Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities.

J H Keen, M H Chestnut, K A Beck

    The Journal of Biological Chemistry
    |March 15, 1987
    PubMed
    Summary

    The 50-kDa component (AP50) of clathrin assembly protein (AP) undergoes autophosphorylation on a threonyl residue. This phosphorylation does not impact its role in clathrin coat assembly.

    Related Concept Videos

    You might also read

    Related Articles

    Articles linked to this work by shared authors, journal, and citation graph.

    Sort by
    Same author

    Changes in visceral yolk sac ultrastructure after exposure of rat embryos to selected teratogens in vitro.

    Toxicology in vitro : an international journal published in association with BIBRA·2010
    Same author

    The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro.

    The Journal of cell biology·2001
    Same author

    The class II phosphoinositide 3-kinase C2alpha is activated by clathrin and regulates clathrin-mediated membrane trafficking.

    Molecular cell·2001
    Same author

    Esophageal motility dysfunction in cats: a study of 44 cases.

    Journal of the American Animal Hospital Association·2000
    Same author

    Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors.

    Journal of cell science·2000
    Same author

    The class II phosphoinositide 3-kinase PI3K-C2alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles.

    The Journal of biological chemistry·2000

    Area of Science:

    • Cell Biology
    • Molecular Biology
    • Protein Biochemistry

    Background:

    • Clathrin-coated vesicles are essential for intracellular trafficking.
    • Clathrin assembly protein (AP) facilitates the formation of clathrin coat structures.
    • The 50-kDa component (AP50) is a subunit of the AP complex.

    Purpose of the Study:

    • To identify the 50-kDa polypeptide rapidly phosphorylated in clathrin-coated vesicles.
    • To characterize the phosphorylation of AP50 and its functional implications.
    • To investigate the potential role of AP50 as a protein kinase.

    Main Methods:

    • Isolation of clathrin-coated vesicles.
    • In vitro phosphorylation assays using [gamma-32P]ATP.
    • Sodium dodecyl sulfate gel electrophoresis (one- and two-dimensional) for protein separation.

    Related Experiment Videos

  • Kinetic studies in solution.
  • Enzyme assays with inorganic and protein phosphatases.

    • Main Results:

    • The rapidly phosphorylated 50-kDa polypeptide was identified as AP50.
    • AP50 autophosphorylation occurs on threonyl residue(s) at 0 degrees C.
    • Phosphorylated AP50 is resistant to inorganic phosphatases but sensitive to protein phosphatases 1 and 2A.
    • AP50 phosphorylation is an intramolecular event, potentially activated intermolecularly.
    • The phosphorylation state of AP50 did not affect its ability to promote in vitro clathrin coat assembly.

    Conclusions:

    • AP50 is an autophosphorylating protein kinase subunit within the clathrin assembly protein complex.
    • A physiological phosphorylation-dephosphorylation cycle for AP50 may exist.
    • Distinct domains of AP are responsible for clathrin assembly (100-kDa components) and autophosphorylation (AP50).
    • AP50 may function as a protein kinase in vivo.