Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21 rho , stimulated tyrosine phosphorylation of focal adhesion kinase (p125 fak ) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22°C. CNF1 and DNT stimulated tyrosine phosphorylation of p125 fak and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics. Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21 rho function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT. In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca 2؉ mobilization. Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42 mapk and p44 mapk providing additional evidence for a novel p21rho -dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3.An increase in the tyrosine phosphorylation of the non-receptor protein tyrosine kinase p125 fak (1, 2) and the cytoskeletal associated protein paxillin (3, 4) has recently been identified as an early event in the action of diverse signaling molecules that mediate cell growth and differentiation (5) fak and paxillin tyrosine phosphorylation are accompanied by profound alterations in the organization of the actin cytoskeleton and in the assembly of focal adhesions (9, 13, 15, 25, 26), the distinct areas of the plasma membrane where p125 fak and paxillin are localized (1, 2, 27). The small G protein p21 rho , a member of the Ras superfamily of small GTP-binding proteins, has been implicated in the mitogen-stimulated formation of focal adhesions and actin stress fibers as well as in the tyrosine phosphorylation of p125 fak and paxillin (22, 25, 28 -30). These findings suggest the existence of a distinct signal transduction pathway in which p21rho is upstream of cytoskeletal reorganization and tyrosine phosphorylation of focal adhesion proteins (5).The mechanism of action of bacterial toxins has provided novel insights into the control of cellular regulatory processes, including signal transduction and cell proliferation. For example, the Clostridium botulinum C3 exoenzyme and the enterotoxins A and B from Clostridium difficile which selectively inactivate members of the Rho subfamily, have provided useful tools to evaluate the role of these small G proteins in signal transduction and cytoskeletal organization (31)(32)(33). In contrast to these clostridial toxins, CNF toxins produced by some pathogenic strains of Escherichia coli (34) and DNT from Bordetell...

Treatment of Swiss 3T3 cells with recombinant Pasteurella multocida toxin (rPMT), a potent intracellularly acting mitogen, stimulated tyrosine phosphorylation of multiple substrates including bands of M(r) 110,000-130,000 and M(r) 70,000-80,000. Tyrosine phosphorylation induced by rPMT occurred after a pronounced lag period (1 h) and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. Focal adhesion kinase (p125FAK) and paxillin are prominent substrates for rPMT-stimulated tyrosine phosphorylation. Tyrosine phosphorylation by rPMT could be dissociated from both protein kinase C activation and the mobilization of calcium from intracellular stores. rPMT stimulated striking actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells. Cytochalasin D, which disrupts the actin cytoskeleton, completely inhibited rPMT-induced tyrosine phosphorylation. In addition, tyrosine phosphorylation of p125FAK and paxillin in response to rPMT was completely abolished when cells were subsequently treated with platelet-derived growth factor at a concentration (30 ng/ml) that disrupted the actin cytoskeleton. Our results demonstrate for the first time that rPMT, a bacterial toxin, induces tyrosine phosphorylation of p125FAK and paxillin and promotes actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells.

Background: Rac1 has a relevant role in signal transduction pathways in T lymphocytes. Results: Rac1 GTPase associates with glycogen phosphorylase and modulates its enzymatic activity to trigger T cell proliferation. Conclusion: This study reveals a new role for Rac1 GTPase in cellular physiology, coordinating metabolism and proliferation. Significance: This new Rac1/PYGM pathway might be essential for an appropriate immune response.

1–42 β-Amyloid (Aβ1–42) peptide is a key molecule involved in the development of Alzheimer's disease. Some of its effects are manifested at the neuronal morphological level. These morphological changes involve loss of neurites due to cytoskeleton alterations. However, the mechanism of Aβ1–42 peptide activation of the neurodegenerative program is still poorly understood. Here, Aβ1–42 peptide-induced transduction of cellular death signals through the phosphatidylinositol 3-kinase (PI3K)/phosphoinositol-dependent kinase (PDK)/novel protein kinase C (nPKC)/Rac 1 axis is described. Furthermore, pharmacological inhibition of PDK1 and nPKC activities blocks Rac 1 activation and neuronal cell death. Our results provide insights into an unsuspected connection between PDK1, nPKCs and Rac 1 in the same signal-transduction pathway and points out nPKCs and Rac 1 as potential therapeutic targets to block the toxic effects of Aβ1–42 peptide in neurons.

Small guanosine triphosphatases (GTPases) of the Ras superfamily are key regulators of many key cellular events such as proliferation, differentiation, cell cycle regulation, migration, or apoptosis. To control these biological responses, GTPases activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some small GTPases also guanine nucleotide dissociation inhibitors (GDIs). Moreover, small GTPases transduce signals by their downstream effector molecules. Many studies demonstrate that small GTPases of the Ras family are involved in neurodegeneration processes. Here, in this review, we focus on the signaling pathways controlled by these small protein superfamilies that culminate in neurodegenerative pathologies, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Specifically, we concentrate on the two most studied families of the Ras superfamily: the Ras and Rho families. We summarize the latest findings of small GTPases of the Ras and Rho families in neurodegeneration in order to highlight these small proteins as potential therapeutic targets capable of slowing down different neurodegenerative diseases.