Protein Kinase Inhibitor-(6-22)-amide Peptide Analogs with Standard and Nonstandard Amino Acid Substitutions for Phenylalanine 10

Glass, David B.; Lundquist, Lucy J.; Katz, Bernice M.; Walsh, Donal A.

doi:10.1016/s0021-9258(18)71718-3

Cited by 107 publications

(23 citation statements)

References 41 publications

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“…3C, that Quin-induced significant changes in I Ca in PC12 cells whose PKA was blocked by the PKA inhibitor fragment 6-22 amide PKI. This small heatstable inhibitory peptide binds to the catalytic subunit of PKA and inactivates the enzyme [11]. Dialysis of cells with a pipette solution containing PKI effectively inhibits the PKAmediated current events [22].…”

Section: Ptx-sensitive G Proteins Mediate the D 2 Receptor-induced Inhibition Of Voltage-dependent Ca 2+ Current In Pc12 Cellsmentioning

confidence: 99%

Role of the D2 dopamine receptor in molecular adaptation to chronic hypoxia in PC12 cells

Kobayashi

Conforti

Zhu

et al. 1999

Pflügers Arch – Eur J Physiol

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We have previously shown that pheochromocytoma (PC12) cells rapidly depolarize and undergo Ca2+ influx through voltage-dependent Ca2+ channels in response to moderate hypoxia and that intracellular free Ca2+ is modulated by activation of dopamine D2 receptors in this cell type. The present study shows that D2 (quinpirole-mediated) inhibition of a voltage-dependent Ca2+ current (ICa) in PC12 cells is dramatically attenuated after chronic exposure to moderate hypoxia (24 h at 10% O2). Pretreatment of cells with pertussis toxin abolished D2-mediated inhibition of ICa. The D2-induced inhibition of ICa did not depend on protein kinase A (PKA), as it persisted both in the presence of a specific PKA inhibitor (PKI) and in PKA-deficient PC12 cells. Prolonged exposure to hypoxia (24 h) significantly reduced the level of Gi/o alpha immunoreactivity, but did not alter G beta levels. Furthermore, dialysis of recombinant G(o) alpha protein through the patch pipette restored the inhibitory effect of quinpirole in cells chronically exposed to hypoxia. We conclude that the attenuation of the D2-mediated inhibition of ICa by chronic hypoxia is caused by impaired receptor-G protein coupling, due to reduced levels of G(o) alpha protein. This attenuated feedback modulation of ICa by dopamine may allow for a more sustained Ca2+ influx and enhanced cellular excitation during prolonged hypoxia.

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Section: Ptx-sensitive G Proteins Mediate the D 2 Receptor-induced Inhibition Of Voltage-dependent Ca 2+ Current In Pc12 Cellsmentioning

confidence: 99%

Role of the D2 dopamine receptor in molecular adaptation to chronic hypoxia in PC12 cells

Kobayashi

Conforti

Zhu

et al. 1999

Pflügers Arch – Eur J Physiol

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“…This potent PKA competitive inhibitor corresponds to residues 7 to 23 of both human and mouse PKA inhibitor and residues 6 to 22 of rabbit PKA inhibitor. 25,26 As shown in Figure 4A, the inhibitor cocktail was able to inhibit the PKA reaction in a dose-dependent manner, showing an IC 50 = 34 nM. This value is comparable to the nanomolar inhibition constant for PKA.…”

Section: Optimization Of Assay Conditionsmentioning

confidence: 68%

“…The main component of the inhibitor cocktail is a 17-mer synthetic peptide Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH2 or PKI-(6-22)-amide. 25,26 ImmunePure avidin from hen egg white (product 21121) was obtained from Pierce (Rockford, IL). Polylysine Ysi beads and γ-33 P-ATP were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).…”

Section: Methodsmentioning

confidence: 99%

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A Generic High-Throughput Screening Assay for Kinases: Protein Kinase A as an Example

Mallari¹,

Swearingen²,

Liu³

et al. 2003

SLAS Discovery

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A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated33P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5-to 9-fold and a Z′ factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors. ( Journal of Biomolecular Screening 2003:198-204)

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“…In addition to the important functions of endogenous PKI, shorter peptides of PKI, for example PKI (6-22) amide, are widely used as pharmaceutical agents to inhibit PKA activity (Cheng et al, 1986;Glass et al, 1989a, Glass et al, 1989b. However, it was unclear if PKI inhibits only PKA or if it inhibits other enzymes as well.…”

Section: Introductionmentioning

confidence: 99%

The Kinase Specificity of Protein Kinase Inhibitor Peptide

Chen

Sabatini

2021

Front. Pharmacol.

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G-protein-coupled-receptor (GPCR) signaling is exquisitely controlled to achieve spatial and temporal specificity. The endogenous protein kinase inhibitor peptide (PKI) confines the spatial and temporal spread of the activity of protein kinase A (PKA), which integrates inputs from three major types of GPCRs. Despite its wide usage as a pharmaceutical inhibitor of PKA, it was unclear whether PKI only inhibits PKA activity. Here, the effects of PKI on 55 mouse kinases were tested in in vitro assays. We found that in addition to inhibiting PKA activity, both PKI (6–22) amide and full-length PKIα facilitated the activation of multiple isoforms of protein kinase C (PKC), albeit at much higher concentrations than necessary to inhibit PKA. Thus, our results call for appropriate interpretation of experimental results using PKI as a pharmaceutical agent. Furthermore, our study lays the foundation to explore the potential functions of PKI in regulating PKC activity and in coordinating PKC and PKA activities.

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Protein Kinase Inhibitor-(6-22)-amide Peptide Analogs with Standard and Nonstandard Amino Acid Substitutions for Phenylalanine 10

Cited by 107 publications

References 41 publications

Role of the D2 dopamine receptor in molecular adaptation to chronic hypoxia in PC12 cells

Role of the D2 dopamine receptor in molecular adaptation to chronic hypoxia in PC12 cells

A Generic High-Throughput Screening Assay for Kinases: Protein Kinase A as an Example

The Kinase Specificity of Protein Kinase Inhibitor Peptide

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