Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination-specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant-negative PKA-R subunits (PKA-RM) and control R-subunits (PKA-RC) were overexpressed in ACA null mutants. Both transformations as well as transformation with an unrelated vector, carrying the same NEOR selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the amino-glycoside phosphotransferase encoded by the very commonly used NEOR selection marker induces ectopic ACG activity in Dictyostelium cells.
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PMID:Transformation with vectors harboring the NEOR selection marker induces germination-specific adenylylcyclase activity in Dictyostelium cells. 755 61

spiA, a marker for sporulation, is expressed during the culmination stage of Dictyostelium development, when the mass of prespore cells has moved partly up the newly formed stalk. Strains containing a full-length spiA promoter/lacZ fusion were stained for beta-galactosidase activity at intervals during development. The results indicate that expression of spiA initiates in prespore cells at the prestalk/prespore boundary (near the apex) and extends downward into the prespore mass as culmination continues. A spatial gradient of staining expands from the top of the prespore mass and intensifies until the front of activation reaches the bottom, whereupon the entire region stains darkly. The spiA promoter can be deleted to within 301 bp of the transcriptional start site with no effect on the relative strength, timing or spatial localization of expression. Further 5' deletions from -301 to -175 reduce promoter strength incrementally, although timing and spatial expression are not affected. Deletions to -159 and beyond result in inactive promoters. Treatment of early developmental structures with 8-Br-cAMP in situ activates the intracellular cAMP-dependent protein kinase (PKA) and precociously induces spiA expression and sporulation. The absence of an apparent gradient of staining in these structures suggest that PKA is equivalently activatable throughout the prespore region and that all prespore cells are competent to express spiA. Thus, we postulate that the pattern of expression of spiA reveals the progression of an inductive signal for sporulation and suggest that this signal may originate from the prestalk cells at the apex.
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PMID:Progression of an inductive signal activates sporulation in Dictyostelium discoideum. 760 79

Throughout vegetative growth, Dictyostelium amoebae secrete an autocrine factor, prestarvation factor, PSF, which accumulates in proportion to cell density. During late exponential growth, PSF induces the expression of several genes whose products are needed for cAMP signaling and cell aggregation. Among these genes are discoidin-I and the 2.4-kb transcript of cyclic nucleotide phosphodiesterase (PDE). We have identified several parameters that modulate expression of one or both of these prestarvation response genes; all effects were monitored in cells growing exponentially on bacteria. Under these conditions, axenic mutants produce higher levels of PSF activity than wild-type cells. Consistent with the high PSF levels, the 2.4-kb PDE transcript is more abundant in axenic strains than wild-type cells at the same cell density. In contrast, the density-dependent induction of discoidin-I is greatly delayed in axenic strains, occurring only at the very end of exponential growth. Analysis of axenic strains of independent origin suggested that this negative effect on discoidin-I expression is attributable to the axenic mutations themselves. The effects of two environmental factors that inhibit the prestarvation response (the bacteria upon which the cells feed and a bacterial product, folic acid) were also analyzed. We found that folate does not account for the inhibitory effect of bacteria. Cells deficient in the G-protein beta subunit, which is thought to be common to all heterotrimeric G-proteins in Dictyostelium, respond to PSF in the same manner as G beta+ cells, and this response is inhibited by bacteria. However, folate has no inhibitory effect on g beta- cells, indicating that folate inhibition is mediated by a heterotrimeric G-protein. In cells lacking the catalytic subunit of protein kinase A, the prestarvation response is severely impaired, but about 3% of the pka- cells manifest an apparently normal density-dependent induction of discoidin-I. This behavior and the heterogeneity of the prestarvation response in wild-type cells lead us to speculate that protein kinase A may not be required for PSF signal transduction per se, but rather may render the cells responsive to PSF. Based on analysis of adenylyl cyclase mutants (aca-), the effect of protein kinase A is not cAMP-dependent.
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PMID:Genetic and physiologic modulation of the prestarvation response in Dictyostelium discoideum. 761 66

Constitutive inhibition of cAMP-dependent protein kinase (PKA) in Dictyostelium cells blocks cell aggregation and development. We investigated the cause of the aggregation defect in transformants overexpressing dominant-negative PKA regulatory subunits (PKA-RM) under an actin 15 promoter. These mutants could not relay pulses of the chemoattractant cAMP, due to a defect in expression of the aggregative adenylyl cyclase (ACA) gene. Unstimulated and cAMP pulse-induced expression of other aggregative genes encoding the cAMP receptor cAR1, adhesive contact sites A and cAMP-phosphodiesterase were also strongly reduced in the mutants. Additionally, the expression of the discoidin I gene, that is expressed early in development in response to cell density sensing factors, was almost completely absent. These data are in interesting contrast with observations that cAMP relay and aggregative gene expression are normal in null mutants for the PKA catalytic (C) subunit and suggest the presence of multiple C subunit genes in Dictyostelium and an almost universal requirement for PKA activity in developmental gene expression.
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PMID:cAMP-dependent protein kinase activity is essential for preaggregative gene expression in Dictyostelium. 762 43

