An essential role for Grk2 in Hedgehog signalling downstream of Smoothened

The G‐protein‐coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock‐down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP‐dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase‐dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C‐terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho‐null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.     

A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.     

Neuromechanical control of the isolated upper airway of mice

We characterized the passive structural and active neuromuscular control of pharyngeal collapsibility in mice and hypothesized that pharyngeal collapsibility, which is elevated by anatomic loads, is reduced by active neuromuscular responses to airflow obstruction. To address this hypothesis, we examined the dynamic control of upper airway function in the isolated upper airway of anesthetized C57BL/6J mice. Pressures were lowered downstream and upstream to the upper airway to induce inspiratory airflow limitation and critical closure of the upper airway, respectively. After hyperventilating the mice to central apnea, we demonstrated a critical closing pressure (Pcrit) of -6.2 +/- 1.1 cmH(2)O under passive conditions that was unaltered by the state of lung inflation. After a period of central apnea, lower airway occlusion led to progressive increases in phasic genioglossal electromyographic activity (EMG(GG)), and in maximal inspiratory airflow (Vi(max)) through the isolated upper airway, particularly as the nasal pressure was lowered toward the passive Pcrit level. Moreover, the active Pcrit fell during inspiration by 8.2 +/- 1.4 cmH(2)O relative to the passive condition (P < 0.0005). We conclude that upper airway collapsibility (passive Pcrit) in the C57BL/6J mouse is similar to that in the anesthetized canine, feline, and sleeping human upper airway, and that collapsibility falls markedly under active conditions. Active EMG(GG) and Vi(max) responses dissociated at higher upstream pressure levels, suggesting a decrease in the mechanical efficiency of upper airway dilators. Our findings in mice imply that anatomic and neuromuscular factors interact dynamically to modulate upper airway function, and provide a novel approach to modeling the impact of genetic and environmental factors in inbred murine strains.     

In vitro reconstitution of a trimeric complex of DivIB, DivIC and FtsL, and their transient co-localization at the division site in Streptococcus pneumoniae

DivIB, DivIC and FtsL are bacterial proteins essential for cell division, which show interdependencies for their stabilities and localization. We have reconstituted in vitro a trimeric complex consisting of the recombinant extracellular domains of the three proteins from Streptococcus pneumoniae. The extracellular domain of DivIB was found to associate with a heterodimer of those of DivIC and FtsL. The heterodimerization of DivIC and FtsL was artificially constrained by fusion with interacting coiled-coils. Immunofluorescence experiments showed that DivIC is always localized at mid-cell, in contrast to DivIB and FtsL, which are co-localized with DivIC only during septation. Taken together, our data suggest that assembly of the trimeric complex DivIB/DivIC/FtsL is regulated during the cell cycle through controlled formation of the DivIC/FtsL heterodimer.