The C terminus of β-tubulin regulates vinblastine-induced tubulin polymerization

Oligoanions such as sodium triphosphate or GTP prevent and/or reverse vinblastine-induced polymerization of tubulin. We now show that the anions of glutamate-rich extreme C termini of tubulin are similarly involved in the regulation of the vinblastine effect. Cleavage of the C termini by limited proteolysis with subtilisin enhances vinblastine-induced tubulin polymerization and abolishes the anion effect. Only the β-tubulin C terminus needs to be removed to achieve these changes and the later cleavage of the α-tubulin C terminus has little additional effect. In fact, vinblastine concentrations >20 μM block cleavage of the α-tubulin C terminus in the polymer, whereas cleavage of the β-tubulin C terminus proceeds unimpeded over the time used. The vinblastine effect on tubulin polymerization is also highly pH-dependent between pH 6.5 and 7.5; this is less marked, but not absent, after subtilisin treatment. A working model is proposed wherein an anionic domain proximal to the extreme C terminus must interact with a cationic domain to permit vinblastine to promote polymerization. Both exogenous and extreme C-terminal anions compete for the cationic domain with the proximal anionic domain to prevent vinblastine-induced polymerization. We conclude that the electrostatic regulation of tubulin polymerization induced by vinblastine resides primarily in the β-tubulin C terminus but that additional regulation proximal in the tubulin molecule also plays a role.     

Rab27b regulates number and secretion of platelet dense granules

The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27a(ash/ash) Rab27b(-/-)) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet alpha-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for delta-storage pool deficiency in humans.     

Receptor for activated C-kinase 1 regulates the cellular localization and function of ABCB4

Aim:  Multidrug resistance protein 3 (MDR3/ABCB4), located on the bile canalicular membrane of hepatocytes, is responsible for the translocation of phosphatidylcholine across the plasma membrane, and its hereditary defect causes liver disorders, such as progressive familial intrahepatic cholestasis type 3. We aimed to identify the proteins responsible for the surface expression of human ABCB4 .
Methods:  We performed yeast two-hybrid screening with the cytoplasmic linker region of ABCB4 against a human liver cDNA library. This screening allowed us to identify the receptor for activated C-kinase 1 ( RACK1 ) as a novel binding partner of ABCB4 . The association of RACK1 with the linker region of ABCB4 was further confirmed by GST-pulldown assay, although we could not find out the interaction of full length of ABCB4 and RACK1 in co-immunoprecipitation assay in HeLa cells.
Results:  Down-regulation of endogenous RACK1 expression by siRNA in HeLa cells resulted in the localization of ABCB4 in the cytosolic compartment as well as reduced protein expression of ABCB4 , although mRNA expression and the protein stability of ABCB4 were not affected by the suppression of endogenous RACK1 . Similar alterations in cellular localization of ABCB4 were also found by suppressing endogenous RACK1 expression in HepG2 cells. Consequently, ABCB4 -mediated phosphatidylcholine translocation activity was significantly reduced when endogenous RACK1 expression was suppressed in HeLa cells. In contrast, the membrane surface localization and the protein expression of ABCB1 were not affected by the suppression of endogenous RACK1 expression.
Conclusion:  These results suggest that RACK1 may have a functional significance as a regulatory cofactor of ABCB4 and is indispensable for the plasma membrane localization and translocation function of ABCB4 .     

Hzf protein regulates dendritic localization and BDNF-induced translation of type 1 inositol 1,4,5-trisphosphate receptor mRNA

The localization of certain mRNAs to dendrites and their local translation in synaptic regions are proposed to be involved in certain aspects of synaptic plasticity. A cis-acting element within the 3' untranslated region (3' UTR) of the targeted mRNAs, which is bound by a trans-acting RNA-binding protein, controls the dendritic mRNA localization. Here, we identified hematopoietic zinc finger (Hzf) as a trans-acting factor that regulates the dendritic mRNA localization of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)RI), a dendritically localized mRNA in cerebellar Purkinje cells, via binding to the 3' UTR. In Hzf-deficient mice, the dendritic localization of IP(3)RI mRNA and brain-derived neurotrophic factor-induced IP(3)RI protein synthesis in the cerebellum were impaired. These findings suggest that Hzf is an RNA-binding protein that controls the dendritic mRNA localization and activity-dependent translation of IP(3)RI, and may be involved in some aspects of synaptic plasticity.     

Ganglioside GM1 binds to the Trk protein and regulates receptor function.