The cotA, cotB, and cotC genes encode the major spore coat proteins of Dictyostelium. All three cot genes are coordinately expressed as aggregation is nearing completion. Induction and maintenance of their expression is dependent upon the presence of extracellular cAMP. We show that expression of a dominant inhibitor of the cAMP dependent protein kinase (PKA) in prespore cells greatly reduces the transcription rates of the cotB and cotC genes. All three cot genes contain, in their upstream regulatory regions, short sequence elements that have a high content of cytosine and adenosine residues. These CA-rich sequences are essential for optimal cot gene transcription. We show that expression of the dominant PKA inhibitor results in a greatly reduced level of the binding activity that recognizes the CA-rich sequences upstream of the cotB gene. Thus PKA acts, either directly or indirectly, to control expression of the cot genes and it may do so by modulating the activity of a DNA binding protein. However, we find that mutant cells where PKA is constitutively active still require exogenous cAMP for optimal cot gene expression in dissociated cells, suggesting that a separate, PKA-independent, signalling pathway is also involved in the regulation of cot gene expression by extracellular cAMP.
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PMID:Protein kinase A is a positive regulator of spore coat gene transcription in Dictyostelium. 771 25

We have isolated a protein from Dictyostelium with a molecular mass of 110 kDa as judged by SDS-gel electrophoresis that can stimulate the actin-activated MgATPase activity of Dictyostelium myosin ID (MyoD). In the presence of MgATP the 110-kDa protein incorporated phosphate into itself and into the heavy chain, but not the light chain, of MyoD. Phosphorylation to 0.5 mol of Pi/mol increased the MyoD actin-activated MgATPase rate from 0.2 to 3 mumol/min/mg. Renaturation following SDS-gel electrophoresis demonstrated that the 110-kDa protein contained intrinsic protein kinase and autophosphorylation activity. Autophosphorylation to 1 mol of Pi/mol enhanced the rate at which the 110-kDa protein kinase phosphorylated MyoD by 40-fold. The rate of autophosphorylation was strongly dependent on the 110-kDa protein kinase concentration, indicating an intermolecular reaction. Synthetic peptides of 9-11 residues corresponding to the heavy chain phosphorylation site of Acanthamoeba myosin IC and the homologous sites in Dictyostelium myosin IB (MyoB) and MyoD were poor substrates for the 110-kDa protein kinase. The 110-kDa protein kinase was unable to phosphorylate the MyoB isozyme suggesting that it may be specific for MyoD.
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PMID:Purification and characterization of a Dictyostelium protein kinase required for actin activation of the Mg2+ ATPase activity of Dictyostelium myosin ID. 774 26

Cell differentiation and proliferation are mutually exclusive processes in many cases. The transition of starving Dictyostelium cells from growth to differentiation phase has been shown to occur at a particular position (putative shift point; PS-point) in the cell cycle of D. discoideum Ax-2. The significance of phosphorylation states of proteins such as 101 kDa, 90 kDa, and 32 kDa phosphoproteins has been argued, particularly around the PS-point. In this study we examined effects of the protein kinase inhibitors and activators on the transition of Ax-2 cells from growth to differentiation. K252a, a potent inhibitor of protein kinases, inhibited growth possibly through the blockage of pinocytotic activity of cells, and promoted the progress of development after starvation when applied to Ax-2 cells at the growth phase. Such a K252a-effect was most pronouncedly exhibited on the cells located near the PS-point. Unexpectedly, however, the development of starved cells was found to be considerably delayed by staurosporine bearing a structural and functional resemblance to K252a when it was applied during the growth phase. Pulse-labelings of growing Ax-2 cells with inorganic 32P (32Pi) showed that K252a induces the disappearance of a 48 kDa phosphoprotein and the appearance of a 50 kDa phosphoprotein, specifically in the cells located around the PS-point. Phosphorylation of 32 kDa and 24 kDa proteins was also inhibited by K252a, but this inhibition was not necessarily specific to the K252a-treatment and occurred independently of the cell-cycle phases. The possible significance of these results is discussed in relation to a breakaway of cells from proliferation to differentiation at the PS-point.
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PMID:K252a, a potent inhibitor of protein kinases, promotes the transition of Dictyostelium cells from growth to differentiation. 776 85

