Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells–including inhibition of the mitochondrial ribosome–there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism.

bHelicobacter pylori infects over 3 billion people worldwide and is the primary risk factor for gastric cancer. Most individuals infected with H. pylori develop only asymptomatic gastritis; however, some develop ulcers or gastric adenocarcinoma. We demonstrate that one previously unappreciated parameter influencing H. pylori disease outcome is variation in the preinfection host microbiota. Utilizing a mouse model, we altered the microbiota by antibiotic treatment and found that these alterations resulted in significantly lowered H. pylori-triggered inflammation. Specifically, antibiotic pretreatment reduced CD4 ؉ T-helper cells and Ifn␥ transcript levels in gastric tissue after H. pylori infection. The bacterial communities in mice with a reduced response to H. pylori displayed many differences from those in untreated mice, including significantly more cluster IV and XIVa Clostridium spp., bacteria known to influence inflammation via regulatory T cell populations. Our findings suggest that microbiota composition, perhaps Clostridium spp., contributes to the variable disease outcome of H. pylori infection by altering the recruitment of CD4 ؉ T cells to the gastric compartment. Our results suggest that gastric microbiota could be used as a diagnostic tool to determine which patients are at risk for developing severe disease.

Unlike many cancers which exhibit glycolytic metabolism, high grade liposarcomas often exhibit low FDG uptake by PET despite rapid tumor growth. Here, we used liquid chromatography tandem mass spectrometry to identify carbon sources taken up by liposarcoma cell lines derived from patient xenograft tumors. Interestingly, we found that liposarcoma cell lines consume nucleosides from culture media, suggesting nucleoside salvage pathway activity. The nucleoside salvage pathway is dependent on deoxycytidine kinase (dCK) (1) and can be imaged in vivo by PET with 1-(2′-deoxy-2′-[18F]fluoroarabinofuranosyl) cytosine (FAC) (2). We found that liposarcoma cell lines and xenograft tumors exhibit dCK activity and dCK-dependent FAC uptake in vitro and in vivo. Additionally, liposarcoma cell lines and xenograft tumors are sensitive to treatment with the nucleoside analog prodrug gemcitabine, and gemcitabine sensitivity is dependent on dCK expression. Elevated dCK activity is evident in 7 out of 68 clinical liposarcoma samples analyzed. This data suggests that a subpopulation of liposarcoma patients have tumors with nucleoside salvage pathway activity that can be identified non-invasively using [18F]-FAC-PET and targeted using gemcitabine.