Receptor association and tyrosine phosphorylation of S6 kinases

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.     

Compensation of large motion sensor displacements during long recordings of limb movements

In motion capture applications using electromagnetic tracking systems the process of anatomical calibration associates the technical frames of sensors attached to the skin with the human anatomy. Joint centers and axes are determined relative to these frames. A change of orientation of the sensor relative to the skin renders this calibration faulty. This sensitivity regarding sensor displacement can turn out to be a serious problem with movement recordings of several minutes duration. We propose the “dislocation distance” as a novel method to quantify sensor displacement and to detect gradual and sudden changes of sensor orientation. Furthermore a method to define a so called fixed technical frame is proposed as a robust reference frame which can adapt to a new sensor orientation on the skin. The proposed methods are applied to quantify the effects of sensor displacement of 120 upper and lower limb movement recordings of newborns revealing the need for a method to compensate for sensor displacement. The reliability of the fixed technical frame is quantified and it is shown that trend and dispersion of the dislocation distance can be significantly reduced. A working example illustrates the consequences of sensor displacement on derived angle time series and how they are avoided using the fixed technical frame.     

How Prof. Burnstock’s enthusiasm supported P2 receptor research in Germany

Purinergic Signalling -     

The K+ Channel KCa3.1 as a Novel Target for Idiopathic Pulmonary Fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that K_Ca3.1 K⁺ channel-dependent cell processes contribute to IPF pathophysiology.

Methods

K_Ca3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of K_Ca3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct K_Ca3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).

Results

Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed K_Ca3.1 channel mRNA and protein. K_Ca3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. K_Ca3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. K_Ca3.1 was expressed strongly in IPF tissue. K_Ca3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca²⁺.

Conclusions

K_Ca3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking K_Ca3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic.     

DEAD-Box RNA Helicases in Bacillus subtilis Have Multiple Functions and Act Independently from Each Other

DEAD-box RNA helicases play important roles in remodeling RNA molecules and in facilitating a variety of RNA-protein interactions that are key to many essential cellular processes. In spite of the importance of RNA, our knowledge about RNA helicases is limited. In this study, we investigated the role of the four DEAD-box RNA helicases in the Gram-positive model organism Bacillus subtilis. A strain deleted of all RNA helicases is able to grow at 37°C but not at lower temperatures. The deletion of cshA, cshB, or yfmL in particular leads to cold-sensitive phenotypes. Moreover, these mutant strains exhibit unique defects in ribosome biogenesis, suggesting distinct functions for the individual enzymes in this process. Based on protein accumulation, severity of the cold-sensitive phenotype, and the interaction with components of the RNA degradosome, CshA is the major RNA helicase of B. subtilis. To unravel the functions of CshA in addition to ribosome biogenesis, we conducted microarray analysis and identified the ysbAB and frlBONMD mRNAs as targets that are strongly affected by the deletion of the cshA gene. Our findings suggest that the different helicases make distinct contributions to the physiology of B. subtilis. Ribosome biogenesis and RNA degradation are two of their major tasks in B. subtilis.