Identification of p130(cas) as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST.

PTP-PEST is a ubiquitously expressed, cytosolic, mammalian protein tyrosine phosphatase (PTP) which exhibits high specific activity in vitro. We have investigated the substrate specificity of PTP-PEST by a novel substrate-trapping approach in combination with in vitro dephosphorylation experiments. We initially identified a prominent 130-kDa tyrosine-phosphorylated protein in pervanadate-treated HeLa cell lysates which was preferentially dephosphorylated by PTP-PEST in vitro. In order to identify this potential substrate, mutant (substrate-trapping) forms of PTP-PEST were generated which lack catalytic activity but retain the ability to bind substrates. These mutant proteins associated in stable complexes exclusively with the same 130-kDa protein, which was identified as p130(cas) by immunoblotting. This exclusive association was observed in lysates from several cell lines and in transfected COS cells, but was not observed with other members of the PTP family, strongly suggesting that p130(cas) represents a major physiologically relevant substrate for PTP-PEST. Our studies suggest potential roles for PTP-PEST in regulation of p130(cas) function. These functions include mitogen- and cell adhesion-induced signalling events and probable roles in transformation by various oncogenes. These results provide the first demonstration of a PTP having an inherently restricted substrate specificity in vitro and in vivo. The methods used to identify p130(cas) as a specific substrate for PTP-PEST are potentially applicable to any PTP and should therefore prove useful in determining the physiological substrates of other members of the PTP family.

Association of PTP-PEST with the SH3 domain of p130cas; a novel mechanism of protein tyrosine phosphatase substrate recognition.

The protein tyrosine phosphatase PTP-PEST displays remarkable substrate specificity, in vitro and in vivo for p130cas a signalling intermediate implicated in mitogenic signalling, cell-adhesion induced signalling, and in transformation by a variety of oncogenes. We have identified a high affinity interaction between the SH3 domain of p130cas and a proline-rich sequence (P335PPKPPR) within the C-terminal segment of PTP-PEST. Mutation of proline 337 within this sequence to alanine significantly impairs the ability of PTP-PEST to recognise tyrosine phosphorylated p130cas as a substrate, without qualitatively affecting the selectivity of the interaction. Thus the highly specific nature of the interaction between PTP-PEST and p130cas appears to result from a combination of two distinct substrate recognition mechanisms; the catalytic domain of PTP-PEST contributes specificity to the interaction with p130cas, whereas the SH3 domain-mediated association of p130cas and PTP-PEST dramatically increases the efficiency of the interaction. Furthermore, our results indicate that one important function of the p130cas SH3 domain is to associate with PTP-PEST and thereby facilitate the dephosphorylation of p130cas, resulting in the termination of tyrosine phosphorylation-dependent signalling events downstream of p130cas.

Hormone-sensitive lipase is involved in the hydrolysis of lipoidal derivatives of estrogens and other steroid hormones.

Long-chain fatty acid esters of 17 beta-estradiol and other steroid hormones, which are formed in hormone-sensitive tissues, can be regenerated to the free hormone by the action of an esterase present in the cytosol. This esterase has now been examined in bovine placenta cotyledons. Activity towards steroid fatty acid esters was accompanied by activity towards a diacylglycerol analogue and cholesteryl oleate. During purification procedures, the ratio of activities towards the diacylglycerol analogue and estradiol 17 beta-oleate remained approximately constant. Activity towards these two substrates was inhibited by increasing concentrations of HgCl2 and phenylmethanesulfonyl fluoride in a parallel manner. Upon treatment with [3H]diisopropyl fluorophosphate, a major labelled species of Mr approx. 84,000 was formed. Activation by ATP and the catalytic subunit of cAMP-dependent protein kinase occurred. These properties were very similar to those of the hormone-sensitive lipase of bovine adipose tissue previously reported and run in parallel in this study. A highly purified preparation of this latter enzyme was found to hydrolyse steroid fatty acid esters and relative activities towards such substrates, diacylglycerol analogue and cholesteryl oleate, were similar to the placenta esterase. When the two esterases were phosphorylated with [gamma-32P]ATP, a labelled species of Mr 84,000 was isolated in both cases by use of an antibody raised against purified hormone-sensitive lipase of bovine adipose tissue. It is concluded that hormone-sensitive lipase is very likely the enzyme responsible for hydrolysis of steroid fatty acid esters in bovine placenta and possibly steroid hormone target tissues in general.

