Self-regulating systems often use robust oscillatory circuits. One such system controls the chemotactic signaling mechanism of Dictyostelium, where pulses of adenosine 3',5'-monophosphate (cAMP) are generated with a periodicity of 7 minutes. We have observed spontaneous oscillations in activation of the mitogen-activated protein (MAP) kinase ERK2 that occur in phase with peaks of cAMP, and we show that ERK2 modulates cAMP levels through the phosphodiesterase RegA. Computer modeling and simulations of the underlying circuit faithfully account for the ability of the cells to spontaneously generate periodic pulses during specific stages of development. Similar oscillatory processes may occur in cells of many different species.

Abstract. Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.

Doc2 has two C2 domains that interact with Ca2؉ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca 2؉ -dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K We have isolated Doc2 as a novel protein having two C2 domains that interact with Ca 2ϩ and PL 1 (1). Doc2 consists of two isoforms, Doc2␣ and Doc2␤ (1, 2). Doc2␣ is specifically expressed in neuronal cells, whereas Doc2␤ is ubiquitously expressed (1-3). Both isoforms have at least the N-terminal Doc2-specific region and C-terminal two C2 domains. We have moreover shown that overexpression of the N-terminal fragment of Doc2␣ or its C-terminal fragment including the C2 domains in PC12 cells inhibits Ca 2ϩ -dependent exocytosis (4).These results suggest that Doc2␣ is involved in Ca 2ϩ -dependent exocytosis and interacts with another component of Ca 2ϩ -dependent exocytotic machinery. To clarify the mode of action of Doc2␣ in Ca 2ϩ -dependent exocytosis, it is important to isolate its interacting protein(s). We have attempted here to isolate a Doc2␣-interacting protein from a rat brain cDNA library by use of the yeast two-hybrid system and isolated Munc13 as a Doc2␣-interacting protein.Munc13 has been isolated as a mammalian homologue of Caenorhabditis elegans unc-13, which is implicated in Ca 2ϩ -dependent neurotransmitter release (5, 6). Munc13 has three isoforms, Munc13-1, -2, and -3. All the isoforms have two C2 domains and Munc13-1 has another atypical C2 domain. They have moreover one C1 domain that interacts with PE or DAG and PL (5-7). Munc13 is specifically expressed in neuronal cells, and Munc13-1 is localized at the presynaptic plasma membrane (6).We describe here that Doc2␣ directly interacts with Munc13-1 in a DAG-dependent manner and that the Doc2␣-Munc13-1 interactions play an important role in Ca 2ϩ -dependent exocytotic machinery. EXPERIMENTAL PROCEDURESTwo-hybrid Assay-The N-terminal fragment (1-90 aa) of human Doc2␣ cDNA (1) was inserted into the pBTM116 (pLexA-Doc2␣N). The yeast reporter strain L40 was transformed with pLexA-Doc2␣N and a rat brain cDNA library constructed in pGAD10 (CLONTECH). Library plasmids from positive clones were analyzed by transformation tests and DNA sequencing. Overlapping clones containing the full-length coding region of Munc13-1 were isolated by screening the rat brain cDNA library. The cDNA fragments encoding several Munc13-1 deletion mutants were constructed from the overlapping clones and inserted into pGAD424. The cDNA fragments encoding several Doc2␣ deletion mutants were inserted into pBTM116. After co-transformation into yeast strain L40, ␤-galactosidase activity was assayed by liquid and filter assays (8,9).Preparation of Recombinant Proteins-The cDNA fragments encoding the N-terminal fragment (1-90 aa) of human Doc2␣ (1) and Munc13-1-...

Mitogen-activated protein (MAP) kinases are involved in controlling a cell's responses to a variety of stimuli and can be activated by both protein tyrosine kinase and G protein-coupled receptors. It was shown previously that Dictyostelium MAP kinase ERK2 is required for normal activation of adenylyl cyclase and erk2 null cells are aggregation-deficient. In this manuscript, we show that the Dictyostelium MAP kinase ERK2 is rapidly and transiently activated in response to the chemoattractant cAMP. This response requires cAMP receptors, but is independent of the coupled G alpha2 subunit and the only known G beta subunit. These data indicate that ligand-mediated receptor activation of adenylyl cyclase requires two receptor-dependent pathways, one of which requires heterotrimeric G proteins, including G alpha2 and the only known G beta subunit, and the second of which requires ERK2. Our results suggest that ERK2 may be activated by a novel receptor-mediated pathway.

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In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.

Two cDNA clones encoding vacuolar H+-inorganic pyrophosphatase (HVP1 and HVP10), one clone encoding the catalytic subunit (68 kDa) of vacuolar H+-ATPase (HvVHA-A), and one clone encoding vacuolar Na+/H+ antiporter (HvNHX1) were isolated from barley (Hordeum vulgare), a salt-tolerant crop. Salt stress increased the transcript levels of HVP1, HVP10, HvVHA-A, and HvNHX1, and osmotic stress also increased the transcript levels of HVP1 and HvNHX1 in barley roots. The transcription of HVP1 in response to salt stress was regulated differently from that of HVP10. In addition, the HVP1 expression changed in a pattern similar to that of HvNHX1 expression. These results indicate that the expression of HVP1 is co-ordinated with that of HvNHX1 in barley roots in response to salt and osmotic stresses.

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer. Surprisingly,however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyperosmotic stress. Microarray analysis identified two genes, gapA and rtoA, that are induced by hyperosmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent on Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress-response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as a second messenger.