The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

MAP kinase phosphatase-1 gene transcription in rat neuroendocrine cells is modulated by a calcium-sensitive block to elongation in the first exon.

Transcriptional elongation of many eukaryotic, prokaryotic, and viral genes is tightly controlled, which contributes to gene regulation. Here we describe this phenomenon for the MAP kinase phosphatase 1 (MKP-1) immediate early gene. In rat GH4C1 pituitary cells, MKP-1 mRNA is rapidly and transiently induced by the thyrotropin-releasing hormone (TRH) and the epidermal growth factor EGF via transcriptional activation of the gene. Ca(2+) signals are necessary for the induction of MKP-1 in response to TRH but not to EGF. Reporter gene analysis with the newly cloned rat promoter sequence shows only limited induction in response to various stimuli, including TRH or EGF. By nuclear run-on assays we demonstrate that in basal conditions, a strong block to elongation in the first exon regulates the MKP-1 gene and that stimulation with either TRH or EGF overcomes the block. Ca(2+) signals are important to release the MKP-1 elongation block in a manner similar to the c-fos oncogene. These results suggest that a common mechanism of intragenic regulation may be conserved between MKP-1 and c-fos in mammalian cells.

Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity.

A Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dock, an SH3/SH2 adaptor protein required for axon guidance. Dscam binds directly to both Dock's SH2 and SH3 domains. Genetic studies revealed that Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, containing a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable Ig and transmembrane domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificity of neuronal connectivity.

Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating "knock-out" phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.

Transradial coronary angioplasty and stenting--immediate results and 3-month clinical follow-up in the first 50 patients performed at the National Heart Institute.

AIM: To explore the safety and feasibility of coronary angioplasty and stenting via the radial artery in a heterogenous group of patients and to report the immediate and 3-month clinical follow-up. BACKGROUND: The use of the transradial approach for coronary angiography was first described by Lucien Campeau in 1989. Based on the favourable initial results, this technique has gained widespread acceptance worldwide. Ferdinand Kiemeneij's work in transradial angioplasty and stenting has taken invasive cardiology into the exciting new era of "minimally invasive coronary intervention". METHODS AND RESULTS: Fifty consecutive patients underwent Transradial Percutaneous Transluminal Coronary Angioplasty (PTCA) with or without stenting from mid March 98-December 98. The right radial approach was utilised in 41 patients (80%) while the left in 9 patients. Ninety percent of the procedures was done on an adhoc basis. Diabetes mellitus was present in 38% of patients. Eighty percent of patients had unstable angina pectoris and 60% had a prior history of acute myocardial infarction. The commonest vessel involved was the LAD (51%) and type B lesions predominated (54%). PTCA was successful in 96%. One patient had a total LAD occlusion, which could not be wired, and another developed severe spasm during catheter manipulation. The latter had successful PTCA via the right femoral route Stents were utilised in 57% of patients. The commonest indication for stenting was suboptimal PTCA results (89%) and dissection (14%). There was no stent embolisation and all stents were successfully deployed (100%). One patient developed acute stent thrombosis necessitating repeat PTCA and another patient sustained an acute anteroseptal myocardial infarction 5 days post procedure as a result of subacute stent thrombosis and died. Two patients had successful primary PTCA. There was no bleeding or vascular complications. 60% of patients were treated on an outpatient basis. At 3-months follow up, 1 patient required CABG's for disease progression. Three patients had absent radial pulses without adverse consequence. No patient required repeat PTCA at follow up. CONCLUSION: In summary, adhoc PTCA and stenting is safe and feasible in our patient population. A study on the cost effectiveness of the procedure compared to conventional femoral PTCA is warranted.

Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.

A human orthologue of the Saccharomyces cerevisiae YVH1 protein-tyrosine phosphatase is able to rescue the slow growth defect caused by the disruption of the S. cerevisiae YVH1 gene. The human YVH1 gene is located on chromosome 1q21-q22, which falls in a region amplified in human liposarcomas. The evolutionary conserved COOH-terminal noncatalytic domain of human YVH1 is essential for in vivo function. The cysteine-rich COOH-terminal domain is capable of coordinating 2 mol of zinc/mol of protein, defining it as a novel zinc finger domain. Human YVH1 is the first protein-tyrosine phosphatase that contains and is regulated by a zinc finger domain.

Calcium signalling and gene expression.

