Role of Abl tyrosine kinase and its targets in the regulation of EGFR endocytosis

The Abl proto-oncoproteins are an important family of non-receptor tyrosine kinases which are required for a number of biological effects such as cell growth, survival, cytoskeletal rearrangements, and signal transduction events downstream of growth factor receptors that direct cellular proliferation and invasion. Aberrant activation of the Abl tyrosine kinases promotes constitutive activation of downstream effectors and leads to cellular transformation. Abi adaptor proteins are binding partners and substrates for Abl tyrosine kinases and can regulate their activity as well as function as downstream effectors of the AN tyrosine kinases. This thesis describes the characterization of the role of Abl tyrosine kinases and their targets in the regulation of EGFR internalization. We show that oncogenic, activated Abl-PP inhibits EGFR internalization and downregulation. Notably, we found that the EGFR is phosphorylated by the Abl kinases. Abl phosphorylated the EGFR primarily on tyrosine 1173 and to a lesser extent on tyrosines 1068 and 992. To investigate a mechanism for the inhibition of EGFR internalization by Abl tyrosine kinases we mutated tyrosine 1173 to phenylalanine in the EGFR. This mutation greatly decreased Abl-mediated phosphorylation of the EGFR and restored EGFR internalization in the presence of oncogenic, constitutively active, Abl-PP. To investigate whether additional mechanisms could mediate the Abl effects in EGFR internalization we focused our attention on the Cbl E3 ligase, which is an Abl substrate. Interestingly, Abl did not phosphorylate tyrosine 1045 on the EGFR, which is the recruitment site for Cbl in the EGFR. Notably, oncogenic Abl inhibited the accumulation of Cbl to the plasma membrane and Cbl recruitment to the EGFR upon addition of EGF. Thus, a decrease in Cbl recruitment to the EGFR suggested that the ubiquitination of the EGFR might be impaired in the presence of kinase-active Abl. Indeed, we found that EGFR ubiquitination decreased in the presence of kinase-active Abl-PP. Further support for a role of Abl in the regulation of endocytosis, was the finding of a novel Abl substrate: the endocytic regulator Endophilin A2. Together, our data suggest that activated Abl and its downstream effectors have a role in the regulation of EGFR internalization. The Abl kinases may interact functionally with the EGFR in tumors characterized by high amounts of activated EGFR on the cell surface and increased Abl activation. Thus, study of the mechanisms and signal transduction events modulated by Abl tyrosine kinases may provide new targets in the treatment of solid tumors.;Abi proteins (Abi-1 and Abi-2) have been shown to modulate the cellular effects of oncogenic Abl. Abi-1 was previously shown to interact with the endocytic regulators Dynamin and Synaptojanin. Additionally, Abi-1 was found to be required for EGFR internalization through an interaction with N-WASP. Here we describe the identification of a novel complex between Abi-1 and Cbl that is induced upon addition of EGF. Notably, expression of the Abi-I DeltaSH3 mutant that fails to interact with Cbl, inhibits EGFR internalization. Thus, the Abi-1/Cbl complex is a strong candidate for the regulation of EGFR internalization by Abi-1 adaptor proteins. In this context, oncogenic Abl tyrosine kinase disrupts the formation of the Abi-1/Cbl complex, highlighting the importance of Abl kinases and downstream effectors in the regulation of EGFR internalization. Thus, this thesis reveals a new role for oncogenic Abl tyrosine kinases in the regulation of EGFR internalization, and identifies Abl targets and mechanisms which may be used to develop therapies for cancer treatment.