Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38

Tyrosine based motifs conforming to the consensus YXX Phi (where Phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes, These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles, We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu 2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2, We have tested whether this phosphorylation has any effect on the interaction of mu 2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu 2. Phosphorylation of mu 2 was shown to have no significant effect on the in vitro interaction of mu 2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu 2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs, In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, me show that mu 2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1, Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis, (C) 1999 Federation of European Biochemical Societies.