Pulling strings below the surface: hormone receptor signaling through inhibition of protein tyrosine phosphatases.

Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important role as therapeutic agents. Most hormone receptors signal through a mechanism that involves phosphorylation of the receptor's tyrosine residues. At any given moment, the receptor's phosphorylation state depends on the balance of kinase and phosphatase activities. Recent findings point to the exciting possibility that receptor signaling can be regulated by inhibition of protein tyrosine phosphatases (PTPs) that specifically hydrolyze receptor tyrosine-phosphates, or their immediate downstream effectors. This strategy has now been firmly validated for the insulin receptor and PTP1B; inhibiting PTP1B activity results in stimulation of the insulin receptor and signaling, even in the absence of insulin. This and similar findings suggest that PTP inhibitors have potential as hormone mimetics. In the present review, we outline this new paradigm for therapeutic regulation of the insulin receptor and discuss evidence that hints at other specific receptor-PTP pairs.

Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting.

Studies of protein-protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so-called "substrate trapping" mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome-wide approach to probe for candidate substrates in cellular extracts that were separated by two-dimensional (2-D) gel electrophoresis and blotted onto membranes. Protein-protein interactions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein-protein interactions.

Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and myositis-specific autoantibodies.

In the human inflammatory myopathies (polymyositis and dermatomyositis), the early, widespread appearance of MHC class I on the surface of muscle cells and the occurrence of certain myositis-specific autoantibodies are striking features. We have used a controllable muscle-specific promoter system to up-regulate MHC class I in the skeletal muscles of young mice. These mice develop clinical, biochemical, histological, and immunological features very similar to human myositis. The disease is inflammatory, limited to skeletal muscles, self-sustaining, more severe in females, and often accompanied by autoantibodies, including, in some mice, autoantibodies to histidyl-tRNA synthetase, the most common specificity found in the spontaneous human disease, anti-Jo-1. This model suggests that an autoimmune disease may unfold in a highly specific pattern as the consequence of an apparently nonspecific event-the sustained up-regulation of MHC class I in a tissue-and that the specificity of the autoantibodies derives not from the specificity of the stimulus, but from the context, location, and probably the duration of the stimulus. This model further suggests that the presumed order of events as an autoimmune disease develops needs to be reconsidered.

MetaBlasts: tracing protein tyrosine phosphatase gene family roots from Man to Drosophila melanogaster and Caenorhabditis elegans genomes.

At increasing speed, sequencing data are being made public from both complex and simple life forms. Although biomedical interests tend to focus on mammalian genes, only simple organisms allow rapid genetic manipulation and functional analysis. A prerequisite for the meaningful extrapolation of gene functional studies from invertebrates to man is that the orthologs under study are unambiguously linked. However, identifying orthologs is not trivial, especially where large gene families are involved. We present here an automated sequence analysis procedure that allows a rapid visualization of most likely ortholog pairs. We illustrate the utility of this approach for the human gene family of protein tyrosine phosphatases (PTPs) as compared with the full set of Caenorhabditis elegans and Drosophila melanogaster conceptual ORFs. The approach is based on a reciprocal series of BLAST searches, which are automatically stored and represented in an HTML-formatted table. We have used this 'MetaBlast' approach to compile lists of human PTPs and their worm and fly orthologs. Many of these PTP orthologs had not been previously identified as such.

Identification of tyrosine phosphatases that dephosphorylate the insulin receptor. A brute force approach based on "substrate-trapping" mutants.

Many pharmacologically important receptors, including all cytokine receptors, signal via tyrosine (auto)phosphorylation, followed by resetting to their original state through the action of protein tyrosine phosphatases (PTPs). Establishing the specificity of PTPs for receptor substrates is critical both for understanding how signaling is regulated and for the development of specific PTP inhibitors that act as ligand mimetics. We have set up a systematic approach for finding PTPs that are specific for a receptor and have validated this approach with the insulin receptor kinase. We have tested nearly all known human PTPs (45) in a membrane binding assay, using "substrate-trapping" PTP mutants. These results, combined with secondary dephosphorylation tests, confirm and extend earlier findings that PTP-1b and T-cell PTP are physiological enzymes for the insulin receptor kinase. We demonstrate that this approach can rapidly reduce the number of PTPs that have a particular receptor or other phosphoprotein as their substrate.

