Glycogen synthase kinase 3 (GSK3) is a key regulator in signaling pathways in both animals and plants. Three Arabidopsis thaliana GSK3s are shown to be related to brassinosteroid (BR) signaling. In a phenotype-based compound screen we identified bikinin, a small molecule that activates BR signaling downstream of the BR receptor. Bikinin directly binds the GSK3 BIN2 and acts as an ATP competitor. Furthermore, bikinin inhibits the activity of six other Arabidopsis GSK3s. Genome-wide transcript analyses demonstrate that simultaneous inhibition of seven GSK3s is sufficient to activate BR responses. Our data suggest that GSK3 inhibition is the sole activation mode of BR signaling and argues against GSK3-independent BR responses in Arabidopsis. The opportunity to generate multiple and conditional knockouts in key regulators in the BR signaling pathway by bikinin represents a useful tool to further unravel regulatory mechanisms.

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.

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Background-Recent studies in mice have established that an endothelial cell protein, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), is essential for the lipolytic processing of triglyceride-rich lipoproteins. Methods and Results-We report the discovery of a homozygous missense mutation in GPIHBP1 in a young boy with severe chylomicronemia. The mutation, p.C65Y, replaces a conserved cysteine in the GPIHBP1 lymphocyte antigen 6 domain with a tyrosine and is predicted to perturb protein structure by interfering with the formation of a disulfide bond. Studies with transfected Chinese hamster ovary cells showed that GPIHBP1-C65Y reaches the cell surface but has lost the ability to bind lipoprotein lipase (LPL). When the GPIHBP1-C65Y homozygote was given an intravenous bolus of heparin, only trace amounts of LPL entered the plasma. We also observed very low levels of LPL in the postheparin plasma of a subject with chylomicronemia who was homozygous for a different GPIHBP1 mutation (p.Q115P). When the GPIHBP1-Q115P homozygote was given a 6-hour infusion of heparin, a significant amount of LPL appeared in the plasma, resulting in a fall in the plasma triglyceride levels from 1780 to 120 mg/dL. Conclusions-We identified a novel GPIHBP1 missense mutation (p.C65Y) associated with defective LPL binding in a young boy with severe chylomicronemia. We also show that homozygosity for the C65Y or Q115P mutations is associated with low levels of LPL in the postheparin plasma, demonstrating that GPIHBP1 is important for plasma triglyceride metabolism in humans. (Circ Cardiovasc Genet. 2010;3:169-178.)