Phosphofructokinase (PFruK) from the slime mold Dictyostelium discoideum has been purified to homogeneity over 15,000-fold with a 29% yield. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the final preparation revealed a single band of 95 kDa. The native molecular mass was determined by gel filtration to be 382 kDa, indicating that the enzyme is a homotetramer. An antibody raised in rabbits against the 95-kDa band immunoprecipitated PFruK activity while it did not react with the enzyme from yeast and mammalian cells. The apparent pI was 6.8 and the pH optimum was 7.6. The enzyme had an activation energy (Ea) of 29.1 kJ/mol. The amino acid composition was distinctive in having high Ser, Gly and Glx and low Ala, Val and Tyr compared with other eukaryotic PFruKs. Enzyme activity did not have a sigmoidal saturation curve for fructose 6-phosphate, was only mildly inhibited by MgATP at acidic pH values, was not affected by enzyme concentration and was insensitive to any of the typical allosteric effectors of PFruKs from other sources. However, the enzyme binds fructose 2,6-bisphosphate as indicated by protection against thermal denaturation. Treatment with cAMP-dependent protein kinase led to phosphorylation of the enzyme without change in activity. The metabolic significance of these properties and their relationship to structure/function are discussed.
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PMID:Purification and properties of phosphofructokinase from Dictyostelium discoideum. 781 55

We report here the cloning and characterization of the gene encoding the 130-kDa myosin heavy chain kinase (MHCK A) from the amoeba Dictyostelium. Previous studies have shown that purified MHCK A phosphorylates threonines in the carboxyl-terminal tail portion of the Dictyostelium myosin II heavy chain and that phosphorylation of these sites is critical in regulating the assembly and disassembly of myosin II filaments in vitro and in vivo. Biochemical analysis of MHCK A, together with analysis of the primary sequence, suggests that the amino-terminal approximately 500 amino acids form an alpha-helical coiled-coil domain and that residues from position approximately 860 to the carboxyl terminus (residue 1146) form a domain with significant similarity to the beta-subunit of heterotrimeric G proteins. No part of the MHCK A sequence displays significant similarity to the catalytic domain of conventional eukaryotic protein kinases. However, both native and recombinant MHCK A displayed autophosphorylation activity following renaturation from SDS gels, and MHCK A expressed in Escherichia coli phosphorylated purified Dictyostelium myosin, confirming that MHCK A is a bona fide protein kinase. Cross-linking studies demonstrated that native MHCK A is a multimer, consistent with the presence of an amino-terminal coiled-coil domain. Southern blot analysis indicates that MHCK A is encoded by a single gene that has no detectable introns.
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PMID:Structural analysis of myosin heavy chain kinase A from Dictyostelium. Evidence for a highly divergent protein kinase domain, an amino-terminal coiled-coil domain, and a domain homologous to the beta-subunit of heterotrimeric G proteins. 782 74

The histidine-rich protein hisactophilin is known to be associated with the inner surface of the plasma membrane and to be present as a soluble protein in the cytoplasm of Dictyostelium discoideum cells. Mass spectrometry of hisactophilin from the cytosol or extracted from a membrane fraction showed that none of the hisactophilin purified from D. discoideum cells had the mass predicted from the known cDNA-derived amino acid sequence of the protein. Electrospray mass spectrometry and liquid secondary ion mass spectrometry of tryptic fragments separated by reversed-phase high performance liquid chromatography (HPLC) identified the most hydrophobic peptide as a myristoylated fragment from the N terminus of hisactophilin. Taken together the analytical data, it is concluded that all hisactophilin in D. discoideum cells is N terminally modified by myristoylation. By reversed-phase HPLC, two isoforms of hisactophilin, HsI and HsII, were recovered from the cytosolic as well as the membrane fraction of D. discoideum cells. Whereas the masses of HsI fragments produced by trypsin fit into the previously published sequence of hisactophilin (myristoylation considered), HsII is another protein distinguished from HsI by several amino acid exchanges. HsI and HsII can form homo- and heterodimers by disulfide bridges. Hisactophilin is phosphorylated in vivo. Both isoforms proved to be substrates of membrane-associated threonine/serine kinase from D. discoideum, which may regulate the interaction of hisactophilin with the plasma membrane.
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PMID:The pH-sensitive actin-binding protein hisactophilin of Dictyostelium exists in two isoforms which both are myristoylated and distributed between plasma membrane and cytoplasm. 782 84


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