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

J. H. Keen, M. H. Chestnut, Kenneth A Beck

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

A 50-kDa polypeptide that is rapidly phosphorylated on addition of [gamma-32P]ATP to isolated clathrin-coated vesicles is shown here to be identical to the 50-kDa component (AP50) of the clathrin assembly protein (AP), a complex that promotes the assembly of clathrin coat structures under physiological conditions of pH and ionic strength. Phosphorylation of the AP50 occurred readily at 0 degrees C, almost exclusively on a threonyl residue(s). This reaction is attributable to autophosphorylation, since the AP50 was able to covalently incorporate 32P from [gamma-32P]ATP after separation by either one- or two-dimensional sodium dodecyl sulfate gel electrophoresis. Kinetic studies in solution were consistent with an intramolecular phosphorylation event; in addition, a concentration-dependent increase in AP50 phosphorylation was observed that may reflect intermolecular AP-AP activation of autophosphorylation. The phosphorylated AP50 was resistant to several inorganic phosphatases tested but was a substrate for protein phosphatases 1 and 2A, suggesting that a physiological phosphorylation-dephosphorylation cycle may exist. The phosphorylation state of the AP50 did not affect the ability of the AP to promote in vitro clathrin coat assembly. These and other data suggest that unique structural domains of the assembly protein are responsible for assembly (the 100-kDa components) and autophosphorylation (the AP50) and that the latter may be active as a protein kinase in the intact cell.

Original languageEnglish (US)
Pages (from-to)3864-3871
Number of pages8
JournalJournal of Biological Chemistry
Volume262
Issue number8
StatePublished - Mar 15 1987
Externally publishedYes

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Clathrin
Phosphorylation
Peptides
Adenosine Triphosphate
Vesicular Transport Adaptor Proteins
Clathrin-Coated Vesicles
Protein Phosphatase 1
Protein Phosphatase 2
Proteins
Phosphoric Monoester Hydrolases
Sodium Dodecyl Sulfate
Osmolar Concentration
Protein Kinases
Electrophoresis
Gels
Ionic strength
Chemical activation
Kinetics
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities. / Keen, J. H.; Chestnut, M. H.; Beck, Kenneth A.

In: Journal of Biological Chemistry, Vol. 262, No. 8, 15.03.1987, p. 3864-3871.

Research output: Contribution to journalArticle

Keen, J. H. ; Chestnut, M. H. ; Beck, Kenneth A. / The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities. In: Journal of Biological Chemistry. 1987 ; Vol. 262, No. 8. pp. 3864-3871.
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