Primary structure of the site on bovine hormone-sensitive lipase phosphorylated by cyclic AMP-dependent protein kinase.

The primary structure of a region on hormone-sensitive lipase was determined to be: Lys-Thr-Glu-Pro-Met-Arg-Arg-Ser- Val-Ser-Glu-Ala-Ala-Leu-Thr-Gln-Pro-Glu-Gly-Pro-Leu-Gly-Thr-Asp-Ser-Leu-Lys. Ser-8 was the only residue in the intact protein phosphorylated by cyclic AMP-dependent protein kinase. However, Ser-10 also appeared to be present in a phosphorylated form, suggesting that it is a target for a distinct protein kinase in vivo.

Evidence for a multi-domain structure for hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) is a multi-functional enzyme involved in several aspects of lipid metabolism. Limited tryptic digestion of HSL led to selective loss of activity against lipid substrates but not against the water-soluble substrate, p-nitrophenyl butyrate. Following labelling of the active site of HSL with either [3H]di-isopropylfluorophosphate or [14C]orlistat, tryptic digestion of HSL generated a stable radiolabelled domain of molecular mass approx. 17.6 kDa, consistent with this representing a catalytic domain of HSL capable of hydrolysing water-soluble but not lipid substrates. Following phosphorylation of HSL by cyclic AMP-dependent protein kinase, limited tryptic digestion produced a stable phosphorylated domain of molecular mass 11.5 kDa. Based on these experimental data a model for a domain structure of HSL is proposed.

Regulation of cholesterol ester hydrolase by cyclic AMP-dependent protein kinase.

Phosphorylation of cholesterol ester hydrolase by cyclic AMP-dependent protein kinase results in activation of both cholesterol ester and triacylglycerol hydrolase activities. Activation against both substrates correlates closely with phosphorylation in time course experiments. Proteolytic digestion of phosphorylated cholesterol ester hydrolase, followed by peptide mapping, indicates the presence of a single phosphorylation site on the enzyme. Phosphoserine is the only phosphoamino acid detected following partial acid hydrolysis of the phosphorylated enzyme.

Further evidence that inhibitor-2 acts like a chaperone to fold PP1 into its native conformation.

The gamma1-isoform of protein phosphatase-1 expressed in Escherichia coli (PP1gamma) and the native PP1 catalytic subunit (PP1C) isolated from skeletal muscle dephosphorylated Ser-14 of glycogen phosphorylase at comparable rates. In contrast, PP1gamma dephosphorylated several tyrosine-phosphorylated proteins at similar rates to authentic protein tyrosine phosphatases (PTPases), but native PP1C was almost inactive towards these substrates. The phosphorylase phosphatase (PhP) and PTPase activities of PP1gamma were inhibited by vanadate with IC50 values (30-100 microM) comparable to authentic PTPases, whereas the PhP activity of native PP1C was insensitive to vanadate. PP1gamma lost its PTPase activity, and its PhP activity became insensitive to vanadate, after interaction with inhibitor-2, followed by the reversible phosphorylation of inhibitor-2 at Thr-72. These findings support and extend the hypothesis that inhibitor-2 functions like a chaperone to fold PP1 into its native conformation, and suggest that the correct folding of PP1 may be critical to prevent the uncontrolled dephosphorylation of cellular phosphotyrosine residues.

Estimation of tube potential in mammography from transmission measurements.

A computer program has been used to study the performance of a method for estimating the potential of mammographic x-ray tubes. The method uses measurements of the transmission of the x-ray beam through a series of filters. The transmissions are fitted to values calculated using tables of mammographic x-ray spectra, thus providing an estimate of tube potential. The performance of the method was studied using a Monte Carlo simulation of experimental errors. The number of measurements and the selection of filter material were both varied, and the precision and accuracy were used as performance indicators. It was found that, for a single filter material, the precision was improved by increasing the number of transmission data, but the accuracy was unaffected. The poorest accuracy and precision were obtained when K-edge filters, or filters with very high or very low atomic number were used. Using combinations of two filter materials, a high transmission (25%) K-edge filter in combination with a low transmission (3%) non-K-edge filter produced results comparable to the best results obtained from seven transmission measurements using a single filter material. By reducing the non-K-edge filter transmission to 1%, an accuracy of +/- 0.4% and a precision of +/- 1.4% were achieved at both 25 kV and 30 kV.

