In vivo and in vitro phosphorylation and subcellular localization of trypanosomatid cytoskeletal giant proteins

Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200), respectively. Polyclonal antibodies to Lt 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. In addition to reacting with giant polypeptides of the Leptomonas species, anti-Ls 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500, Ls 2500, and Lt 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas Lt 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. In addition, there is also evidence that Ps 3500 and Ls 2500, in contrast to Lt 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization.     

In vivo regulation of phosphorylation level and activity of phosphofructokinase by serotonin in Fasciola hepatica

The level of phosphorylation and activation of phosphofructokinase by serotonin (5-hydroxytryptamine) was studied in intact liver flukes Fasciola hepatica. The enzyme was immunoprecipitated with antiserum prepared against pure enzyme from the liver flukes. The resuspended immunoprecipitated enzyme retained most of its original activity and its kinetic properties. The level of phosphorylation was determined by a "back phosphorylation" technique. According to this technique, the immunoprecipitated phosphofructokinase was phosphorylated with the catalytic subunit of pure cAMP-dependent protein kinase. Incubation of intact liver flukes with serotonin caused an increase in the level of enzyme phosphorylation which was concomitant with an increase in enzyme activity. The level of phosphorylation was increased by 0.08 mol per protomer as a result of maximal activation by serotonin. It is proposed that phosphorylation plays, at least in part, a functional role in the regulation of phosphofructokinase from the liver fluke F. hepatica under in vivo conditions.     

In vivo distribution and localization of chorein

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. In this study, we produced an antibody against chorein and examined its protein-level expression and localization in mouse. Immunoblot analysis revealed that chorein was expressed in a gene dose-dependent manner in the VPS13A deletion-mice that we recently developed, which confirms the sensitivity of the antibody. Chorein was highly expressed in testis, kidney, spleen, and brain, and was expressed ubiquitously in various brain regions. Subcellular analysis of the brain showed high levels of chorein in microsomal and synaptosomal fractions. Immunohistochemically, chorein-like immunoreactivity was ubiquitously observed in the brain in the neuronal perinuclear region, cytoplasm and fibers. In testis and kidney, clear cell-specific patterns of chorein-like immunoreactivity were detected. Our findings provide basic information on chorein in vivo and may contribute to taking the first step toward understanding molecular pathogenesis of ChAc.     

In vivo and in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms.

NSP5 (NS26), the product of rotavirus gene 11, is a phosphoprotein whose role in the virus replication cycle is unknown. To gain further insight into its function, we obtained monoclonal antibodies against the baculovirus-expressed protein. By immunoprecipitation and immunoblotting experiments, we showed that (i) NSP5 appears in many different phosphorylated forms in rotavirus-infected cells; (ii) immunoprecipitated NSP5 from rotavirus-infected cells can be phosphorylated in vitro by incubation with ATP; (iii) NSP5, produced either by transient transfection of rotavirus gene 11 or by infection by gene 11 recombinant vaccinia virus or baculovirus, can be phosphorylated in vivo and in vitro; (iv) NSP5 expressed in Escherichia coli is phosphorylated in vitro, and thus NSP5 is a potential protein kinase; and (v) NSP5 forms dimers and interacts with NSP2. The intracellular localization of NSP5 in the course of rotavirus infection and after transient expression in COS7 cells has also been investigated. In rotavirus-infected cells, NSP5 is localized in viroplasms, but it is widespread throughout the cytoplasm of transfected COS7 cells. NSP5 produced by transfected COS7 cells did not acquire the multiphosphorylated forms observed in rotavirus-infected COS7 cells. Thus, there is a tight correlation between the localization of NSP5 in the viroplasms and its protein kinase activity in vivo or in vitro. Our results suggest that cellular or viral cofactors are indispensable to fully phosphorylate NSP5 and to reach its intracellular localization.     