Several lines of evidence have suggested that ganglioside GM1 stimulates neuronal sprouting and enhances the action of nerve growth factor (NGF), but its precise mechanism is yet to be elucidated. We report here that GM1 directly and tightly associates with Trk, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances neurite outgrowth and neurofilament expression in rat PC12 cells elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. The potentiation of NGF activity by GM1 appears to involve tyrosine-autophosphorylation of Trk, which contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain. In the presence of GM1 in culture medium, there is a > 3-fold increase in NGF-induced autophosphorylation of Trk as compared with NGF alone. We also found that GM1 could directly enhance NGF-activated autophosphorylation of immunoprecipitated Trk in vitro. Monosialoganglioside GM1, but not polysialogangliosides, is tightly associated with immunoprecipitated Trk. Furthermore, such tight association of GM1 with Trk appears to be specific, since a similar association was not observed with other growth factor receptors, such as low-affinity NGF receptor (p75NGR) and epidermal growth factor receptor (EGFR). Thus, these results strongly suggest that GM1 functions as a specific endogenous activator of NGF receptor function, and these enhanced effects appear to be due, at least in part, to tight association of GM1 with Trk.     

Leukocyte and platelet function and eicosanoid production in subjects with hypercholesterolaemia

A group of 22 subjects with type IIA hypercholesterolaemia (mean serum cholesterol = 8.3 +/- 0.3 mmol/l) were sex, age and weight matched with 22 control subjects (mean serum cholesterol = 4.5 +/- 0.1 mmol/l). Diastolic blood pressure was significantly higher in hypercholesterolaemic subjects (79.2 +/- 1.4 mm Hg) than in control subjects (71.9 +/- 1.4 mm Hg). While the high cholesterol group had 52% greater thromboxane production in clotted whole blood than controls this difference was not significant, and the platelet aggregation and serotonin secretion response to doses of collagen, ADP and arachidonic acid were similar between the 2 groups. Polymorphonuclear leukocyte (PMN) chemiluminescence (used as a measure of reactive oxygen species production) in response to low doses of the chemotactic-peptide FMLP and opsonized zymosan was significantly greater in high cholesterol subjects compared to their matched controls. The production of platelet activating factor (PAF) by calcium ionophore (2.5 micrograms) stimulated PMN isolated from hypercholesterolaemic subjects (11.5 +/- 1.4 ng/10(6) cells) was significantly greater than PAF production by cells from the control group (8.3 +/- 1.0 ng/10(6) cells). Leukotriene B4 release by PMN in response to calcium ionophore did not differ between the 2 groups. These data suggest a degree of leukocyte activation in hypercholesterolaemic subjects compared to controls with normal cholesterol. In addition, plasma levels of lyso-PAF were higher in high cholesterol subjects (317 +/- 21 ng/ml) compared to their matched controls (271 +/- 18 ng/ml) perhaps indicating increased plasma acetylhydrolase activity in subjects with raised cholesterol levels. Recently described biological activity for lyso PAF suggests a possible role for this substance in atherogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Sumo-targeted ubiquitin ligase RNF4 regulates the localization and function of the HTLV-1 oncoprotein Tax

The Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)-modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax protein results in relocalization of the oncoprotein from the nucleus to the cytoplasm. Overexpression of RNF4, but not the RNF4 RING mutant, resulted in cytoplasmic enrichment of Tax. The RNF4-induced nucleus-to-cytoplasm relocalization was associated with increased NF-κB-mediated and decreased cAMP Response Element-Binding (CREB)-mediated Tax activity. Finally, depletion of RNF4 by RNAi prevented the DNA damage-induced nuclear/cytoplasmic translocation of Tax. These results provide important new insight into STUbL-mediated pathways that regulate the subcellular localization and functional dynamics of viral oncogenes.     

The small GTPase Rap1b regulates the cross talk between platelet integrin alpha2beta1 and integrin alphaIIbbeta3

The involvement of the small GTPase Rap1b in platelet integrin alpha2beta1-dependent outside-in signaling was investigated. Platelet adhesion to 4 different specific ligands for integrin alpha2beta1, monomeric collagen, decorin, and collagen-derived peptides CB8(II) and CB11(II), induced a robust and rapid activation of Rap1b. This process did not require secreted ADP or thromboxane A2 production but was critically regulated by phospholipase C (PLC)-derived second messengers. Both Ca2+ and protein kinase C were found to organize independent but additive pathways for Rap1b activation downstream of integrin-alpha2beta1, which were completely blocked by inhibition of PLC with U73122. Moreover, integrin alpha2beta1 engagement failed to trigger Rap1b activation in murine platelets lacking CalDAG-GEFI, a guanine nucleotide exchange factor regulated by Ca2+ and diacylglycerol, despite normal phosphorylation and activation of PLCgamma2. In addition, CalDAG-GEFI-deficient platelets showed defective integrin alpha2beta1-dependent adhesion and spreading. We found that outside-in signaling through integrin alpha2beta1 triggered inside-out activation of integrin alphaIIbbeta3 and promoted fibrinogen binding. Similarly to Rap1b stimulation, this process occurred downstream of PLC activation and was dramatically impaired in murine platelets lacking the Rap1 exchange factor CalDAG-GEFI. These results demonstrate that Rap1b is an important element in integrin-dependent outside-in signaling during platelet adhesion and regulates the cross talk between adhesive receptors.     