Beam data measurements for dynamic wedges on Varian 600C (6 MV) and 2100C (6 and 10 MV) linear accelerators.

The measurement of beam data for dynamic wedge dosimetry requires the integration of radiation dose at points across the radiation field during the dose delivery. The different measurement techniques required when using a linear diode array to measure beam profiles and when using ionization chambers to measure depth doses and effective wedge factors are described. The segmented treatment tables (STTS), which specify the delivered dose as a function of jaw position, are used by the control algorithm to deliver dynamic wedge fields. The reproducibility achieved using STTS is very consistent (and the stability of the accelerators is very good) so there is no significant variation in effective wedge factor or profile shape. There is a unique set of 132 STTS for each energy and design of treatment machine, which encompasses all the dynamic wedge data. There are significant discontinuities of up to 14% in wedge factors at certain field sizes. This means that wedge factors have to be measured at small increments (0.5 cm) in field size, as it is the width of the dynamic wedge field that determines the STT used. Considerable care must be taken when implementing these data on a current generation treatment planning computer.

Radiological thickness measurement using a liquid ionization chamber electronic portal imaging device.

We present a method of calibrating the Portal Vision electronic portal imaging device to obtain radiological thickness maps for compensator design. In this method, coefficients are derived to describe the relationship between intensity and thickness for a set of water-equivalent blocks. The effects of four parameters were studied: (a) The dose response of the system was measured and found to be describable by a square-root function. (b) The calibration data and images were taken with a wedge in situ. The effects of using different wedges and different wedge orientations were investigated. The intrinsic accuracy of the accelerator/imager system was found to be 1.9 mm, for both 15 degrees and 30 degrees wedges. Changing the wedge orientation between calibration and imaging and rotating the calibration coefficients accordingly led to an error of 3.5 mm. (c) The variation in detector response with gantry angle was measured and corrected. The residual error in this process was 2.4 mm. (d) The use of a model to correct the effects of imaging with different field sizes was investigated and found to yield a residual error of 2.9 mm. The overall error in image calibrations was approximately 4 mm or 2% in dose. This is considered to be sufficiently small for the intended use of designing compensators for tangential breast irradiation.

[Aneurysm formation on dacron prosthesis after 84 months of use. Pathology, structural and chemical evaluation of the explanted device (author's transl)]. / Formation anévrismale sur prothèse artérielle synthétique. Analyse pathologique, structurale et chimique d'une greffe en polyester après 84 mois d'implantation.

A 56 year old patient fitted with a femoro-popliteal Dacron prosthesis (Weavenit-Meadox Medical) 84 months earlier, presented with a severe palpable dilation in the middle part of the graft. The dilated segment of the prosthesis was resected locally and spliced to a new fabric device. The explanted section of prosthesis was analyzed; the dilation was found to extend only over several centimeters near the centre of the prosthesis. The degree of "healing" was found to be satisfactory except in the part which was most distended; this area also revealed mild bacterial colonization. Physico-chemical analysis of the fabric revealed that the dilation had taken place subsequently to hydrolytic degradation of the polyester fibres. Manufacturing conditions may have contributed to predisposing parts of the prosthesis to accelerated biodeterioration in vivo.

[Mechanical fatigue of an arterial prosthesis]. / Fatigue mécanique d'une prothèse artérielle.

