The focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To gain insight into FAK function, we examined the potential interaction of FAK with intracellular signaling molecules containing the Src homology 2 domains. We report here the stable association of FAK with phosphatidylinositol 3-kinase (PI3-kinase; EC 2.7.1.137) in NIH 3T3 mouse fibroblasts. This interaction was stimulated by cell adhesion concomitant with FAK activation. We also found that recombinant FAK bound to the p85 subunit of PI 3-kinase directly in vitro and that autophosphorylation of recombinant FAK in vitro increased its binding to PI 3-kinase. We detected increased tyrosine phosphorylation of the p85 subunit of PI 3-kinase during cell adhesion and observed direct phosphorylation of p85 by FAK in vitro. Together, these results suggest that PI 3-kinase may be a FAK substrate in vivo and serve as an effector of FAK.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

Background. The Delta and Omicron variants of SARS-CoV-2 are currently responsible for breakthrough infections due to waning immunity. We report phase I/II trial results of UB-612, a multitope subunit vaccine containing S1-RBD-sFc protein and rationally designed promiscuous peptides representing sarbecovirus conserved helper T cell and cytotoxic T lymphocyte epitopes on the nucleocapsid (N), membrane (M), and spike (S2) proteins. Method. We conducted a phase I primary 2-dose (28 days apart) trial of 10, 30, or 100 μg UB-612 in 60 healthy young adults 20 to 55 years old, and 50 of them were boosted with 100 μg of UB-612 approximately 7 to 9 months after the second dose. A separate placebo-controlled and randomized phase II study was conducted with 2 doses of 100 μg of UB-612 (n = 3,875, 18–85 years old). We evaluated interim safety and immunogenicity of phase I until 14 days after the third (booster) dose and of phase II until 28 days after the second dose. Results. No vaccine-related serious adverse events were recorded. The most common solicited adverse events were injection site pain and fatigue, mostly mild and transient. In both trials, UB-612 elicited respective neutralizing antibody titers similar to a panel of human convalescent sera. The most striking findings were long-lasting virus-neutralizing antibodies and broad T cell immunity against SARS-CoV-2 variants of concern (VoCs), including Delta and Omicron, and a strong booster-recalled memory immunity with high cross-reactive neutralizing titers against the Delta and Omicron VoCs. Conclusion. UB-612 has presented a favorable safety profile, potent booster effect against VoCs, and long-lasting B and broad T cell immunity that warrants further development for both primary immunization and heterologous boosting of other COVID-19 vaccines. Trial Registration. ClinicalTrials.gov: NCT04545749, NCT04773067, and NCT04967742. Funding. UBI Asia, Vaxxinity Inc., and Taiwan Centers for Disease Control, Ministry of Health and Welfare.