Regulation of constitutive platelet-derived growth factor receptor degradation by the 105 kilodalton isoform of ankyrin3
Deregulation of platelet-derived growth factor receptor (PDGFR) signaling is a driving event in glioblastoma, promotes tumor progression epithelial to mesenchymal transition (EMT) in multiple cancers, modulates the tumor stroma to facilitate tumorigenesis and reduces tumor uptake of chemotherapeutics. Previous studies identified the 105 kDa isoform of ankyrin3 (Ank105) as a binding partner of the PDGFR signaling machinery and demonstrated that expression of Ank105 promoted PDGFR degradation (Ignatiuk et al., 2006)(Ignatiuk et al., 2006)(Ignatiuk et al., 2006). Receptor tyrosine kinases are targeted for degradation via endocytosis and ubiquitin-dependent trafficking to the lysosome. It was hypothesized that Ank105 promoted the constitutive degradation of the PDGFR and attenuation of PDGFR signaling by facilitating endocytosis of the PDGFR and targeting the PDGFR for lysosomal degradation via an ubiquitin-dependent mechanism. The studies in this thesis characterized the effects of Ank105 expression on PDGFR signaling and protein expression levels, determined the endocytic pathways involved in Ank105-mediated PDGFR degradation and studied the role of ubiquitin binding in Ank105 function. The most robust effect of Ank105 expression on the PDGFR was constitutive degradation as PDGFR protein expression levels in Ank105-expressing cells were significantly reduced compared to NIH 3T3 cells in the absence of PDGF ligand. Low constitutive PDGFR levels resulted in attenuated pro-proliferative AKT and mitogen-activated protein kinase (MAPK) signaling in response to ligand stimulation. To determine the endocytic requirements for Ank105-mediated constitutive PDGFR degradation, a constitutive PDGFR degradation assay was developed and the effects of several small molecule endocytosis inhibitors were evaluated. Additionally, the small molecule endocytosis inhibitors were validated by determining the effects of these inhibitors on low density lipoprotein (LDL) uptake and ligand-induced PDGFR degradation in Ank105-expressing cells. Both LDL uptake and ligand induced PDGFR degradation are known to proceed by a clathrin and dynamin dependent mechanism of endocytosis. In Ank105-expressing cells, both LDL uptake and ligand incuded PDGFR degradation were dependent upon clathrin and dynamin function. Interestingly, constitutive PDGFR degradation in Ank105-expressing cells was not dependent upon CME, but required dynamin activity. Expression of Ank105 may promote clathrin-independent, dynamin-dependent, constitutive endocytosis of the PDGFR. Additionally, acute inhibition of either lysosomal or proteasomal degradation strongly impaired constitutive PDGFR degradation, whereas ligand-induced PDGFR degradation was less sensitive to protein degradation inhibitors, while LDL uptake was unaffected. It was unclear if PDGFR was degraded in the proteasome or if the proteasome was involved in sorting of PDGFR to the lysosome for degradation. Ubiquitination of receptors is required to target them for degradation. Ank105 was assayed for the ability to interact with ubiquitin and ubiquitinated proteins. Interestingly, Ank105 bound ubiquitin in vitro via the spectrin binding domain and co-immunoprecipitated with several ubiquitinated proteins, suggesting a role for Ank105 in the sorting of ubiquitinated proteins for degradation. Furthermore, Ank105 co-immunoprecipitated with a number of high and low molecular weight proteins in the absence of PDGF stimulation. Identification of Ank105 binding partners would provide further insight in the mechanism of Ank105-mediated constitutive PDGFR degradation. In summary, Ank105 promoted the attenuation of PDGFR signaling via alteration of constitutive PDGFR endocytosis and targeting of constitutive PDGFR for degradation, potentially through interaction with ubiquitin and ubiquitinated proteins. Reduction of constitutive PDGFR levels in cancers with PDGFR driver mutations, acquired PDGF responsiveness and stromal expression of PDGFR, could significantly reduce tumor proliferation, tumorigenesis and increase effectiveness of chemotherapeutics.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeBonham, Keith; Lee, Jeremy; Chelico, Linda; Geyer, Ron
Copyright DateMarch 2014
Ankyrin, PDGFR, degradation, endocytosis, dynamin, clathrin, constitutive, shRNA, small molecule inhibitors, signaling, ubiquitin