Primary Structural Determinants Essential for Potent Inhibition of cAMP-dependent Protein Kinase by Inhibitory Peptides Corresponding to the Active Portion of the Heat-Stable Inhibitor Protein

Glass, David B.; Cheng, Heung‐Chin; Mende‐Mueller, Liane; Reed, Jennifer; Walsh, Donal A.

doi:10.1016/s0021-9258(18)81864-6

Cited by 264 publications

(101 citation statements)

References 54 publications

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“…The EC 50 for the substrate is 0.091 µM, in close agreement with the reported apparent K m of 0.11 µM. 25 Figure 2B shows a saturation reaction with increasing enzyme concentration. As little as 100 ng PKA per well generates sufficient product that it can be readily detected under the given conditions.…”

Section: Optimization Of Assay Conditionssupporting

confidence: 87%

“…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: 67%

“…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%

“…This value is comparable to the nanomolar inhibition constant for PKA. 25,26 Our next objective was to identify an inhibitor that can be used as a control for the screen campaigns. Because the kinase reaction requires Mg-ATP, we evaluated the effects of EDTA, an effective Mg 2+ chelator, on the enzyme reaction.…”

Section: Optimization Of Assay Conditionsmentioning

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)

show abstract

Section: Optimization Of Assay Conditionssupporting

confidence: 87%

Section: Optimization Of Assay Conditionsmentioning

confidence: 67%

Section: Methodsmentioning

confidence: 99%

Section: Optimization Of Assay Conditionsmentioning

confidence: 99%

See 2 more Smart Citations

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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show abstract

“…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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Regulation of cell adhesion during embryonic compaction of mammalian embryos: Roles for PKC and ?-catenin

Pauken

Capco

1999

Mol. Reprod. Dev.

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Primary Structural Determinants Essential for Potent Inhibition of cAMP-dependent Protein Kinase by Inhibitory Peptides Corresponding to the Active Portion of the Heat-Stable Inhibitor Protein

Cited by 264 publications

References 54 publications

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

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

The Kinase Specificity of Protein Kinase Inhibitor Peptide

Regulation of cell adhesion during embryonic compaction of mammalian embryos: Roles for PKC and ?-catenin

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