F-BAR-containing adaptor CIP4 localizes to early endosomes and regulates Epidermal Growth Factor Receptor trafficking and downregulation.
Cdc42-Interacting Protein-4 (CIP4) family adaptors have been implicated in promoting Epidermal Growth Factor Receptor (EGFR) internalization, however, their unique or overlapping functions remain unclear. Here, we show that although CIP4 was not required for early events in clathrin-mediated endocytosis of EGFR, CIP4 localizes to vesicles containing EGFR and Rab5. Furthermore, expression of constitutively active Rab5 led to accumulation of CIP4 and the related adaptor Toca-1 in giant endosomes. Using a mutagenesis approach, we show that localization of CIP4 to endosomes is mediated in part via the curved phosphoinositide-binding face of the CIP4 F-BAR domain. Downregulation of CIP4 in A431 epidermoid carcinoma cells by RNA interference led to elevated EGFR levels, compared to control cells. Although surface expression of EGFR was not affected by CIP4 silencing, EGF-induced transit of EGFR from EEA1-positive endosomes to lysosomes was reduced compared to control cells. This correlated with more robust activation of ERK kinase and entry to S phase in CIP4-depleted A431 cells, compared to control cells. The combined silencing of CIP4 and Toca-1 was more effective in driving cells into S phase, suggesting a partial redundancy in their functions. Overall, our results implicate CIP4 and Toca-1 in regulating late events in EGFR trafficking from endosomes that serves to limit sustained ERK activation within the endosomal compartment.
Hu
Jinghui
J
Queen's University Cancer Research Institute, Division of Cancer Biology & Genetics and Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Troglio
Flavia
F
Mukhopadhyay
Alka
A
Everingham
Stephanie
S
Kwok
Ester
E
Scita
Giorgio
G
Craig
Andrew W B
AW
D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism; D015536:Down-Regulation; D011992:Endosomes / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D009154:Mutation; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D002352:Carrier Proteins; D002966:Clathrin; C482527:FNBP1L protein, human; D008869:Microtubule-Associated Proteins; D015778:Minor Histocompatibility Antigens; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C406285:TRIP10 protein, human; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D020696:rab5 GTP-Binding Proteins