Akt1 BINDS FOCAL ADHESION KINASE VIA THE Akt1 KINASE DOMAIN INDEPENDENTLY OF THE PLECKSTRIN HOMOLOGY DOMAIN

Akt1 and focal adhesion kinase (FAR) are protein kinases that play key roles in normal cell signaling. Individually, aberrant expression of these kinases has been linked to a variety of cancers. Together, Akt1/FAK interactions facilitate cancer metastasis by increasing cell adhesion under conditions of increased extracellular pressure. Pathological and iatrogenic sources of pressure arise from tumor growth against constraining stroma or direct perioperative manipulation. We previously reported that 15 mmHg increased extracellular pressure causes Aka to both directly interact with FAR and to phosphorylate and activate it. We investigated the nature of the Akt1/FAK binding by creating truncations of recombinant FAR, conjugated to glutathione S-transferase (GST), to pull down full-length Akt1. Western blots probing for Aka showed that FAK/Akt1 binding persisted in FAR truncations consisting of only amino acids 1-126, FAK(NT1), which contains the F1 subdomain of its band 4.1, ezrin, radixin, and moesin (FERM) domain. Using FAK(NT1) as bait, we then pulled down truncated versions of recombinant Aka conjugated to HA (human influenza hemagglutinin). Probes for GST-FAK(NT1) showed Akt1-FAR binding to occur in the absence of the both the Aka (N)-terminal pleckstrin homology (PH) domain and its adjacent hinge region. The Akt1 (C)-terminal regulatory domain was equally unnecessary for Akt1/FAK co-immunoprecipitation. Truncations involving the Akt1 catalytic domain showed that the domain by itself was enough to pull down FAK. Additionally, a fragment spanning from the PH domain to half way through the catalytic domain demonstrated increased FAR binding compared to full length Akt1. These results begin to delineate the Akt1/FAK interaction and can be used to manipulate their force-activated signal interactions. Furthermore, the finding that the N-terminal half of the Aka catalytic domain binds so strongly to FAR when cleaved from the rest of the protein may suggest a means for developing novel inhibitors that target this specific Akt1/FAK interaction.