Abstract: Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.
Abstract: Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase with diverse functions in neuronal development and adult brain physiology. Higher than normal levels of DYRK1A are associated with the pathology of neurodegenerative diseases and have been implicated in some neurobiological alterations of Down syndrome, such as mental retardation. It is therefore important to understand the molecular mechanisms that control the activity of DYRK1A. Here we review the current knowledge about the initial self-activation of DYRK1A by tyrosine autophosphorylation and propose that this mechanism presents an ancestral feature of the CMGC group of kinases. However, tyrosine phosphorylation does not appear to regulate the enzymatic activity of DYRK1A. Control of DYRK1A may take place on the level of gene expression, interaction with regulatory proteins and regulated nuclear translocation. Finally, we compare the properties of small molecule inhibitors that target DYRK1A and evaluate their potential application and limitations. The β-carboline alkaloid harmine is currently the most selective and potent inhibitor of DYRK1A and has proven very useful in cellular assays.
Abstract: Although numerous non-radioactive methods are in use to measure the catalytic activity of protein kinases, most require specialized equipment and reagents and are not sufficiently sensitive for the detection of endogenous kinase activity in biological samples. Kinases of the DYRK family have important functions in developmental and pathophysiological processes in eukaryotic organisms including mammals. We aimed to develop a highly sensitive, low-tech assay suitable to determine the activity of DYRK family kinases in tissues or cells from diverse sources.
Abstract: Leptin is an adipocyte-derived hormone that acts via its hypothalamic receptor (LEPRb) to regulate energy balance. A downstream effect essential for the weight-regulatory action of leptin is the phosphorylation and activation of the latent transcription factor STAT3 by LEPRb-associated Janus kinases (JAKs). Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance). Here we have studied the roles of the intracellular tyrosine residues in the negative feedback regulation of LEPRb-signaling under chronic leptin stimulation.
Abstract: DYRK1A is a dual-specificity protein kinase that autophosphorylates a conserved tyrosine residue in the activation loop but phosphorylates exogenous substrates only at serine or threonine residues. Tyrosine autophosphorylation of DYRKs is a one-off event that takes place during translation and induces the activation of the kinase. Here we characterize the beta-carboline alkaloid harmine as a potent and specific inhibitor of DYRK1A both in vitro and in cultured cells. Comparative in vitro assays of four kinases of the DYRK family showed that harmine inhibited substrate phosphorylation by DYRK1A more potently than it inhibited substrate phosphorylation by the closely related kinase DYRK1B [half maximal inhibitory concentrations (IC(50)) of 33 nm versus 166 nm, respectively] and by the more distant members of the family, DYRK2 and DYRK4 (1.9 microm and 80 microm, respectively). Much higher concentrations of harmine were required to suppress tyrosine autophosphorylation of the translational intermediate of DYRK1A in a bacterial in vitro translation system (IC(50) = 1.9 microm). Importantly, harmine inhibited the phosphorylation of a specific substrate by DYRK1A in cultured cells with a potency similar to that observed in vitro (IC(50) = 48 nm), without negative effects on the viability of the cells. Overexpression of the DYRK1A gene on chromosome 21 has been implicated in the altered neuronal development observed in Down syndrome. Here, we show that harmine interferes with neuritogenesis in cultured hippocampal neurons. In summary, our data show that harmine inhibits DYRK1A substrate phosphorylation more potently than it inhibits tyrosine autophosphorylation, and provide evidence for a role of DYRK1A in the regulation of neurite formation.
Abstract: While in human and rodents lipogenesis occurs predominantly in the liver, adipose tissue is the major site in ruminants. Here we report the nucleotide sequence of the 5'-flanking region of the ovine malic enzyme gene (ME1). The ME1 promoter is located within a GC-rich region fulfilling the criteria of CpG islands and lacks a TATA-box. Deletion analysis identified a region (-231/-170) that suppressed promoter activity in luciferase assays in HepG2 hepatoma cells but not in 3T3-L1 adipocytes. This region contains a putative triiodothyronine response element (T3RE) that differs from the human ME1 T3RE by two nucleotides. When the human ME1 T3RE was introduced into the ovine ME1 promoter context, transcriptional activity was increased in the hepatic cell lines HepG2 and H4IIE but not in differentiated 3T3-L1 cells. Our results suggest that the sequence of the T3RE in the ME1 promoter determines differences in the tissue/species activity of malic enzyme in ruminants and human.
Abstract: The dual-specific kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) is the mammalian orthologue of the Drosophila minibrain (MNB) protein kinase and executes diverse roles in neuronal development and adult brain physiology. DYRK1A is overexpressed in Down syndrome (DS) and has recently been implicated in several neurodegenerative diseases. In an attempt to elucidate the molecular basis of its involvement in cognitive and neurodegeneration processes, we searched for novel proteins interacting with the kinase domain of DYRK1A in the adult mouse brain and identified septin 4 (SEPT4, also known as Pnutl2/CDCrel-2). SEPT4 is a member of the group III septin family of guanosine triphosphate hydrolases (GTPases), which has previously been found in neurofibrillary tangles of Alzheimer disease brains and in alpha-synuclein-positive cytoplasmic inclusions in Parkinson disease brains. In transfected mammalian cells, DYRK1A specifically interacts with and phosphorylates SEPT4. Phosphorylation of SEPT4 by DYRK1A was inhibited by harmine, which has recently been identified as the most specific inhibitor of DYRK1A. In support of a physiological relation in the brain, we found that Dyrk1A and Sept4 are co-expressed and co-localized in neocortical neurons. These findings suggest that SEPT4 is a substrate of DYRK1A kinase and thus provide a possible link for the involvement of DYRK1A in neurodegenerative processes and in DS neuropathologies.
Abstract: BACKGROUND: Overexpression of the human DYRK1A gene due to the presence of a third gene copy in trisomy 21 is thought to play a role in the pathogenesis of Down syndrome. The observation of gene dosage effects in transgenic mouse models implies that subtle changes in expression levels can affect the correct function of the DYRK1A gene product. We have therefore characterized the promoter of the human DYRK1A gene in order to study its transcriptional regulation. RESULTS: Transcription start sites of the human DYRK1A gene are distributed over 800 bp within a region previously identified as an unmethylated CpG island. We have identified a new alternative noncoding 5'-exon of the DYRK1A gene which is located 772 bp upstream of the previously described transcription start site. Transcription of the two splicing variants is controlled by non-overlapping promoter regions that can independently drive reporter gene expression. We found no evidence of cell- or tissue-specific promoter usage, but the two promoter regions differed in their activity and their regulation. The sequence upstream of exon 1A (promoter region A) induced about 10-fold higher reporter gene activity than the sequence upstream of exon 1B (promoter region B). Overexpression of the transcription factor E2F1 increased DYRK1A mRNA levels in Saos2 and Phoenix cells and enhanced the activity of promoter region B three- to fourfold. CONCLUSION: The identification of two alternatively spliced transcripts whose transcription is initiated from differentially regulated promoters regions indicates that the expression of the DYRK1A gene is subject to complex control mechanisms. The regulatory effect of E2F1 suggests that DYRK1A may play a role in cell cycle regulation or apoptosis.
