A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase

BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity

A phantom study investigating the relationship between ground-glass opacity visibility and physical detectability index in low-dose chest computed tomography

In this study, the relationship between ground-glass opacity (GGO) visibility and physical detectability index in low-dose computed tomography (LDCT) for lung cancer screening was investigated. An anthropomorphic chest phantom that included synthetic GGOs with CT numbers of -630 Hounsfield units (HU; high attenuation GGO: HGGO) and -800 HU (low attenuation GGO: LGGO), and three phantoms for physical measurements were employed. The phantoms were scanned using 12 CT systems located in 11 screening centers in Japan. The slice thicknesses and CT dose indices (CTDIvol) varied over 1.0-5.0 mm and 0.85-3.30 mGy, respectively, and several reconstruction kernels were used. Physical detectability index values were calculated from measurements of resolution, noise, and slice thickness properties for all image sets. Five radiologists and one thoracic surgeon, blind to one another\u27s observations, evaluated GGO visibility using a five-point scoring system. The physical detectability index correlated reasonably well with the GGO visibility (R2 = 0.709, p vol. Consequently, the CTDIvol also correlated reasonably well with the GGO visibility (R2 = 0.701, p vol was nearly dominant in the GGO visibility for image sets with different reconstruction kernels and slice thicknesses, used in this study

CCN6 as a profibrotic mediator that stimulates the proliferation of lung fibroblasts via the integrin 1/focal adhesion kinase pathway

Idiopathic pulmonary fibrosis is a progressive and lethal disease of the lung that is characterized by the proliferation of fibroblasts and increased deposition of the extracellular matrix. The CCN6/WISP-3 is a member of the CCN family of matricellular proteins, which consists of six members that are involved in many vital biological functions. However, the regulation of lung fibroblasts mediated by CCN6 protein has not been fully elucidated. Here, we demonstrated that CCN6 induced the proliferation of lung fibroblasts by binding to integrin β1, leading to the phosphorylation of FAKY397. Furthermore, CCN6 showed a weak, but significant, ability to stimulate the expression of fibronectin. CCN6 was highly expressed in the lung tissues of mice treated with bleomycin. Our results suggest that CCN6 plays a role in the fibrogenesis of the lungs mainly by stimulating the growth of lung fibroblasts and is a potential target for the treatment of pulmonary fibrosis

Antifibrotic effects of CXCR4 antagonist in bleomycin-induced pulmonary fibrosis in mice

Circulating fibrocytes had been reported to migrate into the injured lungs, and contribute to fibrogenesis via chemokine-chemokine receptor systems including CXCL12-CXCR4 axis. Here we hypothesized that blockade of CXCR4 might inhibit the migration of fibrocytes to the injured lungs and the subsequent pulmonary fibrosis. To explore the antifibrotic effects of blockade of CXCR4, we used a specific antagonist for CXCR4, AMD3100, in bleomycin-induced pulmonary fibrosis model in mice. Administration of AMD3100 significantly improved the loss of body weight of mice treated with bleomycin, and inhibited the fibrotic lesion in subpleural areas of the lungs. The quantitative analysis demonstrated that treatment with AMD3100 reduced the collagen content and fibrotic score (Aschcroft score) in the lungs. Although AMD3100 did not affect cell classification in bronchoalveolar lavage fluid on day 7, the percentage of lymphocytes was reduced by AMD3100 on day 14. AMD3100 directly inhibited the migration of human fibrocytes in response to CXCL12 in vitro, and reduced the trafficking of fibrocytes into the lungs treated with bleocmycin in vivo. These results suggest that the blockade of CXCR4 might be useful strategy for therapy of patients with pulmonary fibrosis via inhibiting the migration of circulating fibrocytes