In vivo phosphorylation of enolase fromEscherichia coli

The major metabolic route for the synthesis of phosphoenolpyruvate is from 2-phosphoglycerate catalyzed by the enzyme enolase (EC 4.2.1.11). Enolase occurs at the converging point between glycolysis and gluconeogenesis and may be an important regulatory enzyme. Growth ofEscherichia coli JA 200 pLC 11-8 to stationary phase in low-phosphate medium containing³²P-orthophosphate and glucose as the carbon source resulted in incorporation of label into the enzyme. In vivo labeling of enolase was demonstrated by immunoaffinity chromatography of the labeled crude extract. In addition,³²P-enolase was identified with sodium dodecylsulfate polyacrylamide gels, two-dimensional gel electrophoresis, and Western blot analysis, followed by autoradiography.     

In vivo phosphorylation of proteins in Clostridium sphenoides

Abstract Cell contents of Clostridium sphenoides , labeled with [³²P]orthophosphate under strict anaerobic conditions, were analyzed by two-dimensional gel electrophoresis. Autoradiography of these gels demonstrated the presence of at least 15 ³²P-labeled protein species, of which M _r and iso-electric point were determined. Treatment of the radioactively labeled cell contents with alkaline phosphatase and acid phosphatase showed that all these proteins were modified by phosphorylation. These findings demonstrate for the first time the presence of phosphorproteins in a strictly anaerobic bacterium.     

In vivo phosphorylation of sorghum leaf phosphoenolpyruvate carboxylase

The use of immunological techniques allowed us to purify close to homogeneity phosphoenolpyruvate carboxylase (PEPc, EC 4.1.1.31) from sorghum leaf. It was thus established that: 1) this protein is phosphorylated in vivo on seryl residues; 2) in C4-type photosynthesis, the phosphorylation process mainly concerns the PEPC isozyme form G; 3) enzyme phosphorylation displays significant variations through a day-night alternation which therefore suggests light control of the process.     

In vivo and ex vivo regulation of bacterial virulence gene expression

Bacteria are remarkably adaptable organisms that are able to survive and multiply in diverse and sometimes hostile environments. Adaptability is determined by the complement of genetic information available to an organism and by the mechanisms that control gene expression. In general, gene products conferring a growth or survival advantage in a particular situation are expressed, while unnecessary or deleterious functions are not. Expression of virulence gene products that allow pathogenic bacteria to multiply on and within host cells and tissues are no exception to this rule. Being of little or no use to the bacterium except during specific stages of the infectious cycle, these accessory factors are nearly always subject to tight and coordinate regulation. As a result of recent advances, we are beginning to appreciate the complexities of the interactions between bacteria and their hosts. The ability to probe virulence gene regulation in vivo has broadened our perspectives on pathogenesis.     

In vivo phosphorylation of FtsZ2 in Arabidopsis thaliana

The tubulin-like FtsZ protein initiates assembly of the bacterial and plastid division machineries. In bacteria, phosphorylation of FtsZ impairs GTPase activity, polymerization and interactions with other division proteins. Using a proteomics approach, we have shown that AtFtsZ2 is phosphorylated in vivo in Arabidopsis and that PGK1 (phosphoglycerate kinase 1) interacts with AtFtsZ2 in planta, suggesting a possible role in FtsZ phosphorylation.     

In vivo regulation of phosphoinositide 3-kinase in retina through light-induced tyrosine phosphorylation of the insulin receptor beta-subunit

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in bovine rod outer segment (ROS) is activated in vitro by tyrosine phosphorylation of the C-terminal tail of the insulin receptor (Rajala, R. V. S., and Anderson, R. E. (2001) Invest. Ophthal. Vis. Sci. 42, 3110-3117). In this study, we have investigated the in vivo mechanism of PI3K activation in the rodent retina and report the novel finding that light stimulates tyrosine phosphorylation of the beta-subunit of the insulin receptor (IRbeta) in ROS membranes, which leads to the association of PI3K enzyme activity with IRbeta. Retinas from light- or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IRbeta, or the p85 regulatory subunit of PI3K, and PI3K activity was measured using PI-4,5-P(2) as substrate. We observed a light-dependent increase in tyrosine phosphorylation of IRbeta and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IRbeta immunoprecipitates of retinal homogenates. The light effect was localized to photoreceptor neurons and is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IRbeta in outer segment membranes, which leads to the binding of p85 through its N-terminal Src homology 2 domain and the generation of PI-3,4,5-P(3). We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stress-induced apoptosis.     

