The yeast Rab escort protein binds intracellular membranes in vivo and in vitro

In both mammals and yeast, intracellular vesicular transport depends on the correct shuttling between membrane and cytosol of the Rab/Ypt small G proteins. Membrane association of these proteins requires prenylation by the Rab geranylgeranyl transferase that recognizes a complex formed by the Rab/Ypt protein and the Rab escort protein (REP). After prenylation the Rab/Ypt protein is delivered to the target membranes by REP. Little is known about the early steps of the Rab-REP complex formation and where this association occurs in the cell. Although prenylation is believed to take place in the cytosol, we show that the yeast Rab escort protein Mrs6 is present in both soluble and particulate fractions of cell extracts. Mrs6p is associated with the heavy microsomal fraction that contains endoplasmic reticulum-Golgi membranes but is absent in the plasma membrane, vacuoles, mitochondria, and microsomal subfraction associated with mitochondria. The solubilization pattern of the particulate pool of Mrs6p implies that this protein is peripherally but tightly associated with membranes via hydrophobic interactions and metal ions. We also report that the C terminus of Mrs6p is important for maintaining the solubility of the protein because its deletion or replacement with the C terminus of RabGDI results in a protein that localizes only to membranes.     

MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.     

Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro

Fractionation of human cell extracts by cisplatin-DNA affinity chromatography was employed to identify proteins capable of binding cisplatin-damaged DNA. A specific protein-DNA complex, termed DRP-3, was identified in an electrophoretic mobility shift assay (EMSA) using a cisplatin-damaged DNA probe. Using this assay we purified DRP-3 and the final fraction contained proteins of 70, 53, 46, 32, and 14 kDa. On the basis of subunit molecular weights, antibody reactivity, and DNA binding activities, DRP-3 was identified as human replication protein A (hRPA). Therefore, we assessed the binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA in vitro. Global treatment of a highly purified completely duplex 44-bp DNA with cisplatin resulted in a 10-20-fold increase in rhRPA binding compared to the undamaged control. The stability of the RPA-DNA complexes was assessed, and NaCl and MgCl2 concentrations that completely inhibited rhRPA binding to undamaged DNA had only a minimal effect on binding to duplex platinated DNA. We assessed rhRPA binding to a duplex DNA containing a single site-specific 1,2-d(GpG) cisplatin adduct, and the results revealed a 4-6-fold increase in binding to this DNA substrate compared to an undamaged control DNA of identical sequence. These results are consistent with RPA being involved in the initial recognition of cisplatin-damaged DNA, possibly mediating DNA repair events. Therefore, we assessed how another cisplatin DNA binding protein, HMG-1, affected the ability of rhRPA to bind damaged DNA. Competition binding assays show minimal dissociation of either protein from cisplatin-damaged DNA during the course of the reaction. Simultaneous addition experiments revealed that HMG-1 binding to cisplatin-damaged DNA was minimally affected by rhRPA, while HMG-1 inhibited the damaged-DNA binding activity of rhRPA. These data are consistent with HMG-1 blocking DNA repair and possibly having the capability to enhance the cytotoxic efficacy of the drug cisplatin.     

Antisense WT1 transcription parallels sense mRNA and protein expression in fetal kidney and can elevate protein levels in vitro

The intensity of a pain-only decompression sickness (DCS) symptom with respect to time at altitude increases, peaks, and then declines in some cases. A similar pattern is also seen in a graph of the probability density function [f(t)] for DCS. The f(t) is the proportion of DCS per unit time with respect to time at altitude. The integration of f(t) with respect to time provides the cumulative probability of DCS [P(DCS)]. We suspect that the perceived intensity of pain with a given stimulus intensity is related to the P(DCS); it may be related to the intensity of the stimulus to a power (alpha). Our stimuli are defined as pressure ratio [PR = (phi P1N2/ P2)-11] or pressure difference [delta P = phi P1N2-P2], where phi P1N2 is the N2 partial pressure calculated in the 360 min half-time (t1/2) compartment or t1/2 is estimated with other parameters and P2 is ambient pressure after the ascent. Both stimuli represent a potential released volume of gas. We tested the null hypothesis that alpha > 1 was no better than alpha = 1 in PR alpha and delta P alpha in a log logistic survival analysis of 1085 exposures in hypobaric chambers. The log likelihood number increased from -1198 for alpha = 0 for the null model to -724 for PR alpha when alpha = 3.52 with a 42 min t1/2 and -714 for delta P alpha when alpha = 8.44 with a 91 min t1/2. We conclude that the improvement in our expressions for decompression dose with alpha > 1 is not by random chance and that alpha may link the physics of gas evolution to the biology of pain perception. Because of our empirical approach, we do not exclude other possible interpretations.     

Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B

To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, [1+9-76]MCP-1 (MCP-1 lacking residues 2-8), antagonizes wild type MCP-1 by competitive inhibition, or by a dominant negative mechanism wherein heterodimers of MCP-1 and [1+9-76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9-76]MCP-1 over a range of concentrations of both ligands fits well to a simple model in which monomeric [1+9-76]MCP-1 functions as a competitive inhibitor of monomeric MCP-1. These results are crucial for elucidating the molecular details of receptor binding and activation, for interpreting mutagenesis data, for understanding how antagonistic chemokine variants function, and for the design of receptor antagonists.     

The aminoterminus of c-Raf-1 binds a protein kinase phosphorylating Ser259

The kinase negative aminoterminal domain of c-Raf-1 expressed as glutathione S-transferase fusion protein was phosphorylated in vitro after treatment with lysates from A431 cells and subsequent in vitro protein kinase assay. This phosphorylation was independent of stimulation of the cells with EGF; it occurred exclusively on serine and was mapped to Ser259. The identical site of c-Raf-1 was phosphorylated in A431 cells by metabolic labelling in vivo. The kinase binding domain was mapped by various GST-Raf deletion mutants to c-Raf-1 aminoacid residues 181 to 255.     

Factors relating to urinary protein excretion in children with autosomal dominant polycystic kidney disease

Adults with autosomal dominant polycystic kidney disease (ADPKD) who have overt proteinuria (>300 mg/d) have higher mean arterial pressures, lower creatinine clearances, larger renal volumes, and a more aggressive course of renal disease than ADPKD patients without proteinuria. This study examines the relationship between proteinuria and microalbuminuria and similar factors in ADPKD children. A total of 189 children from 81 ADPKD families was included in the analysis. The ADPKD children (n = 103) had significantly greater urine protein excretion rates than the non-ADPKD children (n = 86) (3.9+/-0.3 versus 2.8+/-0.2 mg/m2 per h, P < 0.001). Children with severe renal cystic disease (> 10 cysts; n = 54) had greater protein excretion than those with moderate disease (< or = 10 cysts; n = 49) (4.4+/-0.5 versus 3.3+/-0.2 mg/m2 per h, P < 0.05). The ADPKD children had significantly greater albumin excretion rates than the non-ADPKD children (32+/-6 versus 10+/-2 mg/m2 per 24 h, P < 0.001), and a higher percentage of ADPKD children had significant microalbuminuria (>15 mg/m2 per 24 h in boys and >23 mg/m2 per 24 h in girls) than their unaffected siblings (30% versus 10%, P < 0.05). Thirty percent of ADPKD children had albuminuria and 23% had overt proteinuria. For all ADPKD children, there was no correlation between proteinuria and hypertension. However, there was a significant correlation between urinary protein excretion and diastolic BP among children diagnosed after the first year of life (r = 0.23, P < 0.05). Therefore, proteinuria and albuminuria occur early in the course of ADPKD and may be markers of more severe renal disease.     