A wide variety of compounds acting as extracellular signals cause changes in the free cytosolic Ca2+ concentration. These factors include hormones, growth factors, neurotransmitters, but also nutrient and metabolic activators. Ca2+ signalling is caused by mobilization of Ca2+ from internal stores and by well controlled and timed Ca2+ influx from the extracellular space. Ca2+ signals address Ca2+ dependent enzymes, most importantly Ca2+ sensitive protein kinases and phosphatases. The profound influence of Ca2+ signalling on gene expression has been recognized a long time ago. As Ca2+ signals are short-lived when compared to alterations in differentiated gene expression, it is generally considered that genes coding for short-lived transcription factors (i.e. fos, jun) are the immediate target of Ca2+ signalling. Transcription of these immediate early genes (IEG) can be activated without the need for protein synthesis. Ca2+ signalling affects differentiated gene expression via changes in the absolute and relative abundance of IEG products, which in turn control the expression of differentiated genes. Ca2+ signals can stimulate both transcriptional initiation as well as transcriptional elongation. Initiation of transcription is stimulated by the Ca2+ dependent phosphorylation of binding proteins addressing two response elements in the promoter of IEGs: the cAMP response element, CRE, and the serum response element, SRE. Distinct protein kinases are involved in either case. We study the elongation of transcripts of the IEG c-fos beyond the first intron which is favoured by Ca2+ signals, involving mechanisms which still are poorly understood. We can show that intron sequences contribute to the control of elongation by Ca2+, and that there is a strong interrelation between the transcription control by the promoter and by the intron.

Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase.

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.

The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity.

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.

Induction of the mitogen-activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12.

In PC12 sympathetic neurons activation and nuclear translocation of ERK family MAP kinases plays an essential role in processes underlying nerve growth factor (NGF)-dependent differentiation. We have recently cloned MKP-3 as a novel dual specificity phosphatase displaying selectivity towards inactivation of the ERK1 and ERK2 MAP kinases. Here we report that in PC12 cells, MKP-3 undergoes powerful and specific up-regulation by NGF while a number of mitogens and cellular stresses are ineffective. NGF-stimulated MKP-3 expression appears after 1 h, is maximal at 3 h, and is sustained for 5 days. This coincides with a critical period of neurite outgrowth and terminal differentiation. Consistent with a role mediating inhibition of PC12 cell MAP kinases, NGF-stimulated ERK2 activation was suppressed considerably following pretreatment with fibroblast growth factor and 9-cis-retinal, two additional differentiation factors found to induce powerfully MKP-3 expression. Given the clear cytosolic localization of MKP3 in PC12 cells and sympathetic neurons, these results suggest a critical role for inactivating ERK MAP kinases in non-nuclear compartments during essential stages of NGF-mediated PC12 differentiation.

Regulated expression of dual specificity protein phosphatases in rat brain.

Activated mitogen-activated protein (MAP) kinases play an essential role controlling many neuronal functions. Dual specificity protein phosphatases (DS-PTPs) elicit selective inactivation of MAP kinases and are under tight transcriptional control. We have studied expression of four DS-PTPs (MKP-1, MKP-X, MKP-3 and B23) in rat brain and examined changes during post-natal development and following kainic acid induced seizure activity. In normal adult brain these DS-PTPs exhibit a strikingly different expression pattern. Only MKP-1 was regulated during development with levels increased transiently (P15-P21) within the thalamus and somatosensory cortex. Following kainate treatment, MKP-1, MKP-3 and B23 all exhibit striking changes in expression within hippocampal subfields CA1-3 and dentate gyrus. Regulated transcription of DS-PTPs may play a critical role controlling MAP kinase dependent processes including synaptic remodeling and neuronal death.

Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1.

We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen by both 32PO4 labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.

Induction of the dual specificity phosphatase PAC1 in rat brain following seizure activity.

Recurrent seizure activity leads to delayed neuronal death as well as to inflammatory responses involving microglia in hippocampal subfields CA1, CA3 and CA4. Since mitogen activated protein (MAP) kinases control neuronal apoptosis and trigger generation of inflammatory cytokines, their activation state could determine seizure-related brain damage. PAC1 is a dual specificity protein phosphatase inactivating MAP kinases which we have found to be undetectable in normal brain. Despite this, kainic acid-induced seizure activity lead to rapid (approximately 3 h) but transient appearance of PAC1 mRNA in granule cells of the dentate gyrus as well as in pyramidal CA1 neurons. This pattern changed with time and after 2-3 days PAC1 was induced in dying CA1 and CA3 neurons. At this time PAC1 mRNA was also expressed in white matter microglia as well as in microglia invading the damaged hippocampus. PAC1 may play an important role controlling MAP kinase involvement in both neuronal death and neuro-inflammation following excitotoxic damage.

Chromosomal localization of three human dual specificity phosphatase genes (DUSP4, DUSP6, and DUSP7).

Mitogen-activated protein (MAP) kinase phosphatases constitute a growing family of dual specificity phosphatases thought to play a role in the dephosphorylation and inactivation of MAP kinases and are therefore likely to be important in the regulation of diverse cellular processes such as proliferation, differentiation, and apoptosis. For this reason it has been suggested that MAP kinase phosphatases may be tumor suppressors. We have determined the chromosomal locations of three human dual specificity phosphatase genes by fluorescence in situ hybridization and radiation hybrid mapping. The genes were localized to three different chromosomes, MKP2 (DUSP4) to 8p11-p12, MKP3 (DUSP6) to 12q22-q23, and MKPX (DUSP7) to 3p21. This will allow the potential roles of these genes in disease processes to be evaluated.

Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4.

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.

The dual specificity phosphatases M3/6 and MKP-3 are highly selective for inactivation of distinct mitogen-activated protein kinases.