Abstract: BACKGROUND: Leptin acts not only on hypothalamic centers to control food intake but has additional functions in peripheral tissues, e.g. inhibition of insulin secretion from pancreatic islets. The leptin receptor (LEPRb) is a class I cytokine receptor that mediates activation of STAT transcription factors. In this study, we characterise the regulation of inflammation-related genes by leptin in insulinoma cells and compare the effect of transcriptional regulation by leptin with that of other cytokines. RESULTS: We have used RINm5F insulinoma cells as a model system for a peripheral target cell of leptin. Six transcripts encoding inflammation-related proteins were found to be upregulated by activation of LEPRb, namely lipocalin-2, pancreatitis-associated protein, preprotachykinin-1, fibrinogen-beta, tissue-type plasminogen activator (tPA) and manganese-dependent superoxide dismutase (MnSOD). Four of these transcripts (fibrinogen-beta, lipocalin-2, tPA, MnSOD) were also induced by the proinflammatory cytokine interleukin-1beta (IL-1beta). Interferon-gamma alone had no effect on the leptin-induced transcripts but enhanced the upregulation by IL-1beta of lipocalin-2, tPA and MnSOD mRNA levels. Experiments with LEPRb point mutants revealed that the upregulation of the inflammation-related genes depended on the presence of tyrosine-1138 which mediates the activation of the transcription factors STAT1 and STAT3. Reporter gene assays showed that leptin induced the expression of preprotachykinin-1 and lipocalin-2 on the level of promoter regulation. Finally, leptin treatment increased caspase 3-like proteolytic activity in RINm5F cells. CONCLUSION: The present data show that leptin induces a cytokine-like transcriptional response in RINm5F cells, consistent with the proposed function of leptin as a modulator of immune and inflammatory responses.
Abstract: Cyclin L1 and cyclin L2 are two closely related members of the cyclin family that contain C-terminal arginine- and serine-rich (RS) domains and are localized in the splicing factor compartment (nuclear speckles). Here we applied photobleaching techniques to show that a green fluorescent protein (GFP) fusion protein of cyclin L1, in contrast to cyclin L2, was not mobile within the nucleus of living COS7 cells. The objectives of this study were to 1) characterize the intranuclear localization and mobility properties of cyclin L1 in different cellular states, and 2) dissect the structural elements required for immobilization of cyclin L1. Transcriptional arrest by actinomycin D caused accumulation of GFP-cyclin L2 in rounded and enlarged nuclear speckles but did not affect the subnuclear pattern of distribution of GFP-cyclin L1. Although immobile in most phases of the cell cycle, GFP-cyclin L1 was diffusely distributed and highly mobile in the cytoplasm of metaphase cells. By analysis of a series of chimeras, deletion constructs, and a point mutant, a segment within the RS domain of cyclin L1 was identified to be necessary for the immobility of the protein in nuclear speckles. This study provides the first characterization of an immobile component of nuclear speckles.
Abstract: BACKGROUND: The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells. RESULTS: Overexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo-phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro. Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1. CONCLUSION: The present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing.
Abstract: Hypothalamic leptin receptor signalling plays a central role in weight regulation by controlling fat storage and energy expenditure. In addition, leptin also has direct effects on peripheral cell types involved in regulation of diverse body functions including immune response, bone formation and reproduction. Previous studies have demonstrated the important role of SOCS3 (suppressor of cytokine signalling 3) in leptin physiology. Here, we show that CIS (cytokine-inducible SH2 protein) and SOCS2 can also interact with the leptin receptor. Using MAPPIT (mammalian protein-protein interaction trap), a cytokine receptor-based two-hybrid method operating in intact cells, we show specific binding of CIS with the conserved Y985 and Y1077 motifs in the cytosolic domain of the leptin receptor. SOCS2 only interacts with the Y1077 motif, but with higher binding affinity and can interfere with CIS and STAT5a prey recruitment at this site. Furthermore, although SOCS2 does not associate with Y985 of the leptin receptor, we find that SOCS2 can block interaction of CIS with this position. This unexpected interference can be explained by the direct binding of SOCS2 on the CIS SOCS box, whereby elongin B/C recruitment is crucial to suppress CIS activity.
Abstract: The leptin receptor (LEPR) is a class I cytokine receptor signalling via both the janus kinase/signal transducer and activator of transcription (JAK/STAT) and the MAP kinase pathways. In addition, leptin has been shown previously to activate AMP-activated kinase (AMPK) in skeletal muscle. To enable a detailed analysis of leptin signalling in pancreatic beta cells, LEPR point mutants with single or combined exchanges of the three intracellular tyrosines were expressed in HIT-T15 insulinoma cells. Western blots with activation state-specific antibodies recognizing specific signalling molecules revealed that the wild-type receptor activated STAT1, STAT3, STAT5 and ERK1/2 but failed to alter the phosphorylation of AMPK. Each of the three intracellular tyrosine residues in LEPR exhibited different signalling capacities: Tyr985 was necessary and sufficient for leptin-induced activation of ERK1/2; Tyr1077 induced tyrosyl phosphorylation of STAT5; and Tyr1138 was capable of activating STAT1, STAT3 and STAT5. Consistent results were obtained in reporter gene assays with STAT3 or STAT5-responsive promoter constructs, respectively. Furthermore, the sequence motifs surrounding the three tyrosine residues are conserved in LEPR from mammals, birds and in a LEPR-like cytokine receptor from pufferfish. Mutational analysis of the box3 motif around Tyr1138 identified Met1139 and Gln1141 as important determinants that define specificity towards the different STAT factors. These data indicate that all three conserved tyrosines are involved in LEPR function and define the pleiotropy of signal transduction via STAT1/3, STAT5 or ERK kinases. Activation and inhibition of AMPK appears to require additional components of the signalling pathways that are not present in beta cells.