A rho-associated protein kinase, ROKalpha, binds insulin receptor substrate-1 and modulates insulin signaling

Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptors. These tyrosine phosphorylation sites serve to dock several Src homology 2-containing signaling proteins. In addition, IRS-1 contains a pleckstrin homology domain and a phosphotyrosine binding domain (PTB) implicated in protein-protein and protein-lipid interactions. In a yeast two-hybrid screening using Xenopus IRS-1 (xIRS-1) pleckstrin homology-PTB domains as bait, we identified a Xenopus homolog of Rho-associated kinase alpha (xROKalpha) as a potential xIRS-1-binding protein. The original clone contained the carboxyl terminus of xROKalpha (xROK-C) including the putative Rho binding domain but lacking the amino-terminal kinase domain. Further analyses in yeast indicated that xROK-C bound to the putative PTB domain of xIRS-1. Binding of xROK-C to xIRS-1 was confirmed in Xenopus oocytes after microinjection of mRNA corresponding to xROK-C. Furthermore, microinjection of xROK-C mRNA inhibited insulin-induced mitogen-activated protein kinase activation with a concomitant inhibition of oocyte maturation. In contrast, microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral Ras (v-Ras) mRNA. These results suggest that xROKalpha may play a role in insulin signaling via a direct interaction with xIRS-1.     

Endothelin-induced inositol phosphate formation in rat kidney. Studies on receptor subtypes, G-proteins and regulation during ontogenesis

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12-16 weeks old) ET's concentration-dependently increased IP-formation with an order of potency ET-1 > ET-3. While the non-selective ET-receptor antagonist bosentan (10 microM) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 microM) inhibited it only by 70%, the ETB-receptor antagonist IRL 1038 (1 microM) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal sites from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET-receptor number and the amount of immunodetectable Gq/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET-receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ETA-receptors declined, while that of ETB-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ETA- and ETB-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of Gq/11.     

Molybdenum cofactor biosynthesis. The plant protein Cnx1 binds molybdopterin with high affinity

The molybdenum cofactor is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin (MPT) revealing the same core structure in all organisms. The plant protein Cnx1 from Arabidopsis thaliana is involved in the multi-step biosynthesis of molybdenum cofactor. Previous studies (Stallmeyer, B., Nerlich, A., Schiemann, J., Brinkmann, H., and Mendel, R. R. (1995) Plant J. 8, 751-762) suggested a function of Cnx1 in a late step of cofactor biosynthesis distal to the formation of MPT, i.e. conversion of MPT into molybdenum cofactor. Here we present the first biochemical evidences confirming this assumption. The protein Cnx1 consists of two domains (E and G) homologous to two distinct Escherichia coli proteins involved in cofactor synthesis. Binding studies with recombinantly expressed and purified Cnx1 and with its single domains revealed a high affinity of the G domain to MPT (kD = 0.1 microM) with equimolar binding. In contrast, the E domain of Cnx1 binds MPT with lower affinity (kD = 1.6 microM) and in a cooperative manner (nH = 1. 5). The entire Cnx1 showed a tight and cooperative MPT binding. Based on these data providing a common link between both domains that matches the previous characterization of plant and bacterial Cnx1 homologous mutants, we present a model for the function of Cnx1.     

The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1

BACKGROUND: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway. RESULTS: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis. CONCLUSIONS: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.     

The prevalence of seminal vesicle cysts in autosomal dominant polycystic kidney disease

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a systemic hereditary disorder characterized by bilateral diffuse renal cysts. Extrarenal involvement is a well known manifestation of ADPKD. Data relating to the association between seminal vesicle cysts and ADPKD are limited. The aims of this study are to evaluate the frequency of seminal vesicle cysts in ADPKD and to assess the relationship between seminal vesicle cysts, with cysts in the liver and prostate, and creatininaemia. METHODS: Forty five male patients (mean age 40 years, range 13-67) were included in the study. Each subject underwent a formal interview, physical examination; and abdominal and transrectal ultrasonography. Three patients were infertile, but one of the patients also had varicocele. RESULTS: Seminal vesicle cysts were present in 27 (60%) patients. Liver and prostate cysts were present in 19 (42%) and five (11%) patients, respectively. There was a positive correlation between seminal vesicle cysts, cysts in the liver, and serum creatinine concentrations. CONCLUSION: Our conclusions are: (i) seminal vesicle cysts are not uncommon in ADPKD; (ii) ADPKD should be looked for in patients with seminal vesicle cysts, and (iii) the clinical significance of seminal vesicle cysts in ADPKD remains to be defined.