Properties of CAR-kinase: The enzyme that phosphorylates the cAMP chemotactic receptor ofD. discoideum

The cell surface cAMP chemotactic receptor ofD. discoideum can be phosphorylated in partially purified plasma membrane preparations in a ligand-dependent manner. CAR-kinase, the enzyme responsible for receptor phosphorylation, was shown to be an integral membrane protein. It could utilize either ATP or GTP to phosphorylate the receptor, although ATP was much more efficient. The apparent affinity constant for ATP was approximately 20–25 µM. Maximum CAR-kinase activity was observed betweenpH 6.5 andpH 7, and required the presence of Mg²⁺. Neither Mn²⁺ nor Ca²⁺ could substitute for that divalent cation. The enzyme was found to be sensitive to the ionic strength and temperature of the incubation reaction. Dephosphorylation of the receptor was not observed in the membrane preparations, indicating that the enhanced level of receptor phosphorylation that occurred upon ligand binding was not an indirect reflection of receptor dephosphorylation and subsequent incorporation of radiolabeled phosphate.     

Calcium uptake and gp80 messenger RNA destabilization follows cAMP receptor down regulation in Dictyostelium discoideum

The mechanism by which high concentrations of cAMP selectively destabilize the gp80 mRNA in Dictyostelium discoideum was investigated. This treatment which leads to down-regulation of the cAMP receptor was also found to cause an increase in calcium uptake. Given this observation, we sought a role for calcium as a second messenger in the degradation of the gp80 mRNA. Changes in the mRNA levels were examined after treating cells with compounds known to alter their intracellular Ca²⁺ concentrations. This included the use of A23187, Ca²⁺, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl (TMB-8), LiCl and 8-p-chlorophenylthioadenosine 3′,5′-cyclic monophosphate (ClPhS-Ado-3′:5′-P). The sum of the data suggest that it is the cAMP-induced influx of Ca²⁺ acoss the plasma membrane, as opposed to a cAMP-mediated release of Ca²⁺ from intracellular stores, that initiates gp80 mRNA degradation. Treatment of cells with Concanavalin A (ConA) to induce cAMP receptor down-regulation, also causes a reduction in gp80 mRNA levels and an increase in calcium uptake.     

Induction of post-aggregative differentiation in Dictyostelium discoideum by cAMP: Evidence of involvement of the cell surface cAMP receptor

Exogenous cAMP is known to induce post-aggregative differentiation in Dictyostelium discoideum under conditions that normal development is blocked. We have analysed the cyclic nucleotide specificity, the effect of modulation of the cAMP signal and the dose-response relationship of the induction of two independent markers of post-aggregative differentiation, i.e., a prespore cell-specific antigen detected by a monoclonal antibody, and the activity of glycogen phosphorylase. Our results confirm that high concentrations of cAMP (10(-6)-10(-3)M) are required for the induction of these markers. The cells are shown not to adapt to the cAMP signal. The cyclic nucleotide specificity of induction agrees with the specificity of the cell surface cAMP receptor, but is very dissimilar to the specificity of the intracellular cAMP-dependent protein kinase. It is thus unlikely that cAMP leaks into the cell and activates the cAMP-dependent protein kinase directly. Instead, the induction of post-aggregative differentiation by cAMP seems to be mediated by cell surface cAMP receptors.     

The role of loops 3 and 4 in the interdomains and intersubunits communication of E. coli cAMP receptor protein

Cyclic AMP serves as an intracellular messenger in cells and regulates a variety of biological functions by transmitting information through proteins. These proteins of different functions all consist of a cAMP-binding motif, and the structure of this motif is highly conserved with an exception of the loop 3 and 4. In current study, cAMP receptor protein was employed as a model system to investigate the function of the two loops. The results indicated that the loop 3 involves in the intersubunits communication of CRP, whereas the loop 4 involves in cAMP binding and interdomains communication.     

Signaling pathways mediating chemotaxis in the social amoeba, Dictyostelium discoideum

Chemotaxis, or cell migration guided by chemical cues, is critical for a multitude of biological processes in a diverse array of organisms. Dictyostelium discoideum amoebae rely on chemotaxis to find food and to survive starvation conditions, and we have taken advantage of this system to study the molecular regulation of this vital cell behavior. Previous work has identified phosphoinositide signaling as one mechanism which may contribute to directional sensing and actin polymerization during chemotaxis; a mechanism which is conserved in mammalian neutrophils. In this review, we will discuss recent data on genes and pathways governing directional sensing and actin polymerization, with a particular emphasis on contributions from our laboratory.     