Abstract: Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is involved in a variety of biological functions. It is essential for the signal transduction of interleukin-6 (IL-6) and related cytokines. In response to IL-6 stimulation STAT3 becomes phosphorylated and translocates into the nucleus where it binds to enhancer sequences of target genes. We found that activated STAT3 is enriched in dot-like structures within the nucleus, which we termed STAT3 nuclear bodies. To examine the dynamics of STAT3 nuclear body formation, a fusion protein of STAT3 and yellow fluorescent protein (YFP) was constructed. Studies in living cells have shown that the appearance of STAT3 nuclear bodies is transient, correlating with the timecourse of tyrosine-phosphorylation of STAT3. Furthermore, we show by fluorescence recovery after photobleaching (FRAP) analysis that STAT3 within nuclear bodies consists of a highly mobile and an immobile fraction. Colocalization studies provided evidence that these bodies are accompanied with CREB binding protein (CBP) and acetylated histone H4, which are markers for transcriptionally active chromatin. Moreover, STAT3 nuclear bodies in HepG2 cells are not colocalized with promyelocytic leukemia oncoprotein (PML)-containing bodies; neither is a sumoylation of activated STAT3 detectable. Taken together, our data suggest that STAT3 nuclear bodies are either directly involved in active gene transcription or they serve as reservoirs of activated STAT3.
Abstract: A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.
Abstract: New Zealand obese (NZO) mice exhibit severe insulin resistance of hepatic glucose metabolism. In order to define its biochemical basis, we studied the differential expression of genes involved in hepatic glucose and lipid metabolism by microarray analysis. NZOxF1 (SJLxNZO) backcross mice were generated in order to obtain populations with heterogeneous metabolism but comparable genetic background. In these backcross mice, groups of controls (normoglycemic/normoinsulinemic), insulin-resistant (normoglycemic/hyperinsulinemic) and diabetic (hyperglycemic/hypoinsulinemic) mice were identified. At 22 weeks, mRNA was isolated from liver, converted to cDNA, and used for screening of two types of cDNA arrays (high-density filter arrays and Affymetrix oligonucleotide microarrays). Differential gene expression was ascertained and assessed by Northern blotting. The data indicate that hyperinsulinemia in the NZO mouse is associated with: (i) increased mRNA levels of enzymes involved in lipid synthesis (fatty acid synthase, malic enzyme, stearoyl-CoA desaturase) or fatty acid oxidation (cytochrome P450 4A14, ketoacyl-CoA thiolase, acyl-CoA oxidase), (ii) induction of the key glycolytic enzyme pyruvate kinase, and (iii) increased mRNA levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase. These effects were enhanced by a high-fat diet. In conclusion, the pattern of gene expression in insulin-resistant NZO mice appears to reflect a dissociation of the effects of insulin on genes involved in glucose and lipid metabolism. The data are consistent with a hypothetical scenario in which an insulin-resistant hepatic glucose production produces hyperinsulinemia, and an enhanced insulin- and substrate-driven lipogenesis further aggravates the deleterious insulin resistance of glucose metabolism.
Abstract: Expression of glucose-6-phosphatase (G6Pase), one of the rate-limiting enzymes of hepatic gluconeogenesis, has recently been shown to be transactivated by the transcription factor FKHR. One of the proteins known to directly interact with FKHR is the nuclear protein kinase DYRK1A. In order to study the effects of DYRK1A on G6Pase gene expression, we generated a H4IIEC3 rat hepatoma cell line stably expressing DYRK1A by retroviral infection. Overexpression of DYRK1A increased the expression of G6Pase about threefold, as determined by Northern blotting. In transiently transfected HepG2 cells, co-expression of DYRK1A and a G6Pase promoter construct increased G6Pase promoter activity about twofold. This effect of DYRK1A was independent of its kinase activity, since a kinase-dead DYRK1A mutant as well as a point mutant of the phosphorylation site of DYRK1A in FKHR (Ser329Ala) failed to affect the effect of DYRK1A on the G6Pase expression. The effect of DYRK on the G6Pase promoter activity was produced by the isoforms DYRK1A and DYRK1B, which are localized in the nucleus, but not by DYRK2. Mutations of the FKHR-binding sites in the G6Pase promoter markedly reduced the effect of DYRK1 on the G6Pase promoter activity. In summary, the data suggest that DYRK1 is a specific co-activator of FKHR, independent of its kinase activity.
Abstract: The dual specificity tyrosine phosphorylated and regulated kinase (DYRK) family of protein kinases is a group of evolutionarily conserved protein kinases that have been characterized as regulators of growth and development in mammals, Drosophila and lower eukaryotes. In the present study, we have characterized three splicing variants of DYRK1B (DYRK1B-p65, DYRK1B-p69 and DYRK1B-p75) with different expression patterns and enzymic activities. DYRK1B-p65 and DYRK1B-p69 exhibited similar, but not identical, patterns of expression in mouse tissues, with the highest protein levels found in the spleen, lung, brain, bladder, stomach and testis. In contrast, DYRK1B-p75 was detected specifically in skeletal muscles, in the neuronal cell line GT1-7 and also in differentiated, adipocyte-like 3T3-L1 cells, but not in undifferentiated 3T3-L1 preadipocytes. A comparison of the mouse and human Dyrk1b genomic and cDNA sequences defined the alternative splicing events that produce the variants of DYRK1B. In DYRK1B-p75, transcription starts with exon 1B instead of exon 1A, generating a new translation start, which extends the open reading frame by 60 codons. This gene structure suggests that alternative promoters direct the expression of DYRK1B-p69 and DYRK1B-p75. Both splicing variants exhibited kinase activity in vitro and contained phosphotyrosine when expressed in COS-7 cells. Owing to differential recognition of the 3'-splice site in exon 9, DYRK1B-p65 differs from DYRK1B-p69 by the absence of 40 amino acids within the catalytic domain. DYRK1B-p65 lacked kinase activity in vitro and did not contain phosphotyrosine. DYRK1B-p69 and DYRK1B-p75 stimulated reporter gene activity driven by the f or kh ead in r habdosarcoma (FKHR)-dependent glucose-6-phosphatase promoter more strongly when compared with DYRK1B-p65, indicating that the DYRK1B splicing variants exhibit functional differences.
Abstract: Protein kinases of the DYRK (dual-specificity tyrosine phosphorylation-regulated kinase) family require phosphorylation of a conserved tyrosine residue in the activation loop for full activity. Here we have characterized the role of conserved amino acids that are located in the vicinity of the phosphorylated tyrosine in DYRK1A (Tyr-321). Mutation of Gln-323, but not Asn-365 or Glu-366, to either alanine, glutamate, or asparagine reduced the in vitro-kinase activity of DYRK1A towards the peptide substrate, DYRKtide, to a similar degree (15-37% of wild type) as the mutation of the phosphorylation site itself (Y321F). Similarly, the in vivo-kinase activity of DYRK1A-Q323N and of DYRK1A-Y321F, as assessed by Ser-727 phosphorylation of signal transducer and activator of transcription 3 (STAT3) in COS-7 cells, was markedly reduced in comparison with wild type DYRK1A. These data show that the contribution of Gln-323 and Tyr-321 to the full catalytic activity of DYRK1A is a specific functional characteristic of the DYRK family.
