Phosphorylation of threonine 156 of the μ2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo

Abstract

The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells.

Functional Analysis of AP-2 α and μ2 Subunits

“…This indicates that the conformational change that exposes the YXX⌽ binding site on the 2 subunit is favored but is not driven by phosphorylation, which is entirely consistent with our own results. Our study also agrees with an article by Olusanya et al (2001) on the role of 2 phosphorylation in vivo, in which tagged wild-type or T156A 2 were transiently transfected into cells. Both wild-type and mutant constructs were able to be recruited onto the plasma membrane, but the mutant construct appeared to have a dominant negative effect on the uptake of fluorescent transferrin.…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…Binding of 2 to tyrosine-based sorting signals is proposed to be dependent upon phosphorylation of 2, likely mediated by the kinases AAK1 (Adaptor-Associated Kinase 1) and GAK (cyclin-G-Associated protein Kinase) (Umeda et al, 2000;Olusanya et al, 2001;Collins et al, 2002;Conner and Schmid, 2002;Korolchuk and Banting, 2002;Ricotta et al, 2002;Sorkin, 2004). Other receptors are believed to make use of additional connector proteins coupling their sorting signals to the clathrin coat.…”

Vesicular transport of Fe and interaction with other metal ions in polarized Caco2 Cell monolayers

“…FSBA [5'-( 4 -f l u o r o s u l f o n y l b e n z o y l ) a d e n o s i n e hydrochloride] and nocodazole inhibit the first two steps in endocytosis, respectively. The ATP analogue, FSBA, can interfere with sequestration of receptors into clathrin-coated pits by preventing a specific protein phosphorylation step (Olusanya et al, 2001). Nocodazole depolymerizes microtubules and promotes dispersal and tubulation of the Golgi apparatus (Turner and Tartakoff, 1989).…”