Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin-associated tyrosine kinases, and if integrin signaling targets include NMDA-class glutamate neurotransmitter receptors. The integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, prolinerich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to b1 integrin, and not obtained with control peptides, indicating that kinase activation was integrinmediated. Both GRGDSP and fibronectin caused rapid Src kinase-dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and b1 integrin-dependent increase in NMDA receptor-mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.

Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that a4 and a5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either a4b1-or a5b1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that a5b1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for a5b1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, a4b1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatasea overexpression inhibited a4b1-stimulated NB motility and Src activation consistent with a4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In a4 shRNA-expressing NB cells, a4b1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated a4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that a4b1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during a5b1-mediated NB migration and support the evaluation of inhibitors to a4, Src and FAK in the control of NB tumor progression.

The fibronectin binding integrins ␣5␤1 and ␣4␤1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For ␣5␤1, ␤1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for ␣5␤1-stimulated cell motility and that exogenous expression of human ␣4 in FAK-null fibroblasts forms a functional ␣4␤1 receptor that promotes robust cell motility equal to the ␣5␤1 stimulation of wild-type and FAK-reconstituted fibroblasts. ␣4␤1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the ␣4 cytoplasmic domain, independent of direct paxillin binding to ␣4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. ␣4 cytoplasmic domain-initiated signaling led to a ϳ4-fold activation of c-Src which did not require paxillin binding to ␣4. Notably, ␣4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase ␣ overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. ␣4␤1-stimulated cell motility of triple-null Src ؊/؊ , c-Yes ؊/؊ , and Fyn ؊/؊ fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for ␣5␤1-stimulated FAK activation, our results support the existence of a novel ␣4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.Integrins are a family of heterodimeric ␣/␤ transmembrane cell adhesion receptors that play important roles in the regulation of cell migration during development, wound healing, inflammation, and the spread of tumor cells. Integrins do not possess intrinsic catalytic activity, and thus, signaling events are mediated by either lateral association with other receptors (8,52) or the clustering of signaling proteins with integrin cytoplasmic domains (44). As the composition of integrin signaling complexes is diverse and remains poorly defined (16), it is important to identify the molecular signaling signature of integrins that share a common ␤ subunit, bind to a common substrate such as fibronectin (FN), and function to promote cell motility. The FN binding integrins ␣5␤1 and ␣4␤1 share these properties.␣5␤1 is considered to be the classical FN receptor, with binding occurring within FN repeats III-9 and III-10 (33, 37). Rapid activation of protein-tyrosine kinases (PTKs) is one of the first signaling events associated with ␣5␤1 binding to FN, and signals generated by the ␤1 cytoplasmic domain are important in promoting cell motility (12, 39). Focal adhesion kinase (FAK) is recruited to sites of ␣5␤1 clustering through FAK C-terminal domain interactions with ␤1-integrin binding proteins such as talin and adaptor proteins such as paxillin (34). FN-stimulated FAK activation results in increased F...

After infection with RML murine scrapie agent, transgenic (tg) mice expressing prion protein (PrP) without its glycophosphatidylinositol (GPI) membrane anchor (GPI ؊/؊ PrP tg mice) continue to make abundant amounts of the abnormally folded disease-associated PrPres but have a normal life span. In contrast, all age-, sex-, and genetically matched mice with a GPI-anchored PrP become moribund and die due to a chronic progressive neurodegenerative disease by 160 days after RML scrapie agent infection. We report here that infected GPI ؊/؊ PrP tg mice, although free from progressive neurodegenerative disease of the cerebellum and extrapyramidal and pyramidal systems, nevertheless suffer defects in learning and memory, long-term potentiation, and neuronal excitability. Such dysfunction increases over time and is associated with an increase in gamma aminobutyric acid (