From the results of deletion analyses, the FERM domain of FAK has been proposed to inhibit enzymatic activity and repress FAK signaling. We have identified a sequence in the FERM domain that is important for FAK signaling in vivo. Point mutations in this sequence had little effect upon catalytic activity in vitro. However, the mutant exhibits reduced tyrosine phosphorylation and dramatically reduced Src family kinase binding. Further, the abilities of the mutant to transduce biochemical signals and to promote cell migration were severely impaired. The results implicate a FERM domain interaction in cell adhesion-dependent activation of FAK and downstream signaling. We also show that the purified FERM domain of FAK interacts with full-length FAK in vitro, and mutation of this sequence disrupts the interaction. These findings are discussed in the context of models of FAK regulation by its FERM domain.The focal adhesion kinase, FAK, plays a major role in transducing signals downstream of integrins (40,47). Upon integrinmediated adhesion, FAK becomes tyrosine phosphorylated and activated. Additional signaling molecules, e.g., Src and phosphatidylinositol 3-kinase, are recruited into complexes with FAK, leading to the transduction of biochemical signals that control important biological processes. Integrin signaling via FAK regulates cell migration, proliferation, and survival. FAK-dependent regulation of one or more of these processes is essential, since fak Ϫ/Ϫ mice exhibit embryonic lethality (27). Conversely, enhanced FAK signaling may lead to aberrant cell proliferation, survival, or migration, which may have pathological consequences in humans. For example, aberrant FAK signaling may contribute to cancer development and progression to metastatic disease (40).FAK contains three major domains, an N-terminal domain, a central catalytic domain, and a C-terminal domain (40, 47). The C-terminal domain can be further subdivided into the focal adhesion targeting (FAT) sequence, comprising the Cterminal 140 amino acids of the protein, and the region between the catalytic domain and the FAT sequence. Focal adhesion-associated FAT sequence binding partners have been identified, and insight into the molecular basis of FAT sequence function was recently obtained from crystal and nuclear magnetic resonance structure analyses (3,14,23,26,31). The sequence between the catalytic domain and the FAT sequence contains docking sites for SH3 domain-containing proteins and thus serves as a scaffold for the recruitment of signaling proteins. Several sites of tyrosine phosphorylation play important regulatory roles in FAK. Within the catalytic domain, two tyrosine residues in the activation loop, tyrosines 576 and 577, regulate catalytic activity. The major site of autophosphorylation, tyrosine 397, lies just N terminal to the catalytic domain and serves as a binding site for Src family kinases. While details regarding the function of the catalytic and C-terminal domains have been elucidated, fewer studies have examined the function of ...

Background The limited neurobiological understanding of PTSD has been partially attributed to the need for improved animal models. Stress-enhanced fear learning (SEFL) in rodents recapitulates many PTSD-associated behaviors, including stress-susceptible (SS) and –resilient (SR) subgroups in outbred rats. Identification of subgroups requires additional behavioral phenotyping, a confound to mechanistic studies. Methods We employed a SEFL paradigm in inbred male and female C57BL/6 that combines acute stress with fear conditioning to precipitate “traumatic” memories. Extinction and long-term retention of extinction were examined after SEFL. Further characterization of SEFL effects on male mice was performed with additional behavioral tests, determination of regional activation by Fos immunofluorescence and RNA-sequencing of the basolateral amygdala (BLA). Results Stressed animals displayed persistently elevated freezing during extinction. While more uniform in females, SEFL produced male subgroups with differential susceptibility that were identified without post-training phenotyping. Additional phenotyping of males revealed PTSD-associated behaviors, including extinction-resistant fear memory, hyperarousal, generalization and dysregulated corticosterone in SS males. Altered Fos activation was also seen in the infralimbic cortex and BLA of SS males after remote memory retrieval. Key behavioral outcomes, including susceptibility, were replicated by two independent laboratories. RNA-sequencing of the BLA revealed transcriptional divergence between the male subgroups, including genes with reported polymorphic association to PTSD patients. Conclusions This SEFL model provides a tool for development of PTSD therapeutics that is compatible with the growing number of mouse-specific resources. Furthermore, use of an inbred strain allows for investigation into epigenetic mechanisms that are expected to critically regulate susceptibility and resilience.

Focal adhesion kinase (FAK) is a non-receptor tyro-

Aminoacyl tRNA synthetases--enzymes that catalyze the first step of protein synthesis--in mammalian cells are now known to have expanded functions, including activities in signal transduction pathways, such as those for angiogenesis and inflammation. The native synthetases themselves are procytokines, having no signal transduction activities. After alternative splicing or natural proteolysis, specific fragments that are potent cytokines and that interact with specific receptors on cell surfaces are released. In this manner, a natural fragment of human tyrosyl tRNA synthetase (TyrRS), mini-TyrRS, has been shown to act as a proangiogenic cytokine. The mechanistic basis for the action of mini-TyrRS in angiogenesis has yet to be established. Here, we show that mini-TyrRS is exported from endothelial cells when they are treated with tumor necrosis factor-alpha. Mini-TyrRS binds to vascular endothelial cells and activates an array of angiogenic signal transduction pathways. Mini-TyrRS-induced angiogenesis requires the activation of vascular endothelial growth factor receptor-2 (VEGFR2/Flk-1/KDR). Mini-TyrRS stimulates VEGFR2 phosphorylation in a VEGF-independent manner, suggesting VEGFR2 transactivation. Transactivation of VEGFR2 and downstream angiogenesis require an intact Glu-Leu-Arg (ELR) motif in mini-TyrRS, which is important for its cytokine activity. These studies therefore suggest a mechanism by which mini-TyrRS induces angiogenesis in endothelial cells and provide further insight into the role of mini-TyrRS as a link between translation and angiogenesis.

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) is critical for recruitment of FAK to focal adhesions and contains tyrosine 926, which, when phosphorylated, binds the SH2 domain of Grb2. Structural studies have shown that the FAT domain is a four-helix bundle that exists as a monomer and a dimer due to domain swapping of helix 1. Here, we report the NMR solution structure of the avian FAT domain, which is similar in overall structure to the X-ray crystal structures of monomeric forms of the FAT domain, except that loop 1 is longer and less structured in solution. Residues in this region undergo temperature-dependent exchange broadening and sample aberrant phi and psi angles, which suggests that this region samples multiple conformations. We have also identified a mutant that dimerizes approximately 8 fold more than WT FAT domain and exhibits increased phosphorylation of tyrosine 926 both in vitro and in vivo.