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Related Experiment Videos

Correlation between protein kinase C binding proteins and substrates in REF52 cells

S L Hyatt1, L Liao, A Aderem

  • 1W. Alton Jones Cell Science Center, Lake Placid, New York 12946.

Cell Growth & Differentiation : the Molecular Biology Journal of the American Association for Cancer Research
|May 1, 1994
PubMed
Summary

Protein kinase C (PKC) binding proteins and substrates share properties, with phosphatidylserine-dependent interactions detectable by blot overlay. These interactions are altered in transformed cells, potentially impacting the transformed phenotype.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Protein kinase C (PKC) plays a crucial role in cellular signaling.
  • Identifying PKC binding proteins and substrates is essential for understanding its regulatory mechanisms.
  • Phosphatidylserine is known to be involved in the activation and localization of PKC.

Purpose of the Study:

  • To identify phosphatidylserine-dependent interactions between PKC and its binding proteins using a blot overlay assay.
  • To compare the properties of PKC binding proteins and substrates identified through in vivo and in vitro phosphorylation assays.
  • To investigate the alterations in PKC binding proteins and substrates in SV40-transformed REF52 cells.

Main Methods:

  • Blot overlay assay to detect protein-protein interactions.

Related Experiment Videos

  • Calmodulin-Sepharose chromatography for protein enrichment.
  • In vivo and in vitro phosphorylation assays to identify PKC substrates.
  • Analysis of protein expression and phosphorylation in normal and SV40-transformed REF52 cells.

    • Main Results:

    • Phosphatidylserine-dependent interactions between PKC and several binding proteins/substrates were identified.
    • Major PKC binding proteins and substrates in REF52 cells exhibited similar properties, including calmodulin binding and heat resistance.
    • Several major binding proteins/substrates were coordinately down-modulated in SV40-transformed REF52 cells.
    • The major PKC substrate, MARCKS, was also identified as a PKC binding protein.

    Conclusions:

    • Phosphatidylserine-dependent interactions between PKC and its substrates possess sufficient affinity for detection via blot overlay assays.
    • Down-modulation of these binding proteins/substrates in transformed cells may result from decreased expression or increased basal phosphorylation.
    • These alterations in PKC interacting proteins are likely implicated in the maintenance of the transformed cellular phenotype.