Abstract: Two predominant splice variants of the leptin receptor (LEPR) are coexpressed in leptin-responsive tissues: the long form, LEPRb, characterized as the signal-transducing receptor, and the signaling-defective short form, LEPRa. It is unknown whether heterodimers of these isoforms are capable of signal transduction via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. To address this question, chimeric receptors were constructed consisting of the transmembrane and intracellular parts of LEPRb and LEPRa fused with the extracellular domains of either the alpha- or beta-subunit of the IL-5 receptor. This strategy allows the directed heterodimerization of different LEPR cytoplasmic tails and excludes homodimerization. In COS-7 and HEPG2 cells, chimeric receptor heterodimers of LEPRa and LEPRb failed to activate the JAK/STAT pathway, whereas receptor dimers of LEPRb gave rise to the expected ligand-dependent activation of JAK2, phosphorylation of STAT3, and STAT3-dependent promoter activity. Markedly lower amounts of JAK2 were found to be associated with immunoprecipitated LEPRa chimeras than with LEPRb chimeras. Analysis of a series of deletion constructs indicated that a segment of 15 amino acids in addition to the 29 amino acids common to LEPRa and LEPRb was required for partial restoration of JAK/STAT activation. Site-directed mutagenesis of the critical sequence indicated that two hydrophobic residues (Leu896, Phe897) not present in LEPRa were indispensable for receptor signaling. These findings show that LEPRa/LEPRb heterodimers cannot activate STAT3 and identify sequence elements within the LEPR that are critical for the activation of JAK2 and STAT3.
Abstract: In subgroups of a New Zealand obese mouse-derived backcross population with defined aberrations of glucose homeostasis, a comprehensive study of the hepatic expression of cytochrome P450 and glutathione S-transferase was performed. Three patterns of alterations in response to insulin resistance (normoglycemia/hyperinsulinemia) or diabetes (hyperglycemia/hypoinsulinemia) were observed: mRNA levels of Cyp2b9, Cyp3a16, Cyp4a14, and Gstt2 as assessed by Northern- and dot-blot analysis were increased markedly in liver from diabetic mice with no or only a slight increase in insulin resistant mice. Western-blot analysis detected the corresponding changes of the CYP2B and CYP4A proteins. In contrast, expression of Cyp2c22, Cyp2c29, and Cyp2c40 was reduced in diabetic, but normal in insulin resistant mice. These alterations were correlated with changes in serum free fatty acid levels and, therefore, seem to be mediated by the peroxisome proliferator activated receptor-alpha. Furthermore, expression of Cyp1a2, Cyp7b1, Gstm3, and Gstm6 was reduced in both diabetic and insulin resistant mice. Because this third pattern was not correlated with the alterations of serum free fatty acid levels, it seems to reflect an early alteration in the course of the disease, and may be related to the progression of the syndrome from insulin resistance to the type 2-like diabetes.
Abstract: AIMS/HYPOTHESIS: The diabetes susceptibility locus Nidd/SJL was identified in an outcross of New Zealand obese (NZO) and lean Swiss/Jackson Laboratory mouse strain (SJL) mice. Here we characterise its effects in a NZO x F1(SJLxNZO) backcross population raised on high-fat or standard diet, and describe its interaction with the obesity quantitative trait locus (QTL) Nob1. METHODS: NZO x F1(SJLxNZO) backcross mice were raised on a normal or high fat diet and were monitored (body weight, blood glucose, serum insulin) for 22 weeks. Genotypes of polymorphic markers were determined by PCR, and linkage analysis was done. Pancreas morphology was assessed by conventional staining and immunohistochemistry of insulin. RESULTS: In backcross mice raised on a high-fat diet, Nidd/SJL produced hyperglycaemia (maximum likelihood of the odds (LOD) score 9.9), hypoinsulinaemia, reduction of islet-cell volume, and loss of beta cells. No effect was observed on body weight and serum insulin concentrations before the onset of hyperglycaemia. The development of diabetes in carriers of Nidd/SJL was markedly accelerated and aggravated by the obesity/hyperinsulinaemia QTL Nob1; together, these loci were responsible for approximately 90% of the diabetes observed in the backcross population. When raised on a standard diet, Nidd/SJL carriers exhibited a fivefold higher prevalence of diabetes, but Nob1 failed to enhance the effect of Nidd/SJL. CONCLUSION/INTERPRETATION: Diabetes in this obese mouse model is the result of an interaction of genes responsible for obesity/insulin resistance (e.g. Nob1) and islet cell failure ( Nidd/SJL). The combined diabetogenic effects of Nidd/SJL and Nob1 were markedly enhanced by a high-fat diet, whereas that of Nidd/SJL alone was independent of the dietary fat content.
Abstract: Protein kinases of the DYRK ('dual-specificity tyrosine-regulated kinase') family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319-Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.
Abstract: The substrate specificity of glycogen synthase kinase 3 (GSK3) is unusual in that efficient phosphorylation only occurs if another phosphoserine or phosphothreonine residue is already present four residues C-terminal to the site of GSK3 phosphorylation. One such substrate is the epsilon-subunit of rat eukaryotic protein-synthesis initiation factor 2B (eIF2Bepsilon), which is inhibited by the GSK3-catalysed phosphorylation of Ser(535). There is evidence that GSK3 is only able to phosphorylate eIF2Bepsilon at Ser(535) if Ser(539) is already phosphorylated by another protein kinase. However, no protein kinases capable of phosphorylating Ser(539) have so far been identified. Here we show that Ser(539) of eIF2Bepsilon, which is followed by proline, is phosphorylated specifically by two isoforms of dual-specificity tyrosine phosphorylated and regulated kinase (DYRK2 and DYRK1A), but only weakly or not at all by other 'proline-directed' protein kinases tested. We also establish that phosphorylation of Ser(539) permits GSK3 to phosphorylate Ser(535) in vitro and that eIF2Bepsilon is highly phosphorylated at Ser(539) in vivo. The DYRK isoforms also phosphorylate human microtubule-associated protein tau at Thr(212) in vitro, a residue that is phosphorylated in foetal tau and hyperphosphorylated in filamentous tau from Alzheimer's-disease brain. Phosphorylation of Thr(212) primes tau for phosphorylation by GSK3 at Ser(208) in vitro, suggesting a more general role for DYRK isoforms in priming phosphorylation of GSK3 substrates.
Abstract: Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr(24), Ser(256) and Ser(319)) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser(329)) as a novel in vivo phosphorylation site on FKHR. Ser(329) phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser(329) is phosphorylated in unstimulated HEK-293 cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser(329) specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser(329) in vivo.
