Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.
A.Ott and F.Oehme contributed equally to this work DokA, a homolog of bacterial hybrid histidine kinases, is essential for hyperosmotic stress resistance in Dictyostelium. We show that a transient intracellular cAMP signal, dependent on the presence of DokA, is generated in response to an osmotic shock. This variation of cAMP levels contributes to survival under hypertonic conditions. In contrast to the low cAMP levels observed in dokA ± strains, overexpression of the receiver domain of DokA causes an increase in cAMP levels, resulting in a rapidly developing phenotype. We present biochemical and cell biological data indicating that the DokA receiver domain is a dominantnegative regulator of a phosphorelay, which controls the intracellular cAMP phosphodiesterase RegA. The activity of the DokA receiver domain depends on a conserved aspartate, mutation of which reverses the developmental phenotype, as well as the deregulation of cAMP metabolism. Keywords: cAMP/osmotic stress/phosphatases/ phosphorelay/signal transduction IntroductionThe second messenger cAMP induces a variety of physiological responses in eukaryotic and prokaryotic cells (Robison et al., 1968;Tang and Gilman, 1992). In the amoeba Dictyostelium discoideum, cAMP acts as a morphoregulatory signal, which controls chemotaxis, gene expression and cell differentiation during development (Parent and Devreotes, 1996; van Haastert, 1997;Verkerke-van Wijk and Schaap, 1997). The synthesis and degradation of cAMP are regulated precisely: during aggregation, cAMP is formed by the adenylyl cyclase ACA (van Haastert, 1997), whereas another adenylyl cyclase, ACG, acts as an osmosensor controlling germination in Dictyostelium spores (van Es et al., 1996). More recently, a novel adenylyl cyclase activity has been detected in cell lysates of rapid developing mutants of Dictyostelium (Kim et al., 1998). It is probably attributed to AcrA, a cyclase expressed throughout development with pivotal function during culmination (Soderbom et al., 1999). Extracellular cAMP, which serves as a chemoattractant during aggregation, is hydrolyzed by a phosphodiesterase (PDE) that is either secreted or anchored to the extracellular face of the plasma membrane (Malchow et al., 1972;Gerisch, 1976). In addition, the intracellular phosphodiesterase RegA has also been described . In addition to its morphogenic function, cAMP also acts as an intracellular second messenger activating protein kinase A (PKA), which plays an important role in gene expression and cell differentiation during Dictyostelium development (Verkerke-van Wijk and Schaap, 1997 and references therein). It was shown that mutations causing elevated levels of intracellular cAMP or a constitutively active PKA lead to an accelerated development (Coukell and Chan, 1980;Abe and Yanagisawa, 1983;Simon et al., 1992). In contrast to an increased activity of PKA, the prespore-speci®c inhibition of this cAMP-regulated kinase results in spore heads with a translucent appearance (Hopper et al., 1993), a phenotype resembling Dictyosteli...
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