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J. Biol. Chem., Vol. 283, Issue 9, 5510-5517, February 29, 2008

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Characterization of a Protein Phosphatase 2A Holoenzyme That Dephosphorylates the Clathrin Adaptors AP-1 and AP-2^*

Doris Ricotta¹, Jens Hansen¹², Carolin Preiss, Dominic Teichert^¶, and Stefan Höning³

From the Department of Biomedical Science and Biotechnology, University of Brescia, 25100 Brescia, Italy, Institute for Biochemistry II, University of Göttingen, 37073 Göttingen, Germany, and ^¶Institute for Biochemistry I, University of Cologne, 50931 Cologne, Germany

The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (- and β2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit µ2. Phosphorylation of µ2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for µ2, 25 min for β2, and 70 min for . We were also able to purify a phosphatase that dephosphorylates the µ2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cβ subunit, a scaffolding Aβ subunit, and a regulatory B subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.

Received for publication, August 27, 2007 , and in revised form, December 12, 2007.

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB523, TPA5; SFB635, TPA3) (to S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and a supplemental model.

¹ Both authors contributed equally to this work.

² Present address: Inst. for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany.

³ To whom correspondence should be addressed: Inst. of Biochemistry I, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany. Tel.: 49-221-4783656; Fax: 49-221-4786979; E-mail: shoening{at}uni-koeln.de.

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