Pathways to paediatric urology subspecialisation: a study of casemix, incumbent attitudes and opinions.

OBJECTIVE: To identify any self-reported differences or attitudes towards certification, publication, or practice patterns between adult urology and paediatric general surgery-trained paediatric urology providers. There are no known published differences in clinical/operative/research outcomes in either group. METHODS: An 18-item cross-sectional survey was compiled through the EAU Young Academic Urologists (YAU) office and disseminated to a trans-Atlantic convenience sample of current practising paediatric urologists. This was created using a mini-Delphi method to provide current semi-quantitative data relating to current opinions and attitudes of this cohort. RESULTS: A total of 228 respondents completed the survey, with female respondents representing 37% and 34% for urology and paediatric general surgery, respectively. Nearly 90% overall respondents felt that a full 2-year paediatric fellowship program was very important and 94% endorsed a collaborative dedicated paediatric urology on call service, with 92% supporting the joint development of transitional care. Urology managed higher numbers of bedwetting (p = 0.04), bladder bowel dysfunction (p = 0.02), endourological procedures (p = 0.04), and robotics (p = 0.04). Paediatric general surgery managed higher numbers of laparoscopic reconstruction (p = 0.03), and posterior urethral valve ablation (p = 0.002). CONCLUSION: This study represents the first time that a cross-sectional cohort of paediatric urologists from different training backgrounds were compared to assess their productivity, practice patterns and attitudes. Paediatric urology is in a unique position to have two contributing specialities, with the ability to provide optimal transitional and lifelong care. We believe that there should be a strong emphasis on collaboration and to remove any historically-created barriers under policies of equity, diversity and inclusivity.

Neuropsychiatric Developmental Disorders in Children Are Associated With an Impaired Response to Treatment in Bladder Bowel Dysfunction: A Prospective Multi-Institutional European Observational Study.

PURPOSE: Bladder and bowel dysfunction is a common but underdiagnosed pediatric entity which may represent up to 47% of pediatric urology consults. The objectives of this observational study were to determine functional 1-year outcomes following standard treatment of bladder and bowel dysfunction in both control and neuropsychiatric developmental disorder groups using validated questionnaires, and to perform an initial cost analysis. MATERIALS AND METHODS: This was a prospective observational study conducted across a number of academic European centers (July 2020-November 2022) for new bladder and bowel dysfunction patients. Parents completed a sociodemographic survey, information pertaining to prior neuropsychiatric developmental disorder diagnoses, as well as a number of validated functional scores. RESULTS: A total of 240 patients were recruited. In the control bladder and bowel dysfunction group, the baseline Dysfunctional Voiding Scoring System and Childhood Bladder and Bowel Dysfunction Questionnaire scores were 20% and 17.% lower, respectively, after 1 year compared to the neuropsychiatric developmental disorder group. The change in improvement was diminished for the neuropsychiatric developmental disorder cohort in both Dysfunctional Voiding Scoring System and Childhood Bladder and Bowel Dysfunction Questionnaire scores. The odds ratio of full symptom resolution was 5.7 in the control cohort compared to the neuropsychiatric developmental disorder cohort. A cost analysis on prescribed medications at referral led to a total cost of €32,603.76 (US $35,381.00) in the control group and €37,625.36 (US $40,830.00) in the neuropsychiatric developmental disorder group. CONCLUSIONS: This study demonstrates that pediatric patients with a neuropsychiatric developmental disorder exhibit more severe bladder and bowel dysfunction at baseline and throughout treatment with a lower overall quality of life, as well as 15.4% higher medication costs at referral. It is also important that parents' and caregivers' expectations are managed regarding higher levels of treatment resistance for functional bladder and bowel issues.

A cross-sectional analysis of paediatric urologists' current practices, opinions and areas of perceived importance in the delivery of adolescent & transitional care.