ADAM 20 and 21; two novel human testis-specific membrane metalloproteases with similarity to fertilin-alpha.

Two novel membrane disintegrin-metalloproteases, ADAM 20 and ADAM 21 were cloned from a human testis cDNA library. Their predicted translation products share 50% sequence identity with each other. Among previously characterized ADAMs, the best similarity was to sperm cell-specific fertilins-alpha and -beta, and meltrin-gamma (ADAM 9) which is ubiquitously expressed. Both ADAM 20 and 21 mRNAs are exclusively expressed in testis, presumably, in analogy to all other testis-specific ADAMs, on mature spermatocytes. Both cDNAs were mapped on the genome, and found to be tightly linked to the same marker (SHGC-36001) on chromosome 14q24.1. This region is not syntenic with the loci of mouse sperm-specific ADAMs 1-5. ADAM 20, but not 21, encodes a consensus Zn2+ binding site of active adamalysin metzincin metalloproteases, and both 20 and 21 encode putative cell-fusion peptides, required for sperm-egg fusion. Based on these characteristics it is possible that ADAM 20 and/or 21 is the functional equivalent of sperm fertilin-alpha, as it was recently reported that this gene is non-functional in humans.

Highly controlled gene expression using combinations of a tissue-specific promoter, recombinant adenovirus and a tetracycline-regulatable transcription factor.

Controllable gene expression is a desirable feature both in gene therapy protocols and for the study of gene function in animals and plants. We have exploited the modular character of the tetracycline (tc)-regulatable genetic switch to show that its components can be encoded by any combination of recombinant adenovirus and/or transgenic mice. Transgenic mice were constructed that express the tc-regulatable trans-activator tTA muscle specifically. These were injected with recombinant adenovirus expressing a luciferase reporter controlled by the tTA-regulatable promoter. Virus injected into muscle, but not into a control organ (brain) resulted in luciferase activity. Conversely, injection of tTA producing adenovirus into mice that were transgenic for a trkB/Fc fusion protein gene under tc promoter control resulted in swift expression of serum trkB/Fc receptor-body. Both modes of gene induction were fully inhibited by administration of tc. We demonstrate that a careful choice of these tools allows exquisite in vivo control over transgene expression in a temporal, tc-regulatable, topical and tissue-specific manner.

Protein tyrosine phosphatases: counting the trees in the forest.

The recent identification of many different protein tyrosine phosphatases (PTPs) has led to the recognition that these enzymes match protein tyrosine kinases (PTKs) in importance for intracellular signalling. The total number of PTPs encoded by the mammalian genome has been estimated at between 500 and approx. 2000. These estimates are imprecise due to the large number of sequence database entries that represent different splice forms, or duplicates of the same PTP sequence. A careful analysis of these entries, grouped by identical catalytic domain shows that no more than 48 full-length PTP sequences are currently known, and that their total number in the human genome may not exceed 100. An alignment of all catalytic domains also suggests that during evolution intragenic catalytic domain duplication, as seen in most membrane-bound PTPs, preceded gene duplication.

Distamycin prolongs E-selectin expression by interacting with a specific NF-kappaB-HMG-I(Y) binding site in the promoter.

The E-selectin cell adhesion protein plays a critical role in mediating adherence of leukocytes to endothelium at sites of inflammation. Cytokine-induced E-selectin expression on the surface of endothelial cells is transient; mRNA expression peaks at 3-4 h after induction and returns to basal levels within 24 h. The mechanism for this transcriptional down-modulation is not known. Promoter binding factors responsible for induced gene expression include NF-kappaB, which binds at three sites within the E-selectin promoter, and HMG-I(Y), which binds to the A/T-rich core found at the centre of these binding sites. Distamycin is an antibiotic that also binds A/T-rich DNA and inhibits HMG-I(Y) DNA binding. To study the role of HMG-I(Y) in E-selectin expression, we have examined the effect of distamycin on the cytokine-induced E-selectin expression cycle. We found that distamycin prolonged E-selectin expression, both by sustaining mRNA transcription and by extending the transcript's half-life. The distamycin effect on transcription was mediated through one of the three NF-kappaB-HMG-I(Y) binding sites (NF-kappaBII) within the promoter. This suggests that the NF-kappaB-HMG-I(Y) complex interacting at the NF-kappaBII site plays a role not only in cytokine induction of E-selectin expression, but also in its down-modulation.