Dictyostelium discoideum surface changes elicited by high concentrations of cAMP

The effects of high concentrations of cAMP on both morphological and biochemical development of Dictyostelium discoideum amebae are reported. Observations using light and scanning electron microscopy (SEM) indicate that the cells' response to such treatment varies with the length of time they had been starved prior to cAMP addition. Vegetative and early developmental amebae become rounded within a short period after treatment. Such cells are capable of undertaking a normal aggregation after a delay of a few hours. A substantial induction of phosphodiesterase activity is elicited from these cells by cAMP treatment but their levels of cAMP surface binding sites remain low. cAMP addition to aggregation competent cells causes amebae first to flatten and then to retract into spherical forms and group into small aggregates. No induction of phosphodiesterase activity is observed in such cells and the levels of cAMP binding sites present on the amebae decrease rapidly. The data are discussed in terms of the different states of cAMP-sensitivity between vegative and aggregation-competent amebae.     

PKC phosphorylates GluA1-Ser831 to enhance AMPA receptor conductance

AMPA receptors mediate fast excitatory synaptic transmission in the brain, and are dynamically regulated by phosphorylation of multiple residues within the C-terminal domain. CaMKII phosphorylates Ser831 within the AMPA receptor GluA1 subunit to increase single channel conductance, and biochemical studies show that PKC can also phosphorylate this residue. In light of the discovery of additional PKC phosphorylation sites within the GluA1 C-terminus, it remains unclear whether PKC phosphorylation of Ser831 increases GluA1 conductance in intact receptors. Here, we report that the purified, catalytic subunit of PKC significantly increases the conductance of wild-type GluA1 AMPA receptors expressed in the presence of stargazin in HEK293T cells. Furthermore, the mutation GluA1-S831A blocks the functional effect of PKC. These findings suggest that GluA1 AMPA receptor conductance can be increased by activated CaMKII or PKC, and that phosphorylation at this site provides a mechanism for channel modulation via a variety of protein signaling cascades.     

Dual role of GDP in the regulation of the levels of p36 phosphorylation inDictyostelium discoideum

We examined the dephosphorylation of p36, a protein ofD. discoideum that has previously been shown to be phosphorylated in a GDP-dependent manner (Anschutzet al., 1989). Specific dephosphorylation of p36 was found to occur in cell preparations but the activity responsible was strongly dependent upon the concentration of proteins in those extracts. When preparations were diluted, this activity was no longert detectable and the radiolabeled phosphate incorporated into p36 was stable. In contrast, p36 phosphorylation was seemingly unaffected by this treatment. Under the conditions where endogenous dephosphorylating activity was not detectable, the addition of GDP to the reaction resulted in substantial dephosphorylation of p36. The stimulation of this dephosphorylation process occurred at concentrations of GDP that were distinct from those that led to an increased p36 phosphorylation due to the previously reported stimulation of p36 protein kinase activity. Characterization of the dephosphorylation of p36 indicates that the same enzyme is responsible for the endogenous and GDP-stimulated activities. Additionally, these activities are identical when assayed with p36 that had been phosphortylated with ATP or GTP. In contrast to p36 kinase activity, the dephosphorylation of p36 did not display any developmental changes with respect to its regulatory features.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

Cyclic AMP-dependent protein kinase (PKA) phosphorylates Ser362 and 467 and protein kinase C phosphorylates Ser550 within the M3/M4 cytoplasmic domain of human nicotinic receptor alpha4 subunits