Abstract: DYRK1A is the first member of a novel subfamily of protein kinases with dual specificity. The human gene for DYRK1A is located in the "Down syndrome critical region" (21q22.2). Due to its relationship to the Drosophila gene minibrain (Mnb), whose mutation results in specific defects in neurogenesis, and based on functional experiments on transgenic mice, DYRK1A is discussed as a candidate gene for mental retardation in Down syndrome. The kinase is characterized by its ability to catalyze tyrosine-directed autophosphorylation as well as phosphorylation of serine/threonine residues in substrates. Its exact cellular function is yet unknown. DYRK1A is, however, known to be translocated into the nucleus and supposed to be involved in the control of cell growth and development. The pathogenetic impact of DYRK1A on Down syndrome needs further elucidation.
Abstract: DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K(m) = 35 microM) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogen-activated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P -3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.
Abstract: The DYRK1A gene on human chromosome 21 encodes a protein kinase presumed to be involved in the pathogenesis of mental retardation in Down's syndrome. Here we describe a highly similar homolog, DYRK1B, which is, in contrast to DYRK1A, predominately expressed in muscle and testis. The human DYRK1B gene was mapped to chromosome 19 (19q12-13.11) by radiation hybrid analysis. The amino acid sequences of DYRK1A and DYRK1B are 84% identical in the N-terminus and the catalytic domain but show no extended sequence similarity in the C-terminal region. DYRK1B contains all motifs characteristic for the DYRK family of protein kinases. In addition, the sequence comprises a bipartite nuclear localization motif. A green fluorescent protein (GFP) fusion protein of DYRK1B was found mainly in the nucleus of transfected COS-7 cells. These data suggest that DYRK1B is a muscle- and testis-specific isoform of DYRK1A and is involved in the regulation of nuclear functions.
Abstract: Dyrk-related kinases represent a novel subfamily of protein kinases with unique structural and enzymatic features. Its members have been identified in distantly related organisms. The yeast kinase, Yak1, has been characterized as a negative regulator of growth. Mnb from Drosophila is encoded by the minibrain gene, whose mutation results in specific defects in neurogenesis. Its mammalian homolog, Dyrk1A, is activated by tyrosine phosphorylation in the activation loop between subdomains VII and VIII of the catalytic domain. The human gene for Dyrk1A is located in the "Down syndrome critical region" of chromosome 21 and is therefore a candidate gene for mental retardation in Down syndrome. More recently, six additional mammalian Dyrk-related kinases have been identified (Dyrk1B, Dyrk1C, Dyrk2, Dyrk3, Dyrk4A, and Dyrk4B). All members of the Dyrk family contain in the activation loop the tyrosines that are essential for the full activity of Dyrk1A. Outside their catalytic domains, Dyrk kinases exhibit little sequence similarity except for a small segment immediately preceding the catalytic domain (DH-box, Dyrk homology box). An unusual enzymatic property of Dyrk-related kinases is their ability to catalyze tyrosine-directed autophosphorylation as well as phosphorylation of serine/threonine residues in exogenous substrates. The exact cellular function of the Dyrk kinases is yet unknown. However, it appears reasonable to assume that they are involved in the regulation of cellular growth and/or development.
Abstract: KK obese mice exhibit a multigenic syndrome of moderate obesity, hyperinsulinemia and hyperglycemia. Here we show that the syndrome is accompanied by a marked elevation of leptin protein in adipose tissue, as well as leptin levels in serum, which corresponds with the degree of obesity. The cDNA sequence of leptin is normal in KK mice, whereas three nucleotide polymorphisms were found in the cDNA of the leptin receptor, one of them resulting in exchange of an aspartate residue for asparagine (Asp600Asn) in a highly conserved part of the second extracellular cytokine-receptor homology module. In female (but not male) F2 mice of a C57BL/6JxKK intercross, the weight of gonadal, retroperitoneal and mesenteric adipose tissue was positively correlated with the number of alleles inherited from the KK parental strain at a microsatellite marker (D4Mit175) which maps close (0.7 centimorgan proximal) to the leptin receptor gene. It is suggested that the Asp600Asn leptin receptor variant contributes to the obesity syndrome in KK female mice, but that its contribution is only a part of the multigenic syndrome.
Abstract: ADP-ribosylation factor-like protein 4 (ARL4) is a Ras-related GTPase that has been cloned from the 3T3-L1 preadipocyte cell line as an adipocyte-specific cDNA [Schürmann, Breiner, Becker, Huppertz, Kainulainen, Kentrup and Joost (1994) J. Biol. Chem. 269, 15683-15688]. The Arl4 gene maps to the proximal region of mouse chromosome 12 linked to Lamb1-1, Hfhbf1 and Sos2. Compared with all other known genes of Ras-related GTPases, the genomic organization of Arl4 is unusual in that its entire coding region, the 3' untranslated region (UTR) and most of the 5' UTR are located on a single exon. This structure suggests that Arl4 has evolved by retroposition of an Arf (ADP-ribosylation factor) or Arf-like gene. Isolation of the 5' UTR by rapid amplification of cDNA ends (RACE)-PCR revealed heterogeneous transcription initiation sites in alternative exons 1. Both 5'-flanking regions exhibited promoter activity when expressed in COS-7 cells, indicating that the expression of Arl4 is directed by two separate promoters. mRNA transcribed under the control of the downstream promoter was isolated by RACE-PCR from all investigated tissues. In contrast, the upstream promoter seems to drive specifically the expression of Arl4 in adult testis. Hybridization of rat testis in situ indicated that Arl4 is expressed in germ cells of puberal and adult testis, but not in prepuberal testis, suggesting that Arl4 is involved in sperm production.
Abstract: DYRK1 is a dual specificity protein kinase presumably involved in brain development. Here we show that the kinase belongs to a new family of protein kinases comprising at least seven mammalian isoforms (DYRK1A, DYRK1B, DYRK1C, DYRK2, DYRK3, DYRK4A, and DYRK4B), the yeast homolog Yak1p, and the Drosophila kinase minibrain (MNB). In rat tissues, DYRK1A is expressed ubiquitously, whereas transcripts for DYRK1B, DYRK2, DYRK3, and DYRK4 were detected predominantly in testes of adult but not prepuberal rats. By fluorescence microscopy and subcellular fractionation, a green fluorescent protein (GFP) fusion protein of DYRK1A was found to accumulate in the nucleus of transfected COS-7 and HEK293 cells, whereas GFP-DYRK2 was predominantly detected in the cytoplasm. DYRK1A exhibited a punctate pattern of GFP fluorescence inside the nucleus and was co-purified with the nuclear matrix. Analysis of GFP-DYRK1A deletion constructs showed that the nuclear localization of DYRK1A was mediated by its nuclear targeting signal (amino acids 105-139) but that its characteristic subnuclear distribution depended on additional N-terminal elements (amino acids 1-104). When expressed in Escherichia coli, DYRK1A, DYRK2, DYRK3, MNB, and Yak1p catalyzed their autophosphorylation on tyrosine residues. The kinases differed in their substrate specificity in that DYRK2 and DYRK3, but not DYRK1A and MNB, catalyzed phosphorylation of histone H2B. The heterogeneity of their subcellular localization and substrate specificity suggests that the kinases are involved in different cellular functions.
