The brain-specific activator p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons.

Cell cycle withdrawal in postmitotic cells involves cyclin-dependent kinase (Cdk) inhibitors that repress cell cycle Cdk activity. During mouse neurogenesis, cortical postmitotic neurons are shown here to accumulate high levels of the p27 Cdk inhibitor compared with their progenitor neuroblasts. Elevated p27 levels in staged embryo brain extracts correlate with p27 binding to Cdk2, and Cdk inactivation. Yet, Cdk5, which is associated with the noncyclin activator p35 in neurons, remains active in the presence of high p27 levels. Both in vitro and in vivo, p27 and related inhibitors can recognize a cyclin D-Cdk5 complex but not a p35-Cdk5 complex. The results indicate that the choice of activator determines the susceptibility of Cdk5 to p27 and related Cdk inhibitors, and thus its ability to act in postmitotic cells.

Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide.

To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.

Chemical specificity and physical properties of the lipid bilayer in the regulation of protein kinase C by anionic phospholipids: evidence for the lack of a specific binding site for phosphatidylserine.

The association of protein kinase C (PKC) with membranes was found not to be specific for phosphatidyl-L-serine (PS). In particular, a synthetic phospholipid, dansyl-phosphatidylethanolamine, proved to be fully functional in the association of PKC with lipid bilayers and in mediating the interaction of this enzyme with diacylglycerol. Dansyl-phosphatidylethanolamine was also able to activate the enzyme in a Ca2+-dependent fashion. Differences in the ability to bind and activate PKC observed for an array of anionic lipids were not larger than alterations caused by changes in acyl chain composition. Thus, although different lipids interact to different extents with PKC, there are no specific binding sites for the PS headgroup on the enzyme. We found that lipids with a greater tendency to form inverted phases increased the binding of PKC to bilayers. However, these changes in lipid structure cannot be considered separately from the miscibility of lipid components in the membrane. For pairs of lipids with similar acyl chains, the dependence on PS concentration is sigmoidal, while for dissimilar acyl chains there is much less dependence of binding on PS concentration. The results can be explained in terms of differences in the lateral distribution of components in the membrane.

Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.

The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.

Tissue-specific response of the human platelet-activating factor receptor gene to retinoic acid and thyroid hormone by alternative promoter usage.

We have studied the effects of retinoic acid (RA) and thyroid hormone (3,3',5-triiodothyronine; T3) on platelet-activating factor receptor (PAFR) gene expression in intact rats and the ability of two human PAFR gene promoters (PAFR promoters 1 and 2) to generate two transcripts (PAFR transcripts 1 and 2). Northern blotting showed that RA and T3 regulated PAFR gene expression only in rat tissues that express PAFR transcript 2. Functional analysis of the human PAFR promoter 2 revealed that responsiveness to RA and T3 was conferred through a 24-bp element [PAFR-hormone response element (HRE) located from -67 to -44 bp of the transcription start site, whereas PAFR promoter 1 did not respond to these hormones. The PAFR-HRE is composed of three direct repeated TGACCT-like hexamer motifs with 2-and 4-bp spaces, and the two upstream and two downstream motifs were identified as response elements for RA and T3. Thus, the PAF-PAFR pathway is regulated by the PAFR level altered by a tissue-specific response to RA and T3 through the PAFR-HRE of the PAFR promoter 2.

Identification of an inducible surface molecule specific to fusing macrophages.

Multinucleated giant cells and osteoclasts arise through the fusion of mononuclear phagocyte precursors. To elucidate the mechanism by which cells of monocytic lineage fuse and differentiate into giant cells and osteoclasts, we hypothesized that, as with other cell fusion events, specific surface molecules mediate the adhesion/fusion process. It has been observed that macrophages can be induced to fuse with one another in response to specific stimuli or when placed in a specific microenvironment. The formation of giant cells is primarily associated with chronic inflammatory reactions and tumors, while osteoclasts differentiate on bone which they resorb. The fact that, under normal conditions, macrophages and monocytes fail to fuse in regions and tissues where they are present in large numbers suggests the regulated and transient expression of potential fusion molecules. To identify such a fusion-associated molecule, we established a macrophage fusion assay and generated monoclonal antibodies (mAbs) that alter the fusion of macrophages in vitro. We selected four mAbs that each had the ability to block the fusion but not the aggregation of macrophages in vitro. All four antibodies recognize surface proteins of 150 kDa. The expression of the antigens recognized by all four mAbs is restricted to macrophages that have been induced to fuse in vitro and in vivo and is inducible, transient, and regulated, as neither nonfusing macrophages nor macrophages fused in vitro express these antigens. These results support the hypothesis that macrophage fusion is mediated by specific fusion/adhesion molecules and also provide a means to study the molecular mechanisms of macrophage fusion.