Common neural substrates for the addictive properties of nicotine and cocaine.

Regional brain activation was assessed by mapping of Fos-related protein expression in rats trained to self-administration of intravenous nicotine and cocaine. Both drugs produced specific overlapping patterns of activation in the shell and the core of the nucleus accumbens, medial prefrontal cortex, and medial caudate areas, but not in the amygdala. Thus, the reinforcing properties of cocaine and nicotine map on selected structures of the terminal fields of the mesocorticolimbic dopamine system, supporting the idea that common substrates for these addictive drugs exist.

Bi-directional gene switching with the tetracycline repressor and a novel tetracycline antagonist.

We have screened a panel of tetracycline (tc)-like compounds for their potential use with tc-repressor (tetR) based gene switches. The interaction between tc and tetR appears quite specific, as only tc itself and its close homologues anhydro-tc and doxycycline strongly inhibited DNA binding. However, a single tc-like compound, GR33076X, increased DNA binding of the tetR-VP16 fusion protein, both in eukaryotic cells and in bacteria. We provide evidence that this antagonist of tetracycline is potentially useful for accelerated gene switching, especially in whole animals.

Commonly used cat reporter vectors contain a cAMP-inducible, cryptic enhancer that co-operates with NF-kappa B-sites.

Commercially available and widely used cat expression vectors were found to contain a forskolin (Fs)-inducible element capable of co-operation with NF-kappa B-sites in test promoters. An alternative NF-kappa B-dependent reporter system is presented that allows investigation of the effects of Fs and other agents that augment intracellular cyclic AMP.

Transcription factor-induced, phased bending of the E-selectin promoter.

E-selectin is an endothelial adhesion molecule that is critically involved in neutrophil adhesion and recruitment. All DNA elements required for interleukin-1 inducibility have been located in the proximal promoter: an NF-ELAM1/ATF site, two NF-kappa B sites (I and II), the NF-ELAM2 element and a TATA box. We show here that interleukin-1 induced promoter activity is exquisitely sensitive to the spatial arrangements of these elements. Phasing of the ATF and NF-kappa B II elements indicates that their relative helix orientation is more important than distance per se. This sensitivity is partly due to a requirement for correctly oriented, transcription factor-induced DNA-bending. (i) Band shift analyses with permuted ATF- and NF-kappa B elements show that their associated factors all bend DNA. (ii) One can functionally replace the NF-ELAM1/ATF element by a subset of a panel of DNA fragments that contain defined bends in various planes. We conclude that the main role of the factors binding at the NF-ELAM1/ATF element is to alter the conformation of the E-selectin promoter, presumably looping distant enhancer elements into each other's proximity.

Inhibition of E-selectin gene transcription through a cAMP-dependent protein kinase pathway.

Cytokines induce the expression of E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVECs). We show that expression of these surface proteins is differently affected by cAMP. Increased cAMP levels decrease E-selectin and VCAM-1 but increase ICAM-1 expression. We demonstrate by mRNA half-life analysis and nuclear run-on assays that the cAMP repression of E-selectin occurs at the transcription level. This effect is abolished by protein kinase A inhibition, suggesting that repression is mediated by protein kinase A-driven phosphorylation. We found that a minimal E-selectin promoter sequence necessary to confer cytokine inducibility is also sufficient to mimic the cAMP effect in transfected HUVECs. Previously we characterized two regions (NF-kappa B and NF-ELAM1) of the minimal promoter that bind transcription factors necessary for E-selectin induction, Increased cAMP did not alter the binding of the complexes formed on either the NF-kappa B or NF-ELAM1 site. In contrast, in interleukin-1-treated HUVECs transactivity due to an NF-kappa B site is reduced by elevated cAMP. Increased cAMP in HUVECs appears to induce a protein kinase activity that reduces the cytokine signal for E-selectin and VCAM-1 expression. The reduction in signal may occur through an inhibitory phosphorylation of one or more of the factors responsible for regulating E-selectin expression.

Inducible inhibition of eukaryotic gene expression.

