Tyrosine Phosphorylation of Rac1: A Role in Regulation of Cell Spreading

Rac1 influences a multiplicity of vital cellular- and tissue-level control functions, making it an important candidate for targeted therapeutics. The activity of the Rho family member Cdc42 has been shown to be modulated by tyrosine phosphorylation at position 64. We therefore investigated consequences of the point mutations Y64F and Y64D in Rac1. Both mutations altered cell spreading from baseline in the settings of wild type, constitutively active, or dominant negative Rac1 expression, and were accompanied by differences in Rac1 targeting to focal adhesions. Rac1-Y64F displayed increased GTP-binding, increased association with βPIX, and reduced binding with RhoGDI as compared with wild type Rac1. Rac1-Y64D had less binding to PAK than Rac1-WT or Rac1-64F. In vitro assays demonstrated that Y64 in Rac1 is a target for FAK and Src. Taken together, these data suggest a mechanism for the regulation of Rac1 activity by non-receptor tyrosine kinases, with consequences for membrane extension

KSHV gB associated RGD interactions promote attachment of cells by inhibiting the potential migratory signals induced by the disintegrin-like domain

Background: Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is not only expressed on the envelope of mature virions but also on the surfaces of cells undergoing lytic replication. Among herpesviruses, KSHV gB is the only glycoprotein known to possess the RGD (Arg-Gly-Asp) binding integrin domain critical to mediating cell attachment. Recent studies described gB to also possess a disintegrin-like domain (DLD) said to interact with non-RGD binding integrins. We wanted to decipher the roles of two individually distinct integrin binding domains (RGD versus DLD) within KSHV gB in regulating attachment of cells over cell migration

Computational Analysis of the Spatiotemporal Coordination of Polarized PI3K and Rac1 Activities in Micro-Patterned Live Cells

Polarized molecular activities play important roles in guiding the cell toward persistent and directional migration. In this study, the polarized distributions of the activities of phosphatidylinositol 3-kinase (PI3K) and the Rac1 small GTPase were monitored using chimeric fluorescent proteins (FPs) in cells constrained on micro-patterned strips, with one end connecting to a neighboring cell (junction end) and the other end free of cell-cell contact (free end). The recorded spatiotemporal dynamics of the fluorescent intensity from different cells was scaled into a uniform coordinate system and applied to compute the molecular activity landscapes in space and time. The results revealed different polarization patterns of PI3K and Rac1 activity induced by the growth factor stimulation. The maximal intensity of different FPs, and the edge position and velocity at the free end were further quantified to analyze their correlation and decipher the underlying signaling sequence. The results suggest that the initiation of the edge extension occurred before the activation of PI3K, which led to a stable extension of the free end followed by the Rac1 activation. Therefore, the results support a concerted coordination of sequential signaling events and edge dynamics, underscoring the important roles played by PI3K activity at the free end in regulating the stable lamellipodia extension and cell migration. Meanwhile, the quantification methods and accompanying software developed can provide a convenient and powerful computational analysis platform for the study of spatiotemporal molecular distribution and hierarchy in live cells based on fluorescence images

SSeCKS/Gravin/AKAP12 attenuates expression of proliferative and angiogenic genes during suppression of v-Src-induced oncogenesis

BACKGROUND: SSeCKS is a major protein kinase C substrate with kinase scaffolding and metastasis-suppressor activity whose expression is severely downregulated in Src- and Ras-transformed fibroblast and epithelial cells and in human prostate, breast, and gastric cancers. We previously used NIH3T3 cells with tetracycline-regulated SSeCKS expression plus a temperature-sensitive v-Src allele to show that SSeCKS re-expression inhibited parameters of v-Src-induced oncogenic growth without attenuating in vivo Src kinase activity. METHODS: We use cDNA microarrays and semi-quantitative RT-PCR analysis to identify changes in gene expression correlating with i) SSeCKS expression in the absence of v-Src activity, ii) activation of v-Src activity alone, and iii) SSeCKS re-expression in the presence of active v-Src. RESULTS: SSeCKS re-expression resulted in the attenuation of critical Src-induced proliferative and pro-angiogenic gene expression including Afp, Hif-1α, Cdc20a and Pdgfr-β, and conversely, SSeCKS induced several cell cycle regulatory genes such as Ptpn11, Gadd45a, Ptplad1, Cdkn2d (p19), and Rbbp7. CONCLUSION: Our data provide further evidence that SSeCKS can suppress Src-induced oncogenesis by modulating gene expression downstream of Src kinase activity

Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-β, α-SMA, etc.) followed by the establishment of lung fibrosis