The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1.

The transmembrane protein-tyrosine-phosphatases (PTPases) LAR, PTP delta, and PTP sigma each contain two intracellular PTPase domains and an extracellular region consisting of Ig-like and fibronectin type III-like domains. We describe the cloning and characterization of human PTP sigma (HPTP sigma) and compare the structure, alternative splicing, tissue distribution, and PTPase activity of LAR, HPTP delta, and HPTP sigma, as well their ability to associate with the intracellular coiled-coil LAR-interacting protein LIP.1. Overall, these three PTPases are structurally very similar, sharing 64% amino acid identity. Multiple isoforms of LAR, HPTP delta, and HPTP sigma appear to be generated by tissue-specific alternative splicing of up to four mini-exon segments that encode peptides of 4-16 aa located in both the extracellular and intracellular regions. Alternative usage of these peptides varies depending on the tissue mRNA analyzed. Short isoforms of both HPTP sigma and HPTP delta were also detected that contain only four of the eight fibronectin type III-like domains. Northern blot analysis indicates that LAR and HPTP sigma are broadly distributed whereas HPTP delta expression is largely restricted to brain, as is the short HPTP sigma isoform containing only four fibronectin type III-like domains. LAR, HPTP delta, and HPTP sigma exhibit similar in vitro PTPase activities and all three interact with LIP.1, which has been postulated to recruit LAR to focal adhesions. Thus, these closely related PTPases may perform similar functions in various tissues.

Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells.

We have investigated two regions of the viral RNA of human immunodeficiency virus type 1 (HIV-1) as potential targets for antisense oligonucleotides. An oligodeoxynucleotide targeted to the U5 region of the viral genome was shown to block the elongation of cDNA synthesized by HIV-1 reverse transcriptase in vitro. This arrest of reverse transcription was independent of the presence of RNase H activity associated with the reverse transcriptase enzyme. A second oligodeoxynucleotide targeted to a site adjacent to the primer binding site inhibited reverse transcription in an RNase H-dependent manner. These two oligonucleotides were covalently linked to a poly(L-lysine) carrier and tested for their ability to inhibit HIV-1 infection in cell cultures. Both oligonucleotides inhibited virus production in a sequence- and dose-dependent manner. PCR analysis showed that they inhibited proviral DNA synthesis in infected cells. In contrast, an antisense oligonucleotide targeted to the tat sequence did not inhibit proviral DNA synthesis but inhibited viral production at a later step of virus development. These experiments show that antisense oligonucleotides targeted to two regions of HIV-1 viral RNA can inhibit the first step of viral infection--i.e., reverse transcription--and prevent the synthesis of proviral DNA in cell cultures.

Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability.

We have developed a modified rhodamine (Rho) staining procedure to study uptake and efflux in murine hematopoietic stem cells. Distinct populations of Rho++ (bright), Rho+ (dull), and Rho- (negative) cells could be discriminated. Sorted Rho- cells were subjected to a second Rho staining procedure with the P-glycoprotein blocking agent verapamil (VP). Most cells became Rho positive [Rho-/Rho(VP)+ cells] and some remained Rho negative [Rho-/Rho(VP)- cells]. These cell fractions were characterized by their marrow-repopulating ability in a syngeneic, sex-mismatch transplantation model. Short-term repopulating ability was determined by recipient survival for at least 6 weeks after lethal irradiation and transplantation--i.e., radioprotection. Long-term repopulating ability at 6 months after transplantation was measured by fluorescence in situ hybridization with a Y-chromosome-specific probe, by graft function and recipient survival. Marrow-repopulating cells were mainly present in the small Rho- cell fraction. Transplantation of 30 Rho- cells resulted in 50% radioprotection and > 80% donor repopulation in marrow, spleen, and thymus 6 months after transplantation. Cotransplantation of cells from both fractions in individual mice directly showed that within this Rho- cell fraction, the Rho-/Rho(VP)+ cells exhibited mainly short-term and the Rho-/Rho(VP)- cells exhibited mainly long-term repopulating ability. Our results indicate that hematopoietic stem cells have relatively high P-glycoprotein expression and that the cells responsible for long-term repopulating ability can be separated from cells exhibiting short-term repopulating ability, probably by a reduced mitochondrial Rho-binding capacity.