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.     

In vivo phosphorylation of the yeast uracil permease.

The uptake of uracil by the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. This uracil permease is a multispanning membrane protein that follows the secretory pathway to the plasma membrane. We have used in vivo pulse labeling and immunoprecipitation to show that the uracil permease is phosphorylated. Phosphoamino acid analysis indicates that the phosphorylation occurs on seryl residues. Experiments with temperature sensitive secretory mutants, blocked at successive steps of the secretory pathway, have established that the phosphorylation of the permease takes place at the plasma membrane. Under steady state conditions, Western immunoblotting showed multiple phosphorylated permease species. Their relative abundance appeared susceptible to metabolic conditions. This study is, therefore, a first step toward identifying a molecular mechanism involved in the post-translational control of a yeast transporter.     

Coordinated regulation of endothelial nitric oxide synthase activity by phosphorylation and subcellular localization

Endothelial nitric oxide synthase (eNOS) is regulated by multiple mechanisms including Ca(2+)/calmodulin binding, protein-protein interactions, phosphorylation, and subcellular locations. Emerging evidence suggests that these seemingly independent mechanisms may be closely correlated. In the present study, the interplay between membrane targeting and phosphorylation of eNOS was investigated by using various mutants designed to target specific subcellular locations or to mimic different phospho states. Phospho-mimicking mutations of wild-type eNOS at S635 and S1179 synergistically activated the enzyme. The targeted eNOS mutants to plasma membrane and Golgi complex exhibited higher NO production activities than that of a myristoylation-deficient cytosolic mutant. Phospho-mimicking mutations at S635 and S1179 rescued the activity of the cytosolic mutant and increased those of the plasma membrane- and Golgi-targeted mutants. In contrast, phospho-deficient mutations at these sites led to inactivation of eNOS. Unlike the other targeted mutants, the cytosolic eNOS mutant was unresponsive to cAMP, indicating that membrane association and phosphorylation are required for eNOS activation. These findings suggest that the coordinated interplay between phosphorylation and subcellular localization of eNOS plays an important role in regulating NO production in endothelial cells.     

In vivo and in vitro analysis of cardiac troponin I phosphorylation

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.     

In vivo endothelial gene regulation in diabetes

Background

In comparison to the well established changes in compliance that occur at the large vessel level in diabetes, much less is known about the changes in compliance of the cardiovascular system at the end-organ level. The aim of this study was therefore to examine whether there was a correlation between resistance of the intrarenal arteries of the kidney and compliance of the left ventricle, as estimated by measurements of diastolic function, in subjects with type 2 diabetes.

Methods

We studied 167 unselected clinic patients with type 2 diabetes with a kidney duplex scan to estimate intrarenal vascular resistance, i.e. the resistance index (RI = peak systolic velocity-minimum diastolic velocity/peak systolic velocity) and a transthoracic echocardiogram (TTE) employing tissue doppler studies to document diastolic and systolic ventricular function.

Results

Renal RI was significantly higher in subjects with diastolic dysfunction (0.72 ± 0.05) when compared with those who had a normal TTE examination (0.66 ± 0.06, p < 0.01). Renal RI values were correlated with markers of diastolic dysfunction including the E/Vp ratio (r = 0.41, p < 0.001), left atrial area (r = 0.36, p < 0.001), the E/A ratio (r = 0.36, p < 0.001) and the E/E' ratio (r = 0.31, p < 0.001). These associations were independent of systolic function, hypertension, the presence and severity of chronic kidney disease, the use of renin-angiotensin inhibitors and other potentially confounding variables.

Conclusion

Increasing vascular resistance of the intrarenal arteries was associated with markers of diastolic dysfunction in subjects with type 2 diabetes. These findings are consistent with the hypothesis that vascular and cardiac stiffening in diabetes are manifestations of common pathophysiological mechanisms.