INTRODUCTION: Complex urological anomalies often require continued care as patients reach adulthood. Adequate transition for adolescents with ongoing urological care needs is critical to allow for seamless care in adult hospitals. Studies have shown that this can lead to improved patient and parental satisfaction, and lower utilisation of unplanned inpatient beds and emergency department visits. There is currently no ESPU-EAU consensus on the adequate mechanism and very few individual papers examining the role of urological transition for these patients in a European setting. This study aimed to identify current practice patterns in paediatric urologists providing adolescent/transitional care, to assess their opinions towards formal transition and to look for variations in care. This has implications for long-term patient health and specialist care. METHODS: An 18-item cross-sectional survey was compiled and pre-approved through the EAU-EWPU and ESPU board offices prior to dissemination to all registered ordinary members affiliated with the ESPU. This was created using a mini-Delphi method through the EWPU research meetings to provide current semi-quantitative data relating to current opinions and attitudes of this cohort. RESULTS: A total of 172 respondents (55% paediatric general surgery; 45% urology) across 28 countries completed the survey. The majority of respondents were in practice >10 years and spent >80% time in paediatric urology. There was no formal transition process according to 50% respondents and over half of those that did have less than 1/month, with <10% using validated questionnaires. More than two-thirds respondents continued to provide care after transition, as >70% units had no designated corresponding adult service. Furthermore, 93% paediatric believe a formal transition service to be very important, using a multidisciplinary framework. A pareto chart demonstrated 10 specific conditions to be of most interest in transition to adulthood. CONCLUSION: This is the first study to assess the requirements of paediatric urologists for adequate transitional care, however due to the nature of the survey's distribution, this was a non-scientific poll based on a convenience sample of respondents. It is critical that dual-trained or adult-trained urologists with a specific interest in paediatric urology work with current paediatric urologists in a multidisciplinary fashion to facilitate early transition based on the adolescent's developmental and biopsychosocial requirements. National urological and paediatric surgical societies need to make transitional urology a priority. The ESPU and EAU should collaboratively consider developing transitional urology guidelines to allow a framework by which this can occur.

Activation of insulin-epidermal growth factor (EGF) receptor chimerae regulates EGF receptor binding affinity.

Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which is followed by secondary regulatory processes. The IHE2 cell line is a unique model system to study the regulation of EGF binding to EGF receptors after activation of the EGF receptor kinase. IHE2 cells express both a chimeric insulin-EGF receptor kinase (IER) and a kinase-deficient EGF receptor (HER K721A). We have previously reported that IER is an insulin-responsive EGF receptor tyrosine kinase that activates one or several serine/threonine kinases, which in turn phosphorylate(s) the unoccupied HER K721A. In this article we show that insulin through IER activation induces a decrease in 125I-EGF binding to IHE2 cells. Scatchard analysis indicates that, as for TPA, the effect of insulin can be accounted for by a loss of the high affinity binding of EGF to HER K721A. Since this receptor transmodulation persists in protein kinase C downregulated IHE2 cells, it is likely to be due to a mechanism independent of protein kinase C activation. Using an in vitro system of 125I-EGF binding to transmodulated IHE2 membranes, we illustrate that the inhibition of EGF binding induced by IER activation is related to the phosphorylation state of HER K721A. Further, studies with phosphatase 2A, or at a temperature (4 degrees C) where only IER is functional, strongly suggest that the loss of high affinity EGF binding is related to the serine/threonine phosphorylation of HER K721A after IER activation. Our results provide evidence for a "homologous desensitization" of EGF receptor binding after activation of the EGF receptor kinase of the IER receptor.

Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation.

Regulation of cell proliferation, differentiation, and metabolic homeostasis is associated with the phosphorylation and dephosphorylation of specific tyrosine residues of key regulatory proteins. The phosphotyrosine phosphatase 1D (PTP 1D) contains two amino terminally located Src homology 2 (SH2) domains and is similar to the Drosophila corkscrew gene product, which positively regulates the torso tyrosine kinase signal transduction pathway. PTP activity was found to be regulated by physical interaction with a protein tyrosine kinase. PTP 1D did not dephosphorylate receptor tyrosine kinases, despite the fact that it associated with the epidermal growth factor receptor and chimeric receptors containing the extracellular domain of the epidermal growth factor receptor and the cytoplasmic domain of either the HER2-neu, kit-SCF, or platelet-derived growth factor beta (beta PDGF) receptors. PTP 1D was phosphorylated on tyrosine in cells overexpressing the beta PDGF receptor kinase and this tyrosine phosphorylation correlated with an enhancement of its catalytic activity. Thus, protein tyrosine kinases and phosphatases do not simply oppose each other's action; rather, they may work in concert to maintain a fine balance of effector activation needed for the regulation of cell growth and differentiation.