Abstract: New Zealand Obese (NZO) mice exhibit a polygenic syndrome of hyperphagia, obesity, hyperinsulinemia, and hyperglycemia similar to that observed in young diabetes mutant mice on the C57BLKS/J background (C57BLKS/J-Lepr(db)/Lepr(db)). Here we show that in NZO this syndrome is accompanied by a marked elevation of the leptin protein in adipose tissue and serum. The promoter region and the complementary DNA of the ob gene of NZO mice, including its 5'-untranslated region, are identical with the wild-type sequence (C57BL, BALB/c), except that the transcription start is located 5 bp upstream of the reported site. In contrast to C57BLKS/J+/+ and C57BL/6J-Lep(ob)/Lep(ob) mice, NZO mice failed to respond to recombinant leptin (7.2 microg/g) with a reduction of food intake. Leptin receptor messenger RNA as detected by PCR appears as abundant in hypothalamic tissue of NZO mice as in tissue from lean mice. Ten nucleotide polymorphisms are found in the complementary DNA of the leptin receptor, resulting in two conservative substitutions (V541I and V651I) in the extracellular part of the receptor and one nonconservative substitution (T1044I) in the intracellular domain between the presumed Jak and STAT binding boxes. However, these mutations are also present in the related lean New Zealand Black strain (body fat at 9 weeks: New Zealand Black, 6.2 +/- 1.3%; NZO, 17.0 +/- 1.7%). Thus, the polymorphic leptin receptor seems to play only a minor, if any, role in the obesity and hyperleptinemia of the NZO mouse. It is suggested that the main defect in NZO is located distal from the leptin receptor or at the level of leptin transport into the central nervous system.
Abstract: A novel ras-related GTPase with a unique structure was cloned by PCR-amplification with degenerate primers and screening of a rat fat cell cDNA library. The deduced amino acid sequence of the cDNA comprises all 6 GTP binding motifs which are conserved in Ras-related GTPases. The sequence is similar to that of ADP-ribosylation factors (ARF), and shows several structural features typical for the ARF-family. Because its closest relative is the GTPase ARL1 (49% identical amino acids, 54% identical nucleotides within the coding region), the protein was designated ARL5 (ARF-like protein 5). Low amounts of mRNA were found in most rat tissues examined (heart, skeletal muscle, fat, liver, kidney, lung, spleen, intestine, testis, and thymus) with highest levels in brain, intestine, and thymus.
Abstract: A novel ras-related gene (rab28) was identified by a PCR-based cloning approach and subsequent screening of rat fat cell and brain cDNA libraries. The deduced amino acid sequence of the cDNA is distantly related with members of the Rab family (31-33% sequence identity, mainly restricted to the six GTP-binding motifs). Cloning of the human homologue of Rab28 by a PCR-based approach revealed the existence of two isoforms (hRab28S, hRab28L) which differ only by a 95-bp insertion within the coding region. This insertion generates an alternative sequence of the 30 C-terminal amino acids of the protein. Both C-termini of the human homologues comprise farnesylation motifs, but differ strikingly in a stretch of 13 amino acids. By PCR, mRNA of hRab28S was detected in most tissues investigated (cortex, liver, kidney, skeletal muscle, adipose tissue, testis and urothelium), whereas hRab28L was predominant in testis. Recombinant Rab28 proteins showed specific binding of radiolabeled guanosine 5'-O-[gamma-thio]triphosphate and rapidly hydrolysed [alpha-32P]GTP; there was no difference in the GTP binding characteristics of the two isoforms hRab28S and hRab28L. It is suggested that the isoforms are derived from the same gene by alternative mRNA splicing, and that their functions differ in a parameter unrelated to its basic role as a GTPase.
Abstract: The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent Km of approximately 3.4 microM. Exchange of two tyrosine residues in the "activation loop" between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions.
Abstract: We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by in situ hybridization histochemistry. PPT mRNA was found to be differentially expressed throughout the rat brain. Highest transcript levels were found in specific neuronal populations (hippocampus, piriform cortex, taenia tecta, medial habenula, granular cell layer of the cerebellum) as well as in the choroid plexus of the third and lateral ventricles. In contrast, expression levels in some brain areas, e.g., caudate putamen and white matter, were beyond the detection limit of in situ hybridization. The pattern of expression of PPT in rat brain differs from that of other protein serine/threonine phosphatases and may reflect specific functions of this phosphatase.
Abstract: A cDNA clone of a protein kinase with high similarity to the Clk (Cdc2-like kinases) subfamily was isolated from a rat brain library and characterized. Its deduced amino acid sequence exhibited a 99% identity with human Clk3 and was therefore designated rat Clk3. In addition to the protein kinase domain, the sequence (490 amino acids) comprises an N-terminal domain with a strikingly high portion of basic amino acids. A glutathione S-transferase fusion protein of Clk3 catalyzed autophosphorylation of the kinase but not phosphorylation of the exogenous substrates histone or casein. By Northern blot analysis of different rat tissues, mRNA of Clk3 was detected predominately in testis, suggesting that this kinase regulates a predominately testicular function.
Abstract: Increased levels of mRNA transcribed from the ob gene in adipose tissue of obese/hyperinsulinaemic Zucker (fa/fa) rats were detectable as early as 3 weeks after birth and continued to rise there after in parallel with body weight and serum insulin. mRNA levels of two other fat-specific genes (ARL4, FST44) were unaltered. In C57BL/KsJ db/db mice, ob mRNA levels also increased in parallel with body weight and serum insulin, and remained elevated in older animals when insulin levels decreased. In heterozygous control animals (db/+; fa/Fa), mRNA levels were comparable with those in the homozygous controls. In normal Sprague Dawley rats, the ob mRNA increased continuously, but more slowly than in Zucker rats, in parallel with body weight and insulin levels, and reached 15 times higher levels in the heaviest rats (400 g) studied. In Sprague Dawley rats made diabetic by an injection of streptozotocin, ob mRNA levels were reduced by approximately 50% after 24 h. A 24-h fasting period reduced the ob mRNA by 50% in lean Sprague Dawley and Fa/Fa, but not in obese Zucker fa/fa rats, although insulin levels were reduced in both groups. These data indicate that ob mRNA levels increase in both normal and obese rodents in parallel with age, body weight and serum insulin, reflecting an early (Zucker rats, db-mice) or slowly developing (Sprague Dawley rats) resistance to leptin and insulin. This increase does not appear to be mediated by the recently described rapid regulation of ob mRNA by insulin, but seems to be due to a different, long-term control mechanism which signals the size of the fat depots.