The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPKbeta, p46 SAPKgamma (JNK1) or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKbeta and p38 MAP kinases is inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by constitutive p21(ras) GTPases. Hence, although M3/6 expression effectively blocked p54 SAPKbeta activation by p21(rac) (G12V), ERK1 activated by p21(ras) (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21(ras) was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition of different MAP kinases by two distinct dual specificity phosphatases.

MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase.

MKP-1 (also known as CL100, 3CH134, Erp, and hVH-1) exemplifies a class of dual-specificity phosphatase able to reverse the activation of mitogen-activated protein (MAP) kinase family members by dephosphorylating critical tyrosine and threonine residues. We now report the cloning of MKP-3, a novel protein phosphatase that also suppresses MAP kinase activation state. The deduced amino acid sequence of MKP-3 is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. When expressed in COS-7 cells, MKP-3 blocks both the phosphorylation and enzymatic activation of ERK2 by mitogens. Northern analysis reveals a single mRNA species of 2.7 kilobases with an expression pattern distinct from other dual-specificity phosphatases. MKP-3 is expressed in lung, heart, brain, and kidney, but not significantly in skeletal muscle or testis. In situ hybridization studies of MKP-3 in brain reveal enrichment within the CA1, CA3, and CA4 layers of the hippocampus. Metrazole-stimulated seizure activity triggers rapid (<1 h) but transient up-regulation of MKP-3 mRNA in the cortex, piriform cortex, and some amygdala nuclei. Metrazole stimulated similar regional up-regulation of MKP-1, although this was additionally induced within the thalamus. MKP-3 mRNA also undergoes powerful induction in PC12 cells after 3 h of nerve growth factor treatment. This response appears specific insofar as epidermal growth factor and dibutyryl cyclic AMP fail to induce significant MKP-3 expression. Subcellular localization of epitope-tagged MKP-3 in sympathetic neurons reveals expression in the cytosol with exclusion from the nucleus. Together, these observations indicate that MKP-3 is a novel dual-specificity phosphatase that displays a distinct tissue distribution, subcellular localization, and regulated expression, suggesting a unique function in controlling MAP kinase family members. Identification of a second partial cDNA clone (MKP-X) encoding the C-terminal 280 amino acids of an additional phosphatase that is 76% identical to MKP-3 suggests the existence of a distinct structurally homologous subfamily of MAP kinase phosphatases.

Transfected type II interleukin-1 receptor impairs responsiveness of human keratinocytes to interleukin-1.

Of the two known types of specific receptors for interleukin (IL)-1, the function of the type II IL-1 receptor (IL-1RII) is still elusive. IL-1RII is allegedly devoid of signaling capacity and is therefore thought to act by trapping and inhibiting IL-1. To directly assess the functional role of IL-1RII, a human keratinocyte cell line has been stably transfected with a cDNA coding for IL-1RII, and its responsiveness to IL-1 has been compared with that of nontransfected cells. Parental cells express IL-1RI and are responsive to low doses of IL-1, whereas transfected cells overexpress IL-1RII, both in its membrane and soluble form, and show a dramatically impaired response to IL-1. Selective block of IL-1RII restores the ability of transfected keratinocytes to respond to IL-1, indicating that the overexpressed IL-1RII is in fact uniquely responsible for their refractoriness to IL-1. The main mechanism of unresponsiveness in transfected keratinocytes appears to be the capture and neutralization of IL-1 by the soluble form of IL-1RII.

Differential protein expression in aortic smooth muscle cells cultured from newborn and aged rats.

Atherosclerosis is a complex disease in which smooth muscle cells (SMC) play a fundamental role. Work from several laboratories has suggested that in experimental models of atheromatosis SMC heterogeneity is important in the establishment of intimal thickening. Moreover it has been shown that SMC cultured from different situations in vivo maintain distinct phenotypic features in vitro. In order to find proteins differentially expressed in SMC cultured from newborn and aged rats, total protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), high-resolution maps were built, and differentially expressed spots were identified by automatic computer analysis. Of the 14 differentially expressed protein spots, 4 were present in SMC of newborn and 10 in SMC of old animals; we describe their molecular weights and isoelectric points. One of these proteins (expressed only in cultured SMC of old rats) was successfully microsequenced for 16 amino acids and it was found identical to cellular retinol-binding protein. This results provides, to our knowledge, the first suggestion that retinoids are implicated in the differentiation and aging of vascular SMC.

Role of PhoU in phosphate transport and alkaline phosphatase regulation.

The negative regulatory function of PhoU in alkaline phosphatase (AP) was suggested by the behavior of K10 phoU35 carrying a missense mutation whose product was detected by immunoblotting. To define more clearly the regulatory function of this protein for the synthesis of AP, we constructed a null mutation. The constitutive synthesis of AP in this phoU deletion strain confirmed the negative role of PhoU. However, the expression of the PhoU protein from an isopropyl-beta-D-thiogalactopyranoside-inducible promoter had no effect on the repression of AP synthesis. Furthermore, the involvement of PhoU in free-Pi uptake was demonstrated. These results provide evidence that PhoU participates in Pi transport and in the regulatory role of the phosphate-specific transport system.