A regulatable binary expression system for eukaryotes was recently developed based on the tetracycline repressor and its operator. Here we show that this system can be successfully applied to express antisense RNA and completely inhibit gene expression in a tetracycline-repressible fashion.

RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers.

Members of the superfamily of nuclear receptors share the greatest homology in their DNA-binding domains. We have used reverse transcription-polymerase chain reaction and highly degenerate primers based on the amino acid sequence of the zinc finger motif of known nuclear receptors to identify novel members of the family. Starting with rat brain RNA, we have isolated an orphan receptor that we call RZR beta. The sequence of its nearly full-length complementary DNA shows great similarity to RZR alpha, a receptor we recently identified from human umbilical vein endothelial cells. These RZR subtypes represent members of a new family of orphan nuclear receptors that most likely regulate specific gene expression. Sequence comparison with other known nuclear receptors reveals great similarity for both RZR subtypes to retinoic acid and retinoid-X receptors. By Northern blot analyses, we found RZR beta messenger RNA only in brain, whereas RZR alpha is expressed in many tissues. We show here that the RZRs bind as monomers to natural retinoid response elements formed by (A/G)GGTCA half-sites. However, a T-residue in the -1 position of this motif greatly enhances the DNA binding affinity of RZRs, whereas the -2 position has no influence. We show that RZRs can bind as homodimers on response elements formed by palindromes, inverted palindromes, or direct repeats of two TAGGTCA half-sites. Interestingly, these response elements display dramatically reduced affinity for retinoic acid receptor-retinoid-X receptor heterodimers. Thus, the 5'-flanking sequence of hexameric half-sites appears to be crucial to direct the activity of several nuclear receptors. On monomeric as well as dimeric binding sites, RZRs show constitutive transactivational activity that can be enhanced by unidentified components of fetal calf serum.

ATF-a0, a novel variant of the ATF/CREB transcription factor family, forms a dominant transcription inhibitor in ATF-a heterodimers.

We have isolated a cDNA encoding a variant of the transcription factor ATF-a (called ATF-a0) by screening a HeLa cDNA expression library with a regulatory element of the E-selectin promoter, NF-ELAM1/delta A. Relative to full-length ATF-a, the ATF-a0 cDNA contains a large in-frame deletion of 525 base pairs that removes the P/S/T-rich putative transactivation domain. Using reverse-transcription-polymerase chain reaction and Northern blot hybridization to characterize ATF-a0 expression, we found that putative mRNAs for ATF-a0 and ATF-a are present at varying ratios in different tissues. Full-length ATF-a is a transcriptional activator for the NF-ELAM1/delta A site of the E-selectin promoter. In contrast, we show ATF-a0 has no measurable transactivating function on this element. Moreover, we demonstrate that co-expressed ATP-a0 and ATF-a preferentially heterodimerize. In the heterodimer ATF-a0 is a dominant inhibitor that completely blocks the transactivating activity of ATF-a. Both forms of ATF-a bind the p50 subunit of NF-kappa B as shown by affinity chromatography. ATF-a0 appears to be a splice variant similar to the one found for ATF-2, its closest homologue in structure and function. Taken together, our results suggest that ATF-a0 is an important member of the ATF family with a negative regulatory role in transactivation.

Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines.

We previously reported that NF-kappa B and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-kappa B in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-kappa B to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-kappa B is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

Two distinct NF-kappa B complexes differing in their larger subunit bind the E-selectin promoter kappa B element.

We have investigated the proteins binding the E-selectin promoter NF-kappa B element in its natural DNA context, using probes extending beyond the NF-kappa B recognition decamer. In band shift assays, we detected two distinct NF-kappa B complexes using nuclear extracts from several cytokine-induced cells. Subunit-specific antisera as blockers of complex formation plus DNA-protein cross-linking experiments revealed the faster migrating form to contain the NF-kappa B p50 plus p65 subunits. In contrast, the slower migrating form is composed of p50 plus the p65-related p75 protein. We show as the crucial determinant in generation of the larger complex the presence of more than five basepairs extra DNA sequence downstream of the NF-kappa B-site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact kappa B binding site is. This may be explained by a requirement for activated p50 as part of this complex. The potential for a regulatory role for the p75 containing complex on the E-selectin promoter is discussed.