This case study reports the analysis of a knitted polyester arterial prosthesis that was implanted in an 18-year old patient for five years following a bicycle accident which injured the left external iliac artery. In spite of the prosthesis, the patient continued to participate in competitive cycling. The graft became dilated and stretched by tension from the hypertrophic psoas muscle, and because the patient presented with pain and claudication, a second surgical operation was performed. The central region of the prosthesis was badly kinked. A segment was resected and analyzed in terms of pathological, textile and chemical properties. The analysis showed a poorly healed graft that had suffered from mechanical and chemical degradation. The findings point to the wisdom of implanting woven rather than knitted grafts in young patients and also suggest that in this particular case, where mechanical deterioration was accompanied by chemical degradation, it would have been preferable to remove the whole prosthesis.

Identification and role of the basal phosphorylation site on hormone-sensitive lipase.

Phosphorylation site 2 on bovine hormone-sensitive lipase (HSL), which is phosphorylated in vitro by the AMP-activated protein kinase, has been found also to be phosphorylated in vitro by glycogen synthase kinase-4. Peptide mapping of HSL phosphorylated in vitro and in isolated adipocytes demonstrates that this site corresponds to the basal phosphorylation site on HSL, which is phosphorylated in intact adipocytes in the absence of lipolytic stimuli. Site 2 has been proposed to have an antilipolytic role in that phosphorylation at this site greatly reduces subsequent phosphorylation (at site 1) and activation of HSL by cyclic-AMP-dependent protein kinase. Further evidence for an antilipolytic role of site 2 has been obtained using a synthetic peptide based on the sequence around sites 1 and 2. Phosphorylation of the peptide at site 2 totally prevents the subsequent phosphorylation of site 1 and vice versa.

PTP-PEST: a protein tyrosine phosphatase regulated by serine phosphorylation.

The protein tyrosine phosphatase PTP-PEST is an 88 kDa cytosolic enzyme which is ubiquitously expressed in mammalian tissues. We have expressed PTP-PEST using recombinant baculovirus, and purified the protein essentially to homogeneity in order to investigate phosphorylation as a potential mechanism of regulation of the enzyme. PTP-PEST is phosphorylated in vitro by both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) at two major sites, which we have identified as Ser39 and Ser435. PTP-PEST is also phosphorylated on both Ser39 and Ser435 following treatment of intact HeLa cells with TPA, forskolin or isobutyl methyl xanthine (IBMX). Phosphorylation of Ser39 in vitro decreases the activity of PTP-PEST by reducing its affinity for substrate. In addition, PTP-PEST immunoprecipitated from TPA-treated cells displayed significantly lower PTP activity than enzyme obtained from untreated cells. Our results suggest that both PKC and PKA are capable of phosphorylating, and therefore inhibiting, PTP-PEST in vivo, offering a mechanism whereby signal transduction pathways acting through either PKA or PKC may directly influence cellular processes involving reversible tyrosine phosphorylation.

Participation and interest in leisure activities by adolescent schoolchildren.

Knowledge of the leisure activities and pursuits of adolescents is important as it can provide a greater understanding of the social world of the adolescent. Further, it is necessary not only to know what adolescents actually do in their leisure time, but what they are interested in doing. In this study, the relationship between levels of participation and levels of interest in leisure activities was investigated via analysis of a comprehensive 77 item questionnaire administered to 247 adolescents. Factor analytic techniques were used to summarize the data, enabling comparisons to be made between boys and girls. Adolescents were found to engage in and be interested in activities characterised by their sex stereotyped nature, although in general none of the popular pursuits was either physically or mentally demanding. There was a high correlation between participation and interest, with adolescents participating in activities that interested them. The implications of these findings are discussed.

Leisure activities of adolescent school students: predictors of participation and interest.

Frequency of participation and levels of interest in more than 60 leisure pursuits were measured via a questionnaire administered to 1248 adolescent high school students. The relationship between participation and levels of interest was measured by correlation as well as by asking the students to nominate up to three activities they would like to participate in but cannot and to indicate the reason for their non-participation. Factor analyses reduced the Participation and Interest items to six factors each. Multiple regressions were then conducted on the derived factor-score variables. Sex was the major predictor of participation in sports and vocational activities and of interest in sporting and gregarious activities. Age, school location, ethnicity and SES were lesser predictors for groups of activities such as the social and outdoor pursuits. The results are discussed in terms of the theoretical and practical implications of the relationship between participation and interest as well as the prediction of participation and interest levels by developmental and social factors.