Specific induction of cAMP in Langerhans cells by calcitonin gene-related peptide: relevance to functional effects.

Epidermal Langerhans cells (LC) are associated anatomically with epidermal nerves, and a product of these nerves, calcitonin gene-related peptide (CGRP), inhibits the antigen-presenting capacity of LC and macrophages. As the CGRP receptor appears to be coupled to Gs alpha protein, which in turn activates adenylate cyclase, the ability of CGRP to induce cAMP in LC was examined and correlated with functional effects. LC were isolated from murine epidermal cells using antibodies on magnetic microspheres. Exposure to CGRP induced a significant increase in cAMP content, which could be inhibited by coculture with a truncated form of CGRP [CGRP-(8-37)] that is a specific competitive inhibitor of CGRP. Substance P and calcitonin failed to induce cAMP in LC. Although culture in CGRP reduced the ability of murine epidermal cells enriched for LC content to present pigeon cytochrome c to a responsive clone or to present antigen for elicitation of delayed-type hypersensitivity in immune mice, culture in forskolin had little or no effect on antigen presentation despite increased cAMP content of LC as much or more than that induced by CGRP. The effect of CGRP on antigen presentation in these systems could be blocked with CGRP-(8-37). CGRP inhibited the induction of B7-2 by lipopolysaccharide on peritoneal macrophages and a LC line, whereas calcitonin did not. CGRP induces specific accumulation of cAMP in LC and inhibits LC antigen-presenting function by a receptor-mediated event. However, the induction of cAMP by itself does not account for inhibition of antigen presentation. Suppression of the expression of B7-2 may be one mechanism by which CGRP inhibits antigen presentation.

Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase.

We have developed a strategy to generate mutant genes in mammalian cells in a conditional manner by employing a fusion protein, Cre-ER, consisting of the loxP site-specific Cre recombinase linked to the ligand-binding domain of the human estrogen receptor. We have established homozygous retinoid X receptor alpha-negative (RXR alpha-/-) F9 embryonal carcinoma cells constitutively expressing Cre-ER and have shown that estradiol or the estrogen agonist/antagonist 4-hydroxytamoxifen efficiently induced the recombinase activity, whereas no activity was detected in the absence of ligand or in the presence of the antiestrogen ICI 164,384. Furthermore, using a targeting vector containing a selection marker flanked by loxP sites, we have inactivated one retinoic acid receptor alpha allele in such a line, demonstrating that the presence of the recombinase does not inhibit homologous recombination. Combining this conditional site-specific recombination system with tissue-specific expression of Cre-ER may allow modification of the mammalian genome in vivo in a spatiotemporally regulated manner.

A catalytic mechanism for the dual-specific phosphatases.

Dual-specific protein-tyrosine phosphatases have the common active-site sequence motif HCXXGXXRS(T). The role of the conserved hydroxyl was investigated by changing serine-131 to an alanine (S131A) in the dual-specific protein-tyrosine phosphatase VHR. The pH profile of the kcat/Km value for the S131A mutant is indistinguishable from that of the native enzyme. In contrast, the kcat value for S131A mutant is 100-fold lower than that for the native enzyme, and the shape of the pH profile was perturbed from bell-shaped in the native enzyme to a pH-independent curve over the pH range 4.5-9.0. This evidence, along with results from a previous study, suggests that the S131A mutation alters the rate-limiting step in the catalytic mechanism. Formation of a phosphoenzyme intermediate appears to be rate-limiting with the native enzyme, whereas in the S131A mutant breakdown of the intermediate is rate-limiting. This was confirmed by the appearance of a burst of p-nitrophenol formation when p-nitrophenyl phosphate rapidly reacted with the S131A enzyme in a stopped-flow spectrophotometer. Loss of this hydroxyl group at the active site dramatically diminished the ability of the enzyme to hydrolyze the thiol-phosphate intermediate without exerting any significant change in the steps leading to and including the formation of the intermediate. Consistent with rate-limiting intermediate formation in the native enzyme, the rate of burst in the S131A mutant was 1.5 s-1, which agrees well with the kcat value of 5 s-1 observed for native enzyme. The amplitude of the burst was stoichiometric with final enzyme concentration, and the slow linear rate (0.06 s-1) of p-nitrophenol formation after the burst was in agreement with the steady-state determined value of kcat (0.055 s-1).