Abstract: NON mice exhibit a polygenic syndrome of mild obesity which is less pronounced than that of the ob and db strains. Here, we have shown that the syndrome is accompanied by a rise in leptin mRNA levels in adipose tissue, corresponding with the increase in adipose tissue mass. Surprisingly, levels of the leptin protein in adipose tissue and serum were comparable to those of lean control animals (BL57/Ksj-+/+), and markedly lower than those in db/db-mice. The coding regions of the cDNA sequences of both leptin and the leptin receptor from NON mice were identical with those of the wild-type sequences. We suggested that low levels of leptin in adipose tissue and serum contribute to the obesity of NON mice.
Abstract: The effects of insulin, insulin-like growth factor (IGF)-I, platelet-derived growth factor (PDGF), interleukin (IL)-6 and interferon-gamma on 2-deoxyglucose uptake and insulin receptor substrate (IRS)-1 phosphorylation were compared in 3T3-L1 cells at confluence and after differentiation to the adipocyte-like phenotype. Insulin and IGF-I produced the expected stimulation of glucose transport and tyrosine phosphorylation of IRS-1 in both confluent and differentiated cells. In contrast, IL-6 and interferon-gamma failed to stimulate glucose transport or IRS-1 phosphorylation, although a marked stimulation of the JAK/STAT pathways as shown by acute-phase response factor (APRF)/Stat3 or Stat1 activation was observed in fibroblasts (IL-6, interferon-gamma) and adipocytes (IL-6). PDGF-AA and PDGF-BB stimulated glucose transport in confluent, undifferentiated cells to the same extent as insulin (approximately six-fold stimulation), but produced only a small portion of the effect of insulin in differentiated cells. Similarly, mRNA levels and autophosphorylation of PDGF receptors were much lower in differentiated cells than in confluent fibroblasts. In contrast to insulin and IGF-I, PDGF failed to stimulate tyrosine phosphorylation of IRS-1. All effects of insulin, IGF-I, and PDGF on glucose transport were inhibited by Wortmannin; the half-maximally inhibiting concentration (IC50) of Wortmannin was increased by insulin. These data demonstrate distinct signalling potentials of the investigated receptors, and indicate that the IL-6 and interferon-gamma controlled JAK/STAT pathways lack the potential to stimulate glucose transport. IRS-1 does not appear to be involved in the PDGF receptor-mediated effects, whereas activation of phosphatidylinositol (PI) 3-kinase is a crucial event in all pathways leading to stimulation of glucose transport.
Abstract: The cDNA of a novel protein kinase (referred to as SNRK) was isolated from a rat fat cell cDNA library with a probe generated by a cloning approach based on the polymerase chain reaction. The encoded polypeptide (746 amino acids, Mr=81627) contains all conserved subdomains characteristic of the protein serine/threonine kinase family. A recombinant fusion protein with glutathione S-transferase catalysed autophosphorylation as well as phosphorylation of histone, confirming that SNRK has indeed protein kinase activity. By Northern blot hybridization, a 5-kb mRNA was detected in brain, heart, fat cells, intestine, testis, ovary, adrenal gland and thymus. In 3T3-L1 cells. SNRK was specifically expressed in the differentiated, adipocyte-like phenotype, whereas its mRNA was not detected in fibroblasts. Sequence comparisons of its catalytic domain relate SNRK to the SNF1 family of protein kinases. The noncatalytic domain comprises several intriguing structural features, including a glycine-rich region, two PEST sequences, and a bipartite nuclear localization signal which is preceded by a stretch of ten consecutive acidic residues. This part of the sequence exhibits no extended similarity with other proteins. In addition, we detected a high degree of sequence similarity with other SNF1-related proteinases in a small region (30-35 amino acids) flanking the C-terminus of the catalytic domain. This domain (designated the SNH domain) appears to define the subfamily of SNF1-related protein kinases and might represent a new type of regulatory domain of protein kinases.
Abstract: Two arginine residues (RR333/334) in the conserved GRR motif located in the endofacial loop between helix 8 and 9 of the glucose transporter GLUT4 were substituted for leucine and alanine, respectively. Reconstituted glucose transport activity of the construct (GLUT4-RR333/4LA) expressed in COS-7 or LM(TK-) cells was less than 10% of that of the wild-type GLUT4. In contrast, binding of the inhibitory ligand cytochalasin B and glucose-inhibitable photolabeling with IAPS-forskolin were not significantly affected. Exchange of a histidine residue (H337Q) previously believed to be involved in the binding of inhibitory ligands failed to affect any of the investigated parameters. These data suggest that positive charges in the GRR motif at the cytoplasmic surface of the transporter participate in the conformational changes of the carrier protein during the process of facilitated diffusion.
Abstract: cDNA clones of two novel Ras-related GTP-binding proteins (RagA and RagB) were isolated from rat and human cDNA libraries. Their deduced amino acid sequences comprise four of the six known conserved GTP-binding motifs (PM1, -2, -3, G1), the remaining two (G2, G3) being strikingly different from those of the Ras family, and an unusually large C-terminal domain (100 amino acids) presumably unrelated to GTP binding. RagA and RagB differ by seven conservative amino acid substitutions (98% identity), and by 33 additional residues at the N terminus of RagB. In addition, two isoforms of RagB (RagBs and RagB1) were found that differed only by an insertion of 28 codons between the GTP-binding motifs PM2 and PM3, apparently generated by alternative mRNA splicing. Polymerase chain reaction amplification with specific primers indicated that both long and short form of RagB transcripts were present in adrenal gland, thymus, spleen, and kidney, whereas in brain, only the long form RagB1 was detected. A long splicing variant of RagA was not detected. Recombinant glutathione S-transferase (GST) fusion proteins of RagA and RagBs bound large amounts of radiolabeled GTP gamma S in a specific and saturable manner. In contrast, GTP gamma S binding of GST-RagB1 hardly exceeded that of recombinant GST. GTP gamma S bound to recombinant RagA, and RagBs was rapidly exchangeable for GTP, whereas no intrinsic GTPase activity was detected. A multiple sequence alignment indicated that RagA and RagB cannot be assigned to any of the known subfamilies of Ras-related GTPases but exhibit a 52% identity with a yeast protein (Gtr1) presumably involved in phosphate transport and/or cell growth. It is suggested that RagA and RagB are the mammalian homologues of Gtr1 and that they represent a novel subfamily of Ras-homologous GTP binding proteins.
Abstract: Plant tissues treated with the naturally occurring cyclopentanone compound methyl jasmonate or exposed to stress causing in planta jasmonate accumulation express distinctive proteins and, concomitantly, reduce the synthesis of most preexisting proteins. One of the recently identified jasmonate-induced proteins, designated JIP60, in barley is a ribosome-inactivating protein that cleaves polysomes of both animal and plant origin into their ribosomal subunits. By attacking foreign and self ribosomes, respectively, JIP60 appears to be both a defense protein and a potent regulator of protein synthesis in stressed plant tissues.