Regulation of fibroblast motility by the protein tyrosine phosphatase PTP-PEST.

The protein tyrosine phosphatase PTP-PEST is a cytosolic enzyme that displays a remarkable degree of selectivity for tyrosine-phosphorylated p130(Cas) as a substrate, both in vitro and in intact cells. We have investigated the physiological role of PTP-PEST using Rat1 fibroblast-derived stable cell lines that we have engineered to overexpress PTP-PEST. These cell lines exhibit normal levels of tyrosine phosphorylation of the majority of proteins but have significantly lower levels of tyrosine phosphorylation of p130(Cas) than control cells. Initial cellular events occurring following integrin-mediated attachment to fibronectin (cell attachment and spreading) are essentially unchanged in cells overexpressing PTP-PEST; similarly, the extent and time course of mitogen-activated protein kinase activation in response to integrin engagement is unchanged. In contrast, the reduced phosphorylation state of p130(Cas) is associated with a considerably reduced rate of cell migration and a failure of cells overexpressing PTP-PEST to accomplish the normally observed redistribution of p130(Cas) to the leading edge of migrating cells. Furthermore, cells overexpressing PTP-PEST demonstrate significantly reduced levels of association of p130(Cas) with the Crk adaptor protein. Our results suggest that one physiological role of PTP-PEST is to dephosphorylate p130(Cas), thereby controlling tyrosine phosphorylation-dependent signaling events downstream of p130(Cas) and regulating cell migration.

The presence and role of hormone-sensitive lipase in heart muscle.

Hormone-sensitive lipase (HSL) catalyses the initial, rate-limiting, reaction in adipose-tissue lipolysis. Hormone-stimulated lipolytic activity has also been observed in the heart, where endogenous triacylglycerol is the major energy store. However, the identity of the intracellular lipase responsible has yet to be established. We have partially purified a neutral lipase from bovine heart muscle and compared its properties with those of HSL from bovine adipose tissue. The heart lipase has the same subunit Mr as HSL, is immunoprecipitated by antiserum raised against purified HSL and is phosphorylated by cyclic AMP-dependent protein kinase, apparently at the same site as HSL (as judged by h.p.l.c. of tryptic phosphopeptides). Phosphorylation of the heart lipase was found to result in increased enzyme activity, demonstrating the lipase's potential to respond to hormonal stimuli. The heart lipase was shown to be present in myocytes by its immunoprecipitation from homogenates of rat myocytes by anti-HSL antiserum. These findings are consistent with the conclusion that HSL is responsible for intracellular lipolysis in heart.

The Western Australian child health survey: a pilot study.

A pilot survey of 200 households was undertaken to field test the survey instruments and trial the validation and calibration procedures for the Western Australian Child Health Survey (WACHS) conducted in 1992. This paper describes the background to the WACHS, the development of the instrumentation and the conduct of the pilot study. This survey aims to replicate and extend previous epidemiological surveys conducted in other countries, and to provide Australian norms for mental health morbidity in 4 to 16 year old children. The measurement of mental health was undertaken through the use of the Child Behaviour Checklist (CBCL). This screening instrument provided data on the prevalence of mental health morbidity and of specific mental disorders in 4 to 16 year olds. Its reliability and validity as a diagnostic indicator were checked through a clinical calibration technique. The pilot survey also permitted an examination of the sampling strategy adopted to ensure that the sample selected reflected "normality" in terms of expected trends and results. Modifications to the content of the questionnaires are described in light of both psychometric qualities of the data and comments from field interviewers and professionals who have examined the instrument. Finally, changes to data collection strategies are discussed.

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation.

Using the patch-clamp technique we have identified a Ca2(+)-sensitive, voltage-dependent, maxi-K+ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of approximately 200 pS in excised patches bathed in symmetrical 150 mM K+, and was blocked by 1 mM Ba2+. Channel open-state probability (Po) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted the Po/voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration, Po remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca2+ concentration from 2 mM to 0.1 microM. However, activated channels were still blocked by 1 mM Ba2+. Channel Po was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase. We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K+ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca2+. Physiologically, these K+ channels may contribute to the basolateral K+ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.