The smaller isoforms of ankyrin 3 bind to the p85 subunit of phosphatidylinositol 3'-kinase and enhance platelet-derived growth factor receptor down-regulation.
The Src homology 2 (SH2) domains of the p85 subunit of phosphatidylinositol 3'-kinase have been shown to bind to the tyrosine-phosphorylated platelet-derived growth factor receptor (PDGFR). Previously, we have demonstrated that p85 SH2 domains can also bind to the serine/threonine kinase A-Raf via a unique phosphorylation-independent interaction. In this report, we describe a new phosphotyrosine-independent p85 SH2-binding protein, ankyrin 3 (Ank3). In general, ankyrins serve a structural role by binding to both integral membrane proteins at the plasma membrane and spectrin/fodrin proteins of the cytoskeleton. However, smaller isoforms of Ank3 lack the membrane domain and are localized to late endosomes and lysosomes. We found that p85 binds directly to these smaller 120- and 105-kDa Ank3 isoforms. Both the spectrin domain and the regulatory domain of Ank3 are involved in binding to p85. At least two domains of p85 can bind to Ank3, and the interaction involving the p85 C-SH2 domain was found to be phosphotyrosine-independent. Overexpression of the 120- or 105-kDa Ank3 proteins resulted in significantly enhanced PDGFR degradation and a reduced ability to proliferate in response to PDGF. Ank3 overexpression also differentially regulated signaling pathways downstream from the PDGFR. Chloroquine, an inhibitor of lysosomal-mediated degradation pathways, blocked the ability of Ank3 to enhance PDGFR degradation. Immunofluorescence experiments demonstrated that both small Ank3 isoforms colocalized with the lysosomal-associated membrane protein and with p85 and the PDGFR. These results suggest that Ank3 plays an important role in lysosomal-mediated receptor down-regulation, likely through a p85-Ank3 interaction.
Ignatiuk
Ashley
A
Cancer Research Unit, Health Research Division, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada.
Quickfall
Jeremy P
JP
Hawrysh
Andrea D
AD
Chamberlain
M Dean
MD
Anderson
Deborah H
DH
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D015536:Down-Regulation; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D018909:src Homology Domains
D017487:Ankyrins; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D010982:Platelet-Derived Growth Factor; D020033:Protein Isoforms; D017479:Receptors, Platelet-Derived Growth Factor; C094681:Ank3 protein, mouse