Global water resources: vulnerability from climate change and population growth.

The future adequacy of freshwater resources is difficult to assess, owing to a complex and rapidly changing geography of water supply and use. Numerical experiments combining climate model outputs, water budgets, and socioeconomic information along digitized river networks demonstrate that (i) a large proportion of the world's population is currently experiencing water stress and (ii) rising water demands greatly outweigh greenhouse warming in defining the state of global water systems to 2025. Consideration of direct human impacts on global water supply remains a poorly articulated but potentially important facet of the larger global change question.

The Par6alpha/aPKC complex regulates Akt1 activity by phosphorylating Thr34 in the PH-domain.

A single nucleotide polymorphism in the partitioning defective protein-6alpha (Par6alpha) promoter is coupled with lower Par6alpha expression and better insulin sensitivity, whereas overexpression of Par6alpha in C2C12 myoblasts inhibits insulin-induced protein kinase B/Akt1 activation and glycogen synthesis. Here we show that a direct interaction of Par6alpha with atypical protein kinase C (aPKC) is crucial for this inhibition. A DeltaPB1-Par6alpha deletion mutant that does not interact with aPKC neither increased aPKC activity nor interfered with insulin-induced Akt1 activation in C2C12 cells. Further, T34 phosphorylation of Akt1 through aPKC is important for inhibition of Akt1. When Par6alpha was overexpressed, activation of wild-type Akt1 (-59.3%; p=0.049), but not T34A-Akt1 (+2.9%, p=0.41) was reduced after insulin stimulation. The resistance of T34A-Akt1 to Par6alpha/aPKC-mediated inhibition was also reflected by reconstitution of insulin-induced glycogen synthesis. In summary, Par6alpha-mediated inhibition of insulin-dependent glycogen synthesis in C2C12 cells depends on the direct interaction of Par6alpha with aPKC and on aPKC-mediated T34 phosphorylation of Akt1.

Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.

Association between the melanoma-inducing receptor tyrosine kinase Xmrk and src family tyrosine kinases in Xiphophorus.

Melanoma formation in the fish Xiphophorus is an in vivo model for the function of receptor tyrosine kinases (RTKs) in tumor development. The overexpression and high activity of the RTK Xmrk (Xiphophorus melanoma receptor kinase) is responsible for the formation of hereditary malignant melanoma in this fish, but the mechanism by which Xmrk signals cell proliferation has not been elucidated. Remarkably, in earlier experiments an elevated level of a pp60c-src related kinase activity was found in the melanomas. In order to evaluate the significance of src family SH2 domain interactions in the intracellular signalling of Xmrk, we determined its relative binding affinity to the ubiquitous general RTK substrate, PLC gamma, and to the Xiphophorus cytoplasmic kinases Xsrc, Xfyn and Xyes. Recombinant Xmrk purified from baculovirus infected Sf9 cells bound with high affinity to the SH2 domains of PLC gamma and Xfyn in vitro. The affinity of Xmrk to Xsrc and Xyes SH2 domains was 5- to 10-fold lower. Coprecipitation experiments revealed that the Xmrk/Xfyn interaction occurred also in melanoma cells. Moreover, stimulation of the Xmrk kinase activity was paralleled by an increase in Xfyn activity. These results suggest that in malignant melanoma of Xiphophorus the highly activated Xmrk may enhance the activity of Xfyn through direct interaction and that both kinases are linked in a signal transduction pathway.

Ligand-dependent tumor induction in medakafish embryos by a Xmrk receptor tyrosine kinase transgene.

