Glycogen synthase kinase-3 is required for efficient Dictyostelium chemotaxis.

Glycogen synthase kinase-3 (GSK3) is a highly conserved protein kinase that is involved in several important cell signaling pathways and is associated with a range of medical conditions. Previous studies indicated a major role of the Dictyostelium homologue of GSK3 (gskA) in cell fate determination during morphogenesis of the fruiting body; however, transcriptomic and proteomic studies have suggested that GSK3 regulates gene expression much earlier during Dictyostelium development. To investigate a potential earlier role of GskA, we examined the effects of loss of gskA on cell aggregation. We find that cells lacking gskA exhibit poor chemotaxis toward cAMP and folate. Mutants fail to activate two important regulatory signaling pathways, mediated by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and target of rapamycin complex 2 (TORC2), which in combination are required for chemotaxis and cAMP signaling. These results indicate that GskA is required during early stages of Dictyostelium development, in which it is necessary for both chemotaxis and cell signaling.

A systematic investigation of the protein kinases involved in NMDA receptor-dependent LTD: evidence for a role of GSK-3 but not other serine/threonine kinases.

BACKGROUND: The signalling mechanisms involved in the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term depression (LTD) in the hippocampus are poorly understood. Numerous studies have presented evidence both for and against a variety of second messengers systems being involved in LTD induction. Here we provide the first systematic investigation of the involvement of serine/threonine (ser/thr) protein kinases in NMDAR-LTD, using whole-cell recordings from CA1 pyramidal neurons. RESULTS: Using a panel of 23 inhibitors individually loaded into the recorded neurons, we can discount the involvement of at least 57 kinases, including PKA, PKC, CaMKII, p38 MAPK and DYRK1A. However, we have been able to confirm a role for the ser/thr protein kinase, glycogen synthase kinase 3 (GSK-3). CONCLUSION: The present study is the first to investigate the role of 58 ser/thr protein kinases in LTD in the same study. Of these 58 protein kinases, we have found evidence for the involvement of only one, GSK-3, in LTD.

Use of a penetratin-linked peptide in Dictyostelium.

The plasma membrane is an effective barrier to most macromolecules and hydrophilic molecules. Remarkably, a class of positively charged cell-penetrating peptides (CPPs) has been discovered that can translocate themselves and associated cargoes into the cytoplasm. These have been used to carry oligopeptide- and oligonucleotide-based inhibitors into mammalian cells. A recent report indicates that the same CPPs are internalized by plant protoplasts, suggesting that this may be a universal phenomenon. We report here that the prototypical CPP, penetratin, enters cells of the free-living amoebae Dictyostelium discoideum. To investigate the functionality of this technology, we fused the penetratin sequence to PKI, a peptide inhibitor of the cAMP-dependent protein kinase (PKA). Consistent with its PKA inhibitory action, Penetratin-PKI blocked aggregation in wild-type cells and, at appropriate concentrations, rescued the phenotype of a Dictyostelium mutant that has constitutively high PKA activity. This technology offers an effective method for delivery of oligopeptides and oligonucleotides into Dictyostelium.

Dd-Alix, a conserved endosome-associated protein, controls Dictyostelium development.

We have characterized the Dictyostelium homolog of the mammalian protein Alix. Dd-Alix is encoded by a single gene and is expressed during vegetative growth and multicellular development. We showed that the alx null strain fails to complete its developmental program. Past the tight aggregate stage, morphogenesis is impaired, leading to markedly aberrant structures containing vacuolated and undifferentiated cells but no mature spores. The developmental defect is cell-autonomous as most cells remain of the PstB type even when mixed with wild-type cells. Complementation analysis with different Alix constructs allowed the identification of a 101-residue stretch containing a coiled-coil domain essential for Alix function. In addition, we showed that the protein associates in part with vesicular structures and that its distribution on a Percoll gradient overlaps that of the endocytic marker Vamp7. Dd-Alix also co-localizes with Dd-Vps32. In view of our data, and given the role of Vps32 proteins in membrane protein sorting and multivesicular body formation in yeast and mammals, we hypothesize that the developmental defects of the alx null strain result from abnormal trafficking of cell-surface receptors.

The interaction of glycogen synthase kinase-3 (GSK-3) with the cell cycle.

GSK-3 is a multifunctional protein kinase known to play a pivotal role in the regulation of metabolism, the cytoskeleton and gene expression. It also interacts with the cell cycle in a number of ways. GSK-3 forms part of both Wnt and Hh signalling pathways and hence controls expression of a number of cell cycle regulatory genes. Prominent among these is cyclin D1. GSK-3 also phosphorylates cyclin D1 to promote its nuclear export and subsequent degradation. In this chapter we examine how GSK-3 mediates these effects and consider how therapeutic strategies may be developed to specifically target these pathways.

Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites.

Lithium inhibits (Li(+)) glycogen synthase kinase-3 (GSK-3) by competition for magnesium (Mg(2+)), but not ATP or substrate. Here, we show that the group II metal ion beryllium (Be(2+)) is a potent inhibitor of GSK-3 and competes for both Mg(2+) and ATP. Be(2+) also inhibits the related protein kinase cdc2 at similar potency, but not MAP kinase 2. To compare the actions of Li(+) and Be(2+) on GSK-3, we have devised a novel dual inhibition analysis. When Be(2+) and ADP are present together each interferes with the action of the other, indicating that both agents inhibit GSK-3 at the ATP binding site. In contrast, Li(+) exerts no interference with ADP inhibition or vice versa. We find, however, that Li(+) and Be(2+) do interfere with each other. These results suggest that Be(2+) competes for two distinct Mg(2+) binding sites: one is Li(+)-sensitive and the other, which is Li(+)-insensitive, binds the Mg:ATP complex.