Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.Src family kinases (SFKs) are a group of ubiquitously expressed nonreceptor tyrosine kinases that are essential for integrin-mediated cell motility and adhesion signaling (17). SFKs contain at least two distinct protein-protein interaction sequences, the eponymously named Src homology 2 and 3 (SH2 and SH3) domains. First identified in vSrc, SH2 domains have since been found in an array of proteins having catalytic, adapter, or regulatory roles.The SH2 domain is an ϳ100-amino-acid module that binds to particular phosphorylated tyrosine residues. SH2 binding specificity is determined by flanking sequences C terminal to the target phosphotyrosine, for Src usually the YEEI motif (16). In SFKs, the SH2 domain functions in both intra-and intermolecular binding. The intramolecular association is with its C-terminal phosphotyrosine, and this interaction maintains the enzyme in a closed, catalytically inactive conformation while also blocking intermolecular SH2 interactions and sequestering the SH3 domain (3, 39). Dephosphorylation of Src's C-terminal tail by tyrosine phosphatases (30, 32, 40) opens and activates the kinase while also allowing the SH2 domain to bind to its downstream substrates.The determination of unambiguous biological functions for SFKs was impeded for a number of years by redundancy among family members. The development of fibroblasts with the three ubiquitously expressed enzymes, Src, Yes, and Fyn, deleted (SYF cells), identified SFKs as crucial mediators of adhesion signaling (17). In adhesion, an important Src substrate is another nonreceptor tyrosine kinase, focal adhesion kinase (FAK) (22). FAK localizes to structures called focal adhesions (FAs) that form the link between the extracellular matrix (ECM) and the actin cytoskeleton. FAs are plaques containing accumulations of integrins, the cell's ECM receptor, and cytoplasmic proteins that mediate the connection between integrins and actin and regulate important biological functions such as apoptosis and ...

The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.The actin cytoskeleton plays key roles in cell motility and morphology, intracellular organization, membrane trafficking, and the intracellular movement of a variety of pathogens (1, 2). Many actin-based structures, especially those involved in membrane protrusion, are assembled through the coordinated polymerization and cross-linking of actin monomers into actin filaments that in turn form orthogonal or parallel filament networks (3). In that regard, the dynamics of actin stress fibers, filopodia, and lamellipodia (or membrane ruffles) are tightly regulated by various nucleation-promoting factors (WASP 4 family proteins) and actin nucleation proteins (e.g. the Arp2/3 complex) (4, 5). Among these components, the Arp2/3 complex localizes at the leading edge of cells, where the actin cytoskeleton is nucleated and reorganized (6, 7). The major activators of the complex include the WASP family proteins, which contain a conserved VCA domain composed of one or two VPH domains, which bind actin monomers, a central region, and an A domain, which binds to the Arp2/3 complex (8). In addition, cortactin, a filamentous actin-associated protein, binds to the Arp2/3 complex via an A domain at its N terminus and stimulates nucleation of actin filaments, ultimately promoting formation and stabilization of actin filament networks (9). In similar fashion, Abp1, an F-actin binding protein, also associates with the Arp2/3 complex and stimulates actin nucleation (10).The Arp2/3 complex also plays a role in the intracellular motility of pathogens and vesicles. For instance, the pathogenic bacterium Listeria monocytogenes utilizes actin polymerization mediated by the Arp2/3 complex to move within the cytoplasm of infected host cells. Likewise, movement of intracellular vesicles (i.e. endosomes) is dependent upon actin polymerization mediated by the Arp2/3 complex, which localizes al...

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin-mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase-linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell-extracellular matrix interactions.

Tussilagone, extracted from Tussilago farfara is an oriental medicine used for asthma and bronchitis. We investigated its mechanism of action, its inhibitory effects on lipopolysaccharide-induced inflammation in macrophages, and its impact on viability in a cecal ligation and puncture (CLP)-induced mouse model of sepsis. Tussilagone suppressed the expression of the inflammatory mediators, nitric oxide and prostaglandin E2, and the inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and high-mobility group box 1 (HMGB1), in lipopolysaccharide-stimulated RAW 264.7 cells and peritoneal macrophages. Tussilagone also reduced the activation of the mitogen-activated protein kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) involved in the activation of various inflammatory mediators in activated macrophages. Moreover, tussilagone administration (1 mg/kg and 10 mg/kg) produced decreased mortality and lung injury in CLP-activated septic mice. Augmented expression of cyclooxygenase (COX)-2 and TNF-α in pulmonary alveolar macrophages of septic mice were attenuated by tussilagone administration. Tussilagone also suppressed the induction of nitric oxide, prostaglandin E2, TNF-α and HMGB1 in the serum of the septic mice. Overall, tussilagone exhibited protective effects against inflammation and polymicrobial sepsis by suppressing inflammatory mediators possibly via the inhibition of NF-κB activation and the MAP kinase pathway. These results suggest the possible use of tussilagone for developing novel therapeutic modalities for sepsis and other inflammatory diseases.

We examined the consequences of v-Crk expression in mouse embryo fibroblasts deficient Src family kinases or p130CAS. We found that Src kinases are essential for p130CAS/v-Crk signaling leading to FAK phosphorylation and cell migration in which Src is likely to mediate the focal adhesion targeting of v-Crk. SYF cells showed only low levels of FAK phosphorylation and cell migration, even in the presence of v-Crk. Expression of v-Crk restored migration of p130CAS-deficient cells to the level of wild-type cells, most likely through the targeting of v-Crk to focal adhesions by cSrc. In addition, we identified a new v-Crk-interacting protein that mediates v-Crk signaling in p130CAS-deficient cells. Using RT-PCR and caspase cleavage assays, we confirmed that this protein is not p130CAS and is responsible for maintaining v-Crk/Src signaling and migration in these. These findings suggest that focal adhesion targeting of v-Crk is essential in v-Crk-mediated cellular signaling and that v-Crk must form a complex with p130CAS or a p130CAS substitute to transduce signaling from the extracellular matrix.