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 repres ents a bulky hydrophobic residue) have been shown to interact with the medi um chain subunit of clathrin adaptor complexes, These medium chains are tar gets for phosphorylation by a kinase activity associated with clathrin coat ed vesicles, We have used the clathrin coated vesicle associated kinase act ivity to specifically phosphorylate a soluble recombinant fusion protein of mu 2, the medium chain subunit of the plasma membrane associated adaptor p rotein complex AP-2, We have tested whether this phosphorylation has any ef fect on the interaction of mu 2 with the tyrosine based motif containing pr otein, TGN38, that has previously been shown to interact with mu 2. Phospho rylation of mu 2 was shown to have no significant effect on the in vitro in teraction of mu 2 with the cytosolic domain of TGN38, indicating that rever sible phosphorylation of mu 2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs, In addition, altho ugh a casein kinase II-like activity has been shown to be associated with c lathrin coated vesicles, me show that mu 2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coat ed vesicles. Furthermore the kinase activity associated with clathrin coate d vesicles was shown to be capable of phosphorylating dynamin 1, Phosphoryl ation of dynamin 1 has previously been shown to regulate its interaction wi th other proteins involved in clathrin mediated endocytosis, (C) 1999 Feder ation of European Biochemical Societies.