Rational Redesign of a Functional Protein Kinase-Substrate Interaction

Eukaryotic protein kinases typically phosphorylate substrates in the context of specific sequence motifs, contributing to specificity essential for accurate signal transmission. Protein kinases recognize their target sequences through complementary interactions within the active site cleft. As a step toward the construction of orthogonal kinase signaling systems, we have re-engineered the protein kinase Pim1 to alter its phosphorylation consensus sequence. Residues in the Pim1 catalytic domain interacting directly with a critical arginine residue in the substrate were substituted to produce a kinase mutant that instead accommodates a hydrophobic residue. We then introduced a compensating mutation into a Pim1 substrate, the pro-apoptotic protein BAD, to reconstitute phosphorylation both <i>in vitro</i> and in living cells. Coexpression of the redesigned kinase with its substrate in cells protected them from apoptosis. Such orthogonal kinase–substrate pairs provide tools to probe the functional consequences of specific phosphorylation events in living cells and to design synthetic signaling pathways

Additional file 1: Supplemental figures. of Neural stem cells for disease modeling of Wolman disease and evaluation of therapeutics

Figure S1. Immunocytochemical characterization of WD iPSCs. Figure S2. STR DNA analysis of WD fibroblasts, iPSCs, and NSCs. Figure S3. LysoTracker staining and Nile red staining of LDL loaded NSCs. Figure S4. HT144B NSCs show increased LysoTracker staining. Figure S5. Chemical structures. Figure S6. DT and HPBCD treatment reduces lysosomal staining in HT144B NSCs. Figure S7. DT and HPBCD do not significantly reduce neutral lipid accumulation in WD NSCs. Figure S8. High concentrations of DT and HPBCD affect cell viability. Figure S9. DT and HPBCD combination treatment have an additive effect on reducing lysosomal staining in HT144B NSCs. (PDF 652 kb

Sizing Ocean Giants Data and Scripts

Balaenoptera musculus and Physeter macrocephalus data were provided by the International Whaling Commission (IWC). These data are not included in sizingoceangiants.zip, and must be obtained from IWC directly