The localization of a platelet GpIIb-IIIa-related protein in endothelial cell adhesion structures

Previous studies have shown that human endothelial cells (ECs) adhere to fibrinogen (fg) and fibronectin (fn) and organize their cytoskeleton on these substrata. However, the mechanism governing this chain of events is poorly known. In ECs glycoproteins immunologically and biochemically similar to the platelet membrane GpIIb-IIIa complex have been described. The functional role of this complex in ECs remains to be established. In this study we show that the antigens recognized by polyclonal antibodies raised against human platelet GpIIb-IIIa and crossreacting with the EC form have a discrete and well-organized distribution at cell adhesion structures. Indeed these antigens are located at vinculin-rich focal contacts found at the membrane insertion of microfilament bundles of the stress fiber type. They are also found at cell-to-cell contacts and, with a diffuse pattern, at the dorsal surface of ECs. GpIIb-IIIa antibodies, added to EC suspensions prior to plating, inhibit EC spreading on fg and vitronectin (vn) substrata in a concentration-dependent way. In contrast, the antibodies are very poorly active when the cells are seeded on fn-coated glass. The same antibodies, added to adherent cells, disrupt cell-to-cell contacts and cause their rounding and detachment. Overall these results indicate that EC GpIIb-IIIa complex is involved in controlling the adhesion mechanism of these cells to extracellular matrix proteins.     

A multipurpose transposon system for analyzing protein production, localization, and function in Saccharomyces cerevisiae

Analysis of the function of a particular gene product typically involves determining the expression profile of the gene, the subcellular location of the protein, and the phenotype of a null strain lacking the protein. Conditional alleles of the gene are often created as an additional tool. We have developed a multifunctional, transposon-based system that simultaneously generates constructs for all the above analyses and is suitable for mutagenesis of any given Saccharomyces cerevisiae gene. Depending on the transposon used, the yeast gene is fused to a coding region for β-galactosidase or green fluorescent protein. Gene expression can therefore be monitored by chemical or fluorescence assays. The transposons create insertion mutations in the target gene, allowing phenotypic analysis. The transposon can be reduced by cre–lox site-specific recombination to a smaller element that leaves an epitope tag inserted in the encoded protein. In addition to its utility for a variety of immunodetection purposes, the epitope tag element also has the potential to create conditional alleles of the target gene. We demonstrate these features of the transposons by mutagenesis of the SPA2, ARP100, SER1, and BDF1 genes.     

Thrombin-induced platelet prostaglandin and cyclic AMP production and a possible intrinsic modulation of platelet function.

In previous studies, we observed an increase in intracellular cyclic AMP in platelets during thrombin-induced nucleotide and calcium release. Our present work suggests that platelet prostaglandins are directly involved in this phenomenon. Washed human platelets were incubated at 37 degrees C with human thrombin for times ranging between 5 s and 5 min. The release of nucleotides and calcium in response to thrombin, determined spectrophotometrically, reached 40-60% of maximal plateau levels by 5 s, 70-80% by 10 s, and 90-100% by 15 s. The production and secretion of prostaglandin E (PGE), measured by radioimmunoassay, parelleled this rapid time course. After 10 s, however, the amount of extracellular PGE began to decrease, continuing to do so through 5 min of incubation. Intracellular cyclic AMP accumulation, determined by radioimmunoassay, lagged behind the time course of nucleotide and calcium release, and that of PGE secretion, by several seconds. This lag in cyclic AMP accumulation appeared to correlate closely with the disappearance of PGE from the supernate in the same platelet samples. In view of the inhibitory effects of cyclic AMP and PGE on platelet release and aggregation, these interrelated time course curves suggest the existence within platelets of a mechanism for the modulation of platelet activity.     

Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers

The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 microg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 +/- 41 x 10(3)/microL to 522 +/- 90 x 10(3)/microL (P <.0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P >.4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P >.6 in both cases); or density of platelet TPO-receptors (P >.5). Platelet counts normalized by day 28. The life span of autologous (111)In-labeled platelets increased from 205 +/- 18 hours (baseline) to 226 +/- 22 hours (P <.01) on day 8. Platelet life span decreased from 226 +/- 22 hours (day 8) to 178 +/- 53 hours (P <.05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 +/- 11.9 x 10(3) platelets/microL/d (baseline) to 101 +/- 27.6 x 10(3) platelets/microL/d (P =.0009), and marrow megakaryocyte mass expanded from 37.4 +/- 18.5 fL/kg to 62 +/- 17 x 10(10) fL/kg (P =. 015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks. (Blood. 2000;95:2514-2522)     

Endosomal localization and function of sorting nexin 1

There are 17 human members of the sorting nexin (SNX) family of proteins that contain Phox (PX) domains. Yeast orthologs function in vesicular trafficking and mammalian proteins have been implicated in endocytic trafficking of cell surface receptors. The first member of this family, SNX1, was identified via interaction with the epidermal growth factor receptor. The present studies indicate that SNX1 and SNX2 are colocalized to tubulovesicular endosomal membranes and this localization depends on PI 3-kinase activity. Point mutations in the PX domain that abolish recognition of phosphorylated phosphatidylinositol (PtdIns) in vitro abolish vesicle localization in vivo indicating that lipid binding by the PX domain is necessary for localization to vesicle membranes. Deletion of a predicted coiled-coil region in the COOH terminus of SNX1 also abolished vesicle localization, indicating that this helical domain, too, is necessary for SNX1 localization. Thus, both PX domain recognition of PtdIns and COOH terminal helical domains are necessary for localization of SNX1 with neither alone being sufficient. Regulated overexpression of the NH(2) terminus of SNX1 containing the PX domain decreased the rate of ligand-induced epidermal growth factor receptor degradation, an effect consistent with inhibition of endogenous SNX1 function in the endosome compartment. SNX1 thus functions in regulating trafficking in the endosome compartment via PX domain recognition of phosphorylated PtdIns and via interaction with other protein components.     

Nuclear localization of cyclin B1 regulates DNA damage-induced apoptosis

Some cells undergo apoptosis in response to DNA damage, whereas others do not. To understand the biochemical pathways controlling this differential response, we have studied the intracellular localization of cyclin B1 in cell types sensitive or resistant to apoptosis induced by DNA damage. We found that cyclin B1 protein accumulates in the nucleus of cells that are sensitive to gamma radiation-induced apoptosis (thymocytes, lymphoid cell lines), but remains cytoplasmic in apoptosis-resistant cells (primary and transformed fibroblasts). Treatment of both cell types with leptomycin B, an inhibitor of CRM1-dependent cyclin B1 nuclear export, induces apoptosis. Furthermore, ectopic expression of cyclin B1-5xE, a protein that preferentially localizes to the nucleus, is sufficient to trigger apoptosis. Conversely, expression of cyclin B1-5xA, a predominantly cytoplasmic protein, fails to induce apoptosis. This suggests that nuclear accumulation is necessary for cyclin B1-dependent apoptosis. Our observations are consistent with the idea that localization of cyclin B1 is among the factors determining the cellular decision to undergo apoptosis in response to DNA damage.     

The oxysterol binding protein Kes1p regulates Golgi apparatus phosphatidylinositol-4-phosphate function

The Saccharomyces cerevisiae phosphatidylcholine/phosphatidylinositol transfer protein Sec14p is required for Golgi apparatus-derived vesicular transport through coordinate regulation of phospholipid metabolism. Sec14p is normally essential. The essential requirement for SEC14 can be bypassed by inactivation of (i) the CDP-choline pathway for phosphatidylcholine synthesis or (ii) KES1, which encodes an oxysterol binding protein. A unique screen was used to determine genome-wide genetic interactions for the essential gene SEC14 and to assess whether the two modes of "sec14 bypass" were similar or distinct. The results indicate that inactivation of the CDP-choline pathway allows cells with inactivated SEC14 to live through a mechanism distinct from that of inactivation of KES1. We go on to demonstrate an important biological function of Kes1p. Kes1p regulates Golgi apparatus-derived vesicular transport by inhibiting the function of Pik1p-generated Golgi apparatus phosphatidylinositol-4-phosphate (PI-4P). Kes1p affects both the availability and level of Golgi apparatus PI-4P. A set of potential PI-4P-responsive proteins that include the Rab GTPase Ypt31p and its GTP exchange factor are described.