specificity only for the free catalytic unit of cAMP-dependent protein kinase (4,14,16) . It does not bind to either form of the holoenzyme nor to the cyclic 3':5' guanosine monophosphate(cGMP)-dependent protein kinase (15,16).…”

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confidence: 99%

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Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor.

Fletcher¹,

Byus²

1982

The Journal of Cell Biology

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A specific and sensitive procedure has been developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC conjugate (F:PKI) of affinity column-purified heat-stable protein inhibitor (PKI) of free C. The fidelity of this cytochemical probe was determined using cultures of Reuber H-35 hepatoma cells that had been stimulated for 2 h with 0.1 mM DBcAMP, or with diluent, then fixed with anhydrous acetone at -30 degrees C. In these preparations the F:PKI probe complexed with free C in cytoplasm, nucleolus, and, to a minor extent, in nucleoplasm. Binding of the F:PKI molecule to free C was competitively diminished by arginine analogues, guanidinium HCI and polyarginine, each used over a 2-log dose range. When the inhibitor's arginine residues were blocked by reaction with cyclohexanedione it no longer inhibited phosphotransferase activity of free C, and when fluorescinated it failed to localize C in stimulated cells. Similarly, when F:PKI was preabsorbed with excess pure C it no longer functioned as a cytochemical stain. Affinity column-purified antibody to free C also reduced significantly the ability of F:PKI to complex with C in cell cultures stimulated with 0.1 mM DBcAMP. 1 microgram of antibody reduced by approximately 10% the binding of F:PKI to all cell compartments while 5 microgram of antibody diminished binding by greater than 50%. Together, these results indicate that the F:PKI binds specifically, perhaps exclusively, to the catalytic units of cAMP-dependent protein kinase. The cytochemical procedure, unlike its biochemical counterparts, is able to locate the dissociation of cAMP-dependent protein kinase in individual cells of functionally or histologically complex cultures. Also, it reveals variations in the time- and dose-dependent activation of the kinase amongst clonal cells stimulated with cyclic nucleotide analogues or hormones.

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Isolation and properties of the bovine brain protein inhibitor of adenosine 3′:5′‐monophosphate‐dependent protein kinases

Cited by 34 publications

References 19 publications

Multiplicity of the β Form of the cAMP-dependent Protein Kinase Inhibitor Protein Generated by Post-translational Modification and Alternate Translational Initiation

Multiplicity of the β Form of the cAMP-dependent Protein Kinase Inhibitor Protein Generated by Post-translational Modification and Alternate Translational Initiation

Regulation of microtubule cold stability by calmodulin-dependent and -independent phosphorylation.

Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor.

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