PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

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

ECM receptors in neuronal structure, synaptic plasticity, and behavior

During central nervous system development, extracellular matrix (ECM) receptors and their ligands play key roles as guidance molecules, informing neurons where and when to send axonal and dendritic projections, establish connections, and form synapses between pre- and postsynaptic cells. Once stable synapses are formed, many ECM receptors transition in function to control the maintenance of stable connections between neurons and regulate synaptic plasticity. These receptors bind to and are activated by ECM ligands. In turn, ECM receptor activation modulates downstream signaling cascades that control cytoskeletal dynamics and synaptic activity to regulate neuronal structure and function and thereby impact animal behavior. The activities of cell adhesion receptors that mediate interactions between pre- and postsynaptic partners are also strongly influenced by ECM composition. This chapter highlights a number of ECM receptors, their roles in the control of synapse structure and function, and the impact of these receptors on synaptic plasticity and animal behavior

Scrapie-Induced Defects in Learning and Memory of Transgenic Mice Expressing Anchorless Prion Protein Are Associated with Alterations in the Gamma Aminobutyric Acid-Ergic Pathway▿ †

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 (GABA) inhibition but not loss of excitatory glutamate/N-methyl-d-aspartic acid. Enhanced deposition of abnormally folded infectious PrP (PrPsc or PrPres) in the central nervous system (CNS) localizes with GABAA receptors. This occurs with minimal evidence of CNS spongiosis or apoptosis of neurons. The use of monoclonal antibodies reveals an association of PrPres with GABAA receptors. Thus, the clinical defects of learning and memory loss in vivo in GPI−/− PrP tg mice infected with scrapie agent may likely involve the GABAergic pathway

Integrin α4β1 Promotes Focal Adhesion Kinase-Independent Cell Motility via α4 Cytoplasmic Domain-Specific Activation of c-Src

The fibronectin binding integrins α5β1 and α4β1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For α5β1, β1-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 α5β1-stimulated cell motility and that exogenous expression of human α4 in FAK-null fibroblasts forms a functional α4β1 receptor that promotes robust cell motility equal to the α5β1 stimulation of wild-type and FAK-reconstituted fibroblasts. α4β1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the α4 cytoplasmic domain, independent of direct paxillin binding to α4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. α4 cytoplasmic domain-initiated signaling led to a ∼4-fold activation of c-Src which did not require paxillin binding to α4. Notably, α4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase α overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. α4β1-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 α5β1-stimulated FAK activation, our results support the existence of a novel α4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway

Learning induces neurotrophin signaling at hippocampal synapses

Learning-induced trophic activity is thought to be critical for maintaining health of the aging brain. We report here that learning, acting through an unexpected pathway, activates synaptic receptors for one of the brain's primary trophic factors. Unsupervised learning, but not exploratory activity alone, robustly increased the number of postsynaptic densities associated with activated (phosphorylated) forms of BDNF's TrkB receptor in adult rat hippocampus; these increases were blocked by an NMDA receptor antagonist. Similarly, stimulation of hippocampal slices at the learning-related theta frequency increased synaptic TrkB phosphorylation in an NMDA receptor–dependent fashion. Theta burst stimulation, which was more effective in this regard than other stimulation patterns, preferentially engaged NMDA receptors that, in turn, activated Src kinases. Blocking the latter, or scavenging extracellular TrkB ligands, prevented theta-induced TrkB phosphorylation. Thus, synaptic TrkB activation was dependent upon both ligand presentation and postsynaptic signaling cascades. These results show that afferent activity patterns and cellular events involved in memory encoding initiate BDNF signaling through synaptic TrkB, thereby ensuring that learning will trigger neurotrophic support