Masakazu Oyama

A novel role of differentiation-inducing factor-1 in Dictyostelium development, assessed by the restoration of a developmental defect in a mutant lacking mitogen-activated protein kinase ERK2

It has been previously reported that the differentiating wild‐type cells of Dictyostelium discoideum secrete a diffusible factor or factors that are able to rescue the developmental defect in the mutant lacking extracellular signal‐regulated kinase 2 (ERK2), encoded by the gene erkB. In the present study, it is demonstrated that differentiation‐inducing factor‐1 (DIF‐1) for stalk cells can mimic the role of the factor(s) and the mechanism of the action of DIF‐1 in the erkB null mutant is also discussed. The mutant usually never forms multicellular aggregates, because of its defect in cyclic adenosine monophosphate (cAMP) signaling. In the presence of 100 n M DIF‐1, however, the mutant cells formed tiny slugs, which eventually developed into small fruiting bodies. In contrast, DIF‐1 never rescued the developmental arrest of other Dictyostelium mutants lacking adenylyl cyclase A (ACA), cAMP receptors cAR1 and cAR3, heterotrimeric G‐protein, the cytosolic regulator of ACA, or the catalytic subunit of cAMP‐dependent protein kinase (PKA‐C). Most importantly, it was found that DIF‐1 did not affect the cellular cAMP level, but rather elevated the transcriptional level of pka during the development of erkB null cells. These results suggest that DIF‐1 may rescue the developmental defect in erkB null cells via the increase in PKA activity, thus giving the first conclusive evidence that DIF‐1 plays a crucial role in the early events of Dictyostelium development as well as in prestalk and stalk cell induction.

Regulation of guanylate cyclase by a guanine nucleotide binding protein, Gα2, in Dictyostelium discoideum

Binding of cyclic AMP (cAMP) to the cell surface receptor induces a transient activation of guanylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) which lacks G alpha 2, a guanine nucleotide binding protein, does not respond to cAMP. We found that 2,3-dimercapto-1-propanol (BAL) induced a continuous activation both in the frigid and in its parents. Therefore, the BAL-induced continuous activation of guanylate cyclase is independent of G alpha 2. We also found that cAMP enhanced the BAL-induced continuous activation in the frigid mutant. This result suggests that an unidentified signal transduction mechanism from the cAMP-receptor besides the one involving G alpha 2 plays a role in the enhancement of activation. Lastly, we found that the BAL-induced continuous activation was terminated by cAMP in the parental strain, but not in the frigid mutant. Therefore, the cAMP-induced suppression on the BAL-induced continuous activation is mediated through G alpha 2.

Inhibition by EDTA and enhancement by divalent cations or polyamines of the dithiothreitol-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of cAMP to cell surface receptors evokes the transient activation of of adenylate cyclase in Dictyostelium discoideum. Dithiothreitol is also known as an activator of this enzyme. We found that the dithiothreitol-induced activation was specifically enhanced by extracellular polyamines or divalent cations. Furthermore, EDTA, a chelating agent of divalent cations, completely inhibited the dithiothreitol-induced activation of adenylate cyclase while EDTA did not inhibit the cAMP-induced activation. The inhibition was nullified by addition of polyamines or divalent cations. These results suggest that extracellular polyamines and divalent cations play a specific role in the dithiothreitol-induced activation of adenylate cyclase.

Defect in peroxisomal multifunctional enzyme MFE1 affects cAMP relay in Dictyostelium.

We have previously reported that cells of Dictyostelium discoideum lacking the fatty acid oxidation enzyme MFE1 accumulate excess cyclopropane fatty acids from ingested bacteria. Cells in which mfeA– is disrupted fail to develop when grown in association with bacteria but form normal fruiting bodies when grown in axenic media. Bacterially grown mfeA– cells express the genes for the cyclic AMP (cAMP) receptor (carA) and adenylyl cyclase (acaA) but fail to respond to a cAMP pulse by synthesis of additional cAMP which normally relays the signal. Moreover, they do not accumulate the adhesion protein, gp80, which is encoded by the cAMP‐induced gene, csaA. As a consequence, they do not acquire developmentally regulated EDTA‐resistant cell–cell adhesion. When mutant cells are mixed with wild‐type cells and allowed to develop together, they co‐aggregate and differentiate into both spores and stalk cells. Thus, most of the developmental consequences of excess cyclopropane fatty acids appear to result from impaired cAMP relay.

Role of a guanine nucleotide binding protein, Gα 2, in regulation of adenylate cyclase in Dictyosteliumdiscoideum

Two substances, cAMP and 2,3-dimercapto-1-propanol (BAL) are known to induce transient activation of adenylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) has a deletion in a gene for G alpha 2, a guanine nucleotide binding protein and cannot activate the cyclase in response to cAMP. We found that BAL induced activation in the frigid mutant. This result suggests that the BAL-induced activation is independent of G alpha 2 and that BAL mimics a role of activated G alpha 2. We also found that cAMP promoted the BAL-induced activation. This result suggests that cAMP plays a role in activation through a mechanism in which G alpha 2 is not involved. We lastly showed that continuous cAMP stimulation could not inhibit the BAL-induced activation in the frigid mutant. Since the cAMP-induced inhibition observed in the wild type strain (NC4) proceeds with the time course identical to the cAMP-induced adaptation (Oyama, submitted), this result suggests that G alpha 2 is involved in adaptation of adenylate cyclase.

INVOLVEMENT OF PROTEIN KINASE(S) IN THE INTRACELLULAR SIGNAL TRANSDUCTION PATHWAYS FOR ACTIVATION AND ADAPTATION OF ADENYLATE CYCLASE IN DICTYOSTELIUM DISCOIDEUM

Binding of cyclic AMP (cAMP) to cell surface receptors induces activation and adaptation of adenylate cyclase while 2,3-dimercapto-1-propanol (BAL) acts only on the activation pathway. Here we show that an inhibitor of protein kinase (K252a) inhibits the cAMP-induced activation of the cyclase but not (rather enhances) the BAL-induced activation. These results suggest that protein kinase is involved in transduction of the activation signal and that phosphorylation might take place between the receptor and the action site of BAL. Since adaptation causes cessation of the activation, the enhancement of the BAL-induced cAMP accumulation by K252a might imply that K252a also blocks transduction of the adaptation signal.