Research Article The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis Vibeke Bertelsen a , Kamilla Breen a , Kirsten Sandvig b , Espen Stang a , Inger Helene Madshus a, ⁎ a Institute of Pathology, Faculty Division Rikshospitalet, University of Oslo, Rikshospitalet-Radiumhospitalet Medical Centre, 0027 Oslo, Norway b Department of Biochemistry, Rikshospitalet-Radiumhospitalet Medical Centre, Montebello, 0310 Oslo, Norway ARTICLEINFORMATION ABSTRACT Article Chronology: Received 5 October 2006 Revised version received 21 February 2007 Accepted 22 February 2007 Available online 28 February 2007 The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin- dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin. © 2007 Elsevier Inc. All rights reserved. Keywords: TULA/Sts-2 Endocytosis Dynamin Clathrin Ubiquitin Cbl EGFR Introduction Activation of the epidermal growth factor receptor (EGFR) initiates signal transduction important for gene expression, as well as endocytic downregulation of the EGFR. The ubiquitin ligase Cbl is recruited to the activated EGFR directly via binding of its tyrosine kinase binding domain to pY1045 of the EGFR or indirectly through the adaptor protein Grb2. Both direct and indirect binding of Cbl to activated EGFR are reported to result in ubiquitination of the EGFR [1]. Ubiquitination of the EGFR has been implicated in both its ligand-mediated endocytosis and in endosomal sorting [2]. The role of Cbl in EGF-induced endocytosis and downregulation of the EGFR is further supported by the finding that overexpression of the Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) (also known as suppressor of T-cell signalling-2, Sts-2) inhibits EXPERIMENTAL CELL RESEARCH 313 (2007) 1696 – 1709 ⁎ Corresponding author. Fax: +47 23071511. E-mail address: i.h.madshus@medisin.uio.no (I.H. Madshus) Abbreviations: EGFR, EGF receptor; LDL, low-density lipoprotein; MEM, minimal essential medium; MHC-I, major histocompatibility complex class I; SH3, Src homology 3; Tf, Transferrin; TULA, T-cell ubiquitin ligand; wt, wild type 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.02.017 available at www.sciencedirect.com www.elsevier.com/locate/yexcr