Abstract: A polymerase chain reaction-based cloning approach was employed in order to identify ADP-ribosylation factors (ARF) in murine 3T3-L1 cells and to study their expression before and after differentiation of cells to the adipocyte-like phenotype. Partial sequences comprising the effector domains of ARF were amplified with degenerate primers and cloned. Five of these sequences were identified as murine homologues of known human ADP-ribosylation factors (ARF 1, 2, 4, 5, and 6). In addition, partial sequences of two previously unknown isoforms were found, and complete cDNA clones were isolated from a rat fat cell library and were sequenced. Both sequences harbor a putative myristoylation site in position 2, the known consensus sequences presumably involved in GTP binding and hydrolysis, and lack cysteine residues in the C terminus. Their amino acid sequences share a 56 and 41% identity, respectively, with human ARF 1. Based on a comparison with the known ARF isoforms, the first clone appears to represent the mammalian homologue of a known sequence from Drosophila (dARL 1, 79% identity) and was therefore designated rARL 1. The second clone resembled none of the known ARF-like proteins and was designated rARL 4. mRNA of ARL 4 was undetectable in the fibroblasts but abundant in the adipocyte-like phenotype, its expression starting on day 6 of the differentiation. In contrast, ARF 1, 2, and 5 were unaltered by differentiation of the 3T3-L1 cells; mRNA levels of ARF 6, and also of ARL 1 and ARF 4, were reduced after differentiation. It is suggested that the function of ARL 4 is related to the adipocyte-like phenotype of 3T3-L1 cells.
Abstract: Six tyrosine residues (Y28, Y143, Y292, Y293, Y308, Y432(1)) which are conserved in all mammalian glucose transporters were substituted for phenylalanine by site-directed mutagenesis, and mutant glucose transporters were transiently expressed in COS-7 cells. Glucose transport activity as assessed by reconstitution of the solubilized transporters into lecithin liposomes was reduced by 70% in the mutant Y143F and appeared to be abolished in Y293F, but was not affected by substitution of Y28, Y292, Y308 and Y432. In contrast, covalent binding of the photolabel 125IAPS-forskolin was normal in all mutants. Stable expression of the mutants Y143F, Y293F, and Y292F in LTK cells yielded identical results. These data indicate that only two of the 6 conserved helical tyrosine residues, located in helices 4 and 7, are essential for full activity, but not for IAPS-forskolin binding of the GLUT4.
Abstract: Two novel protein serine/threonine phosphatases were cloned from a rat fat cell library with probes generated by a polymerase chain reaction-based cloning approach. One of these cDNAs encoded a protein presumably representing the rat homologue of PPV from Drosophila (75% identity of amino acids). The other novel cDNA encoded a protein phosphatase of 499 amino acids and was designated PPT. Its catalytic domain contains motifs typical for protein phosphatases but is only distantly related with PP1, PP2A, and PP2B (38-42% identical amino acids). When expressed in Escherichia coli, the catalytic domain of PPT exhibited protein phosphatase activity (dephosphorylation of phosphorylase a) that was inhibitable by okadaic acid. As a unique feature among other members of this gene family, PPT has an amino-terminal extension of 200 amino acids harboring three tandemly arranged tetratricopeptide repeat (TPR) motifs. This domain has previously been found in other proteins involved in the regulation of RNA synthesis or mitosis. mRNA of PPT was predominantly found in brain and, in lower levels, in testis, but was nearly undetectable in spleen, lung, skeletal muscle, kidney, and liver. It is suggested that the TPR domain of PPT may be involved in the regulation of the function of this novel protein phosphatase.
Abstract: Using the insulin receptor partial agonist B29,B29'-suberoyl-insulin, a covalently dimerized insulin derivative, we previously demonstrated a heterogeneity of signal transduction by insulin receptors in two cell systems. The present study was designed to characterize the heterogeneity of insulin receptors in different rat tissues with this agent. Binding of 125I-insulin to insulin receptors and its inhibition by B29,B29'-suberoyl-insulin or by unlabeled insulin were assayed in plasma membranes from brain, spleen, adipocytes, and liver. IC50 values of B29,B29'-suberoyl-insulin were different in all tissues investigated (brain < spleen < adipocytes < liver). In contrast, IC50 values of insulin were identical, with the exception of spleen membranes (spleen < brain = adipocytes = liver). Furthermore, the IC50 ratios (B29 dimer/insulin) were significantly different, ranging from 0.7 (brain) to 12.8 (liver). Solubilization and partial purification of insulin receptors failed to abolish the marked difference between brain and liver (IC50 ratios of 1.8 and 7.1, respectively). The apparent molecular masses of the alpha subunits of insulin receptors, as labeled with a photoreactive insulin derivative, appeared identical in liver and spleen but were significantly lower in adipocytes and brain (liver = spleen > adipocytes > brain). The tissue-specific expression of the known insulin receptor isoforms generated by alternative splicing (insulin receptor types A and B), as assessed by polymerase chain reaction amplification with oligonucleotide primers flanking exon 11, was not correlated with the differences in the IC50 values and ratios for insulin and B29,B29'-suberoyl-insulin. Furthermore, IC50 values of both insulin and the B29 dimer were 3-fold lower in membranes from Rat1 cells overexpressing insulin receptor type A, compared with membranes with insulin receptor type B; the IC50 ratios were identical. No additional alternative splicing of insulin receptor mRNA was found by polymerase chain reaction amplification and digestion with HaeIII and AluI of seven overlapping domains of the receptor alpha subunit. These data suggest a heterogeneity of insulin receptors in rat tissues that is unrelated to alternative splicing of the insulin receptor gene.
Abstract: Jasmonic acid (JA) and its methyl ester (JA-Me) are able to introduce the accumulation of several specific polypeptides in cut leaf segments of barley. Two of the most prominent JA-induced proteins of M(r) 15,000 and 23,000 have been characterized by isolating and sequencing complete cDNA sequences. While the sequence of the M(r) 23,000 polypeptide shows no similarity to published sequences, the sequence of the M(r) 15,000 polypeptide corresponds to the higher-molecular-weight precursor of a leaf thionin previously characterized. Transcripts for the M(r) 23,000 and M(r) 15,000 polypeptides accumulate in leaf segments shortly after the beginning of JA treatment. JA and JA-Me induce the appearance of the two proteins not only in leaf segments but also in intact barley seedlings. However, in seedlings the accumulation of JA-induced proteins occurs much more slowly and requires high concentrations of volatile JA-Me. Thus, in barley it seems unlikely that volatile JA-Me is involved in the interaction between different members of this species, as has been proposed recently for tomato seedlings.