Xmrk encodes a subclass I receptor tyrosine kinase (RTK) which has been cloned from the melanoma-inducing locus Tu of the poeciliid fish Xiphophorus. To demonstrate a high oncogenic potential in vivo we transferred the gene into early embryos of the closely related medakafish. Ectopic expression of the Xmrk oncogene under the control of a strong, constitutive promoter (CMVTk) led to the induction of embryonic tumors with high incidence, after short latency periods, and with a specific pattern of affected tissues. We demonstrate ligand-dependent transformation in vivo using a chimeric receptor consisting of the extracellular and transmembrane domains of the human EGF receptor (HER) and the cytoplasmatic domain of Xmrk. Expression of the chimeric receptor alone does not lead to kinase activation or induction of tumors. Coexpression of the chimera with its corresponding ligand, human transforming growth factor alpha (hTGF alpha), however, results in the activation of the chimeric RTK. In injected fish embryos the induction of the neoplastic growth is observed with similar incidence and tissue distribution as in embryos carrying the native Xmrk oncogene suggesting that the ligand as well as factors downstream of the RTK are required for tumor formation. In this study we show single-step induction of tumors by ectopic expression of RTKs in vivo substantiating the significance of autocrine stimulation in RTK induced tumors in vertebrates.

Characterization of the PEST family protein tyrosine phosphatase BDP1.

Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues.

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

Circulating and liver-bound salt-resistant hepatic lipases in the golden hamster.

The serum of male golden hamsters was found to contain a circulating triacylglycerol hydrolase activity (serum lipase). In vitro, the enzyme activity was slightly activated by 1 M NaCl (+20%) and inhibited by rat serum (-29%). The hamster liver contained an enzyme with similar characteristics (liver lipase). This enzyme was released into the circulation after intravenous administration of heparin. Both lipase activities were further characterized and compared. The serum lipase had a pH optimum of 9, which was higher than that of the liver enzyme (pH 8.0). The serum enzyme did not bind to Sepharose-heparin columns in contrast to the liver lipase, which could be eluted from the column with 0.75 M NaCl. A polyclonal antibody preparation raised against the heparin-releasable salt-resistant lipase from rat liver inhibited both the hamster serum enzyme and the liver enzyme completely. The affinity of the antibodies towards the hamster enzymes was lower than the affinity towards the rat liver enzyme, but similar with that towards the hamster enzymes in the serum and the liver. A panel of five monoclonal antibodies raised against the rat enzyme did not bind either of the hamster enzymes. If the hamsters were fed a normal lab chow, the lipase activity in the serum amounted up to 110 +/- 20 mU (mean +/- S.D., n = 16) per ml serum (about 600 mU per animal), the liver contained 200 +/- 41 mU per g tissue (total about 800 mU per animal). In animals fed a cholesterol-enriched diet, the serum activity increased by 82% and the liver activity by 27%.

Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation.

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

Inhibition of Ret oncogene activity by the protein tyrosine phosphatase SHP1.

Germline mutations in the Ret protooncogene give rise to the inherited endocrine cancer syndromes MEN types 2A and 2B and familiar medullary thyroid carcinoma. Although it is well accepted that the constitutive active tyrosine kinase of Ret oncogenes ultimately leads to malignant transformation, it is not clear whether a decrease in the autophosphorylation of oncogenic Ret forms can affect the mitogenic and transforming activities of Ret. Potential modulators of the tyrosine kinase activity of Ret could be tyrosine phosphatases that are expressed in human thyroid tissue. Therefore, we investigated the impact of the tyrosine phosphatases SHP1 and SHP2 on the intrinsic tyrosine kinase activity and oncogenic potency of Ret with a 9-bp duplication in the cysteine-rich domain (codons 634-636), which was described in a patient with MEN type 2A recently. SHP1 and SHP2 were stably overexpressed in NIH3T3 fibroblasts together with Ret-9bp. Coexpression of SHP1 with Ret-9bp reduced the autophosphorylation of Ret-9bp by 19 +/- 7% (P = 0.01, n = 4), whereas no effect was seen with SHP2. Furthermore, Ret-9bp could be coimmunoprecipitated with SHP1 but not with SHP2 antibodies. Suppression of the Ret-9bp tyrosine kinase activity by SHP1 caused a decrease in activation of Erk2 (extracellular signal-regulated kinase) and abolished PKB/Akt (protein kinase B) phosphorylation. In addition, diminished Ret-9bp autophosphorylation led to reduced phosphorylation of the transcription factor jun-D. Finally, the inhibitory effect on Ret-9bp signaling resulted in a 40-60% reduction of [(3)H]thymidine incorporation and in reduced ability of NIH3T3 cells to form colonies in soft agar. In conclusion, the data suggest that SHP1 caused a moderate reduction of Ret autophosphorylation, which led to a strong suppression of the Ret oncogene activity.

Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes.

The cDNA sequences encoding mature human interleukin 2 (IL2) and beta-interferon (INF beta), respectively, were fused with various translational initiation regions and inserted into two different types of expression vector. The relative levels of expression of the two genes and the functional stability of their respective mRNAs were examined in vivo in Escherichia coli hosts. The addition of the 30-bp sequence, found immediately upstream of the E. coli atpE gene Shine-Dalgarno (SD) sequence, to the translational initiation regions of IL2 and INF beta increased the expression of both these genes by a factor of 6-10. Thus this sequence, which naturally acts within the E. coli atp operon to enhance the translational initiation frequency of the atpE gene, can increase the expression of other genes in E. coli. It may exemplify a specific type of recognition signal for the E. coli translational apparatus.

The transmembrane protein tyrosine phosphatase alpha dephosphorylates the insulin receptor in intact cells.

Protein tyrosine phosphatases (PTPs) are key regulators in a variety of signal transduction processes. However, substrates for most PTPs have not been determined. In a previous report, we demonstrated that in a transient expression system the intracellular phosphatases PTPs 1B and TC preferentially dephosphorylated the precursor form of several receptor tyrosine kinases. In this paper we show that the dephosphorylation of kinase precursors is a specific feature of PTPs 1B and TC that is not shared by two other intracellular PTPs, PTPH1 or SHP-1. By contrast, the receptor phosphatase PTP alpha preferentially dephosphorylated the beta-subunit of the insulin receptor localized on the cell surface. The insulin receptor was a better substrate for PTP alpha than for other receptor type PTPs. We conclude that the intracellular PTPs 1B and TC regulate the autophosphorylation of receptor tyrosine kinases during their posttranslational processing while receptor type PTPs regulate the mature, cell surface localized receptor tyrosine kinases.

Multisite serine phosphorylation of the insulin and IGF-I receptors in transfected cells.

Serine phosphorylation of insulin/IGF-I receptors in transfected fibroblasts was analysed by peptide mapping. PMA stimulated the phosphorylation of 5 distinct insulin receptor phosphopeptides: a single major phosphothreonine peptide containing Thr-1348, one major and 3 minor phosphoserine peptides. The major insulin-stimulated phosphoserine peptides were the same as those after PMA, with the exception of 2 minor phosphoserine peptides. PMA stimulated phosphorylation of a single major IGF-I receptor phosphoserine peptide which was phosphorylated to a lesser extent after IGF-I. We conclude that insulin/IGF-I and PMA stimulate phosphorylation of the same sites, but differ in the extents of phosphorylation.

The inhibitory effect of 2-deoxyglucose on insulin receptor autophosphorylation does not depend on known serine phosphorylation sites or other conserved serine residues of the receptor beta-subunit.

Hyperglycemia induces insulin resistance in diabetic patients. It is known that supraphysiological levels of D-glucose or 2-deoxyglucose inhibit the insulin receptor and it is speculated that this effect is mediated by serine phosphorylation of the insulin receptor beta-subunit and other proteins of the insulin signaling chain. To test this hypothesis we prepared point mutations of the human insulin receptor where serine was exchanged to alanine at 16 different positions, either at known phosphorylation sites or at positions which are conserved in different tyrosine kinase receptors. These receptor constructs were expressed in HEK 293 cells and the effect of 2-deoxyglucose (25 mM) on insulin (100 nM) induced receptor autophosphorylation was studied. 2-Deoxyglucose consistently inhibits insulin stimulated autophosphorylation of all constructs to the same degree as observed in wild-type human insulin receptor. The data suggest that none of the chosen serine positions are involved in 2-deoxyglucose induced receptor inhibition.