Studies have suggested that the expression, translocation, and function of alpha4beta2 nicotinic receptors may be modulated by alpha4 subunit phosphorylation, but little direct evidence exists to support this idea. The objective of these experiments was to identify specific serine/threonine residues on alpha4 subunits that are phosphorylated in vivo by cAMP-dependent protein kinase and protein kinase C (PKC). To accomplish this, DNAs coding for human alpha4 subunits containing alanines in place of serines/threonines predicted to represent phosphorylation sites were constructed, and transiently transfected with the DNA coding for wild-type beta2 subunits into SH-EP1 cells. Cells were pre-incubated with (32)Pi and incubated in the absence or presence of forskolin or phorbol 12,13-dibutyrate. Immunoprecipitated alpha4 subunits were subjected to immunoblot, autoradiographic and phosphoamino acid analyses, and two-dimensional phosphopeptide mapping. Results confirmed the presence of two alpha4 protein bands, a major band of 71/75 kDa and a minor band of 80/85 kDa. Phosphoamino acid analysis of the major band indicated that only serine residues were phosphorylated. Phosphopeptide maps demonstrated that Ser362 and 467 on the M3/M4 cytoplasmic domain of the alpha4 subunit represent major cAMP-dependent protein kinase phosphorylation sites, while Ser550 also contained within this major intracellular loop is a major site for protein kinase C phosphorylation.     

Modulation of allosteric behavior through adjustment of the differential stability of the two interacting domains in E. coli cAMP receptor protein

The communication mechanism(s) responsible for the allosteric behavior of E.coli cAMP binding receptor protein, CRP, is still a subject of intense investigation. As a tool to explore the communication mechanism, the mutations at various positions in the cAMP-binding (K52N, D53H, S62F and T127L) or the DNA- binding (H159L) domain or both (K52N/H159L) were generated. The sites and specific nature of side chain substitutions were defined by earlier genetic studies, the results of which show that these mutants have a similar phenotype i.e. they are activated without exogenous cAMP. Presently, no significant changes in the structures of WT and mutant CRPs have been observed. Hence, the pressing issue is to identify a physical parameter that reflects the effects of mutations. In this study, the stability of these various CRP species in the presence of GuHCl was monitored by three spectroscopic techniques, namely, CD, tryptophan fluorescence and FT-IR which could provide data on the stability of α-helices and β-strands separately. Results of this study led to the following conclusions: 1. The α-helices can be grouped into two families with different stabilities. Mutations exert a differential effect on the stability of helices as demonstrated by a biphasic unfolding curve for the helices. 2. Regardless of the locations of mutations, the effects can be communicated to the other domain resulting in a perturbation of the stability of both domains, although the effects are more significantly expressed in the stability of the helices. 3. Although in an earlier study [Gekko, et al. Biochemistry 43 (2004) 3844] we showed that cooperativity of cAMP binding is generally correlated to the global dynamics of the protein and DNA binding affinity, in this study we found that generally there is no clear correlation between functional energetics and stability of secondary structures. Thus, results of this study imply that modulation of allostery in CRP is entropic in nature.     

Roles of cAMP and cAMP-dependent protein kinase in the progression of prostate cancer: Cross-talk with the androgen receptor

Prostate carcinomas are among the most frequently diagnosed and death causing cancers affecting males in the developed world. It has become clear that the molecular mechanisms that drive the differentiation of normal prostate cells towards neoplasia involve multiple signal transduction cascades that often overlap and interact. A critical mediator of cellular proliferation and differentiation in various cells (and cancers) is the cAMP-dependent protein kinase, also known as protein kinase A (PKA), and its activating secondary messenger, cAMP. PKA and cAMP have been shown to play critical roles in prostate carcinogenesis and are the subject of this review. In particular we will focus on the cross-talk between PKA/cAMP signaling and that of the androgen receptor.     

Cytochrome b of the Dictyostelium discoideum plasma membrane

Cytochrome b of the plasma membrane of Dictyostelium discoideum was investigated in purified plasma membranes and in solubilized form. The membrane-bound cytochrome b can be reduced by NADH. This reduction is inhibited by p-hydroxymercuribenzoate. The reduced cytochrome b does not react with carbon monoxide. Its apparent molecular weight lies between 13000 and 16000. Tryptic digestion yields a large, heme-containing peptide with an apparent molecular weight between 12000 and 15000. After solubilization with cholate, cytochrome b can be enriched by reversed-phase HPLC, indicating that it contains also a hydrophobic component. With these properties, cytochrome b of the D. discoideum plasma membrane resembles microsomal cytochrome b₅.     

The role of hexose transport and phosphorylation in cAMP signalling in the yeast Saccharomyces cerevisiae

Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.     

Membrane trafficking of AQP5 and cAMP dependent phosphorylation in bronchial epithelium

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.