Ack1 is a non-receptor tyrosine kinase that participates in tumorigenesis, cell survival and migration. Relatively little is known about mechanisms that regulate Ack1 activity. First, this dissertation will address the effect of naturally occurring somatic mutations on Ack1 function and activity and the use of these mutations to gain insight into the regulation of Ack1. Second, I will describe the study of the role of the N-terminal domain on Ack1 regulation.

As my first aim, I studied four somatic missense mutations of Ack1 that were recently identified in cancer tissue samples. The effects of these cancer-associated mutations on Ack1 activity and function have not been described. These mutations occur in the N-terminal region, the C-lobe of the kinase domain, and the SH3 domain. Here, I show that the cancer-associated mutations increase Ack1 autophosphorylation in mammalian cells without affecting localization, and increase Ack1 activity in immune complex kinase assays. The cancer-associated mutations potentiate the ability of Ack1 to promote proliferation and migration, suggesting that point mutation is a mechanism for Ack1 deregulation. I propose that the C-terminal Mig6 homology region (MHR) (residues 802 to 990) participates in inhibitory intramolecular interactions. The isolated kinase domain of Ack1 interacts directly with the MHR, and the cancer-associated E346K mutation prevents binding. Likewise, mutation of a key hydrophobic residue in the MHR (F820) prevents the MHR-kinase interaction, activates Ack1, and increases cell migration. Thus, the cancer-associated mutation E346K appears to destabilize an autoinhibited conformation of Ack1, leading to constitutively high Ack1 activity.

The second set of experiments focused on the role of the N-terminal domain on Ack1 regulation. When overexpressed in mammalian cells, NKD (N-terminus and Kinase domain) is autophosphorylated while KD (Kinase domain alone) is not, indicating that the N-terminus is needed for Ack1 autophosphorylation. The purified kinase domain is autophosphorylated and active towards a peptide substrate in vitro and can be activated by increasing its local concentration, suggesting that an intermolecular interaction promotes activation. NKD localizes at the plasma membrane while KD shows a more diffuse cytosolic localization. Co-immunoprecipitation data show an interaction between NKD and full-length wild type Ack1. Taken together these studies suggest that the N-terminal domain of Ack1 is involved in a dimerization or self-association event that is required for autophosphorylation.