Iterative tyrosine phosphorylation controls non-canonical domain utilization in Crk.

Crk, the prototypical member of a class of Src homology-2 (SH2) and Src homology-3 (SH3) domain containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-N-terminal SH3 domain (SH3N) signaling. Here, we show using LC-MS/MS and by generating phospho-specific antibodies that, iteratively with Y221, the Crk C-terminal SH3 domain (SH3C) is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. Although phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind polyproline type II ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high-throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix-based bioinformatics approaches, and unbiased mass spectometry, we found that the phosphoSH3C binds several SH2 domain containing proteins, including specific non-receptor tyrosine kinases-Abl via pY251 and C-terminal Src kinase via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator.

Phosphorylation of Crk on tyrosine 251 in the RT loop of the SH3C domain promotes Abl kinase transactivation.

Here, we report the identification and characterization of a novel tyrosine phosphorylation site in the carboxy-terminal Src Homology 3 (SH3) (SH3C) domain of the Crk adaptor protein. Y251 is located in the highly conserved RT loop structure of the SH3C, a region of Crk involved in the allosteric regulation of the Abl kinase. Exploiting kinase assays to show that Y251 is phosphorylated by Abl in vitro, we generated affinity-purified antisera against phosphorylated Y251 in Crk and showed that Abl induces phosphorylation at Y251 in vivo, and that the kinetics of phosphorylation at Y251 and the negative regulatory Y221 site in vitro are similar. Y251 on endogenous Crk was robustly phosphorylated in chronic myelogenous leukemia cell lines and in A431 and MDA-MB-468 cells stimulated with epidermal growth factor. Using streptavidin-biotin pull downs and unbiased high-throughput Src Homology 2 (SH2) profiling approaches, we found that a pY251 phosphopeptide binds specifically to a subset of SH2 domains, including Abl and Arg SH2, and that binding of pY251 to Abl SH2 induces transactivation of Abl 1b. Finally, the Y251F Crk mutant significantly abrogates Abl transactivation in vitro and in vivo. These studies point to a yet unrealized positive regulatory role resulting from tyrosine phosphorylation of Crk, and identify a novel mechanism by which an adaptor protein activates a non-receptor tyrosine kinase by SH2 domain displacement.

Centile reference charts for total energy expenditure in infants from 1 to 12 months.

BACKGROUND: A knowledge of energy expenditure in infancy is required for the estimation of recommended daily amounts of food energy, for designing artificial infant feeds, and as a reference standard for studies of energy metabolism in disease states. OBJECTIVES: The objectives of this study were to construct centile reference charts for total energy expenditure (TEE) in infants across the first year of life. METHODS: Repeated measures of TEE using the doubly labeled water technique were made in 162 infants at 1.5, 3, 6, 9 and 12 months. In total, 322 TEE measurements were obtained. The LMS method with maximum penalized likelihood was used to construct the centile reference charts. Centiles were constructed for TEE expressed as MJ/day and also expressed relative to body weight (BW) and fat-free mass (FFM). RESULTS: TEE increased with age and was 1.40,1.86, 2.64, 3.07 and 3.65 MJ/day at 1.5, 3, 6, 9 and 12 months, respectively. The standard deviations were 0.43, 0.47, 0.52,0.66 and 0.88, respectively. TEE in MJ/kg increased from 0.29 to 0.36 and in MJ/day/kg FFM from 0.36 to 0.48. CONCLUSIONS: We have presented centile reference charts for TEE expressed as MJ/day and expressed relative to BW and FFM in infants across the first year of life. There was a wide variation or biological scatter in TEE values seen at all ages. We suggest that these centile charts may be used to assess and possibly quantify abnormal energy metabolism in disease states in infants.

Comparison of measured sleeping metabolic rate and predicted basal metabolic rate during the first year of life: evidence of a bias changing with increasing metabolic rate.

OBJECTIVE: To compare measurements of sleeping metabolic rate (SMR) in infancy with predicted basal metabolic rate (BMR) estimated by the equations of Schofield. METHODS: Some 104 serial measurements of SMR by indirect calorimetry were performed in 43 healthy infants at 1.5, 3, 6, 9 and 12 months of age. Predicted BMR was calculated using the weight only (BMR-wo) and weight and height (BMR-wh) equations of Schofield for 0-3-y-olds. Measured SMR values were compared with both predictive values by means of the Bland-Altman statistical test. RESULTS: The mean measured SMR was 1.48 MJ/day. The mean predicted BMR values were 1.66 and 1.47 MJ/day for the weight only and weight and height equations, respectively. The Bland-Altman analysis showed that BMR-wo equation on average overestimated SMR by 0.18 MJ/day (11%) and the BMR-wh equation underestimated SMR by 0.01 MJ/day (1%). However the 95% limits of agreement were wide: -0.64 to +0.28 MJ/day (28%) for the former equation and -0.39 to +0.41 MJ/day (27%) for the latter equation. Moreover there was a significant correlation between the mean of the measured and predicted metabolic rate and the difference between them. CONCLUSIONS: The wide variation seen in the difference between measured and predicted metabolic rate and the bias probably with age indicates there is a need to measure actual metabolic rate for individual clinical care in this age group.

Fast-track office-based anesthesia: a comparison of propofol versus desflurane with antiemetic prophylaxis in spontaneously breathing patients.

IMPLICATIONS: Compared to propofol, maintenance of anesthesia with desflurane provided significantly better intraoperative conditions during office-based surgery. In addition, desflurane with routine antiemetic prophylaxis was associated with a faster early recovery and similar incidence of postoperative side effects.

Delay in serum stimulation of Erk activity caused by oncogenic transformation.

Mitogenic growth factor stimulation activates several signal transduction pathways, including the well-characterized Ras-Erk pathway, resulting in transient activation of Erk1 and Erk2. Oncogenic transformation, however, causes constitutive activation of growth signalling pathways, resulting in an accelerated rate of cell division. We investigated the effects of transformation on serum and growth factor stimulation of Erk1 and Erk2, and show that stimulation of these MAP kinases, as well as the Erk activator Mek, is delayed in oncogene transformed cells. Possible mechanisms of this delay are explored. In addition, our data indicate that prolonged mitogenic stimulation does not necessarily result in constitutive activation of Erk1 and Erk2.

Relative contributions of voluntary apnoea, exposure to cold and face immersion in water to diving bradycardia in humans.

1. Diving or face immersion bradycardia is a well recognized but incompletely understood reflux which occurs in man and other mammals. 2. In order to investigate the contributions made by voluntary apnoea, face immersion in water and cold exposure, 18 normal subjects were exposed to these challenges separately and in various combination. 3. Tested individually, cold and apnoea caused significant reductions in heart beat (P < 0.01 and 0.002, respectively). Face immersion in thermoneutral water had no effect on heart rate. 4. The bradycardic effect of apnoea at maximal inspiration may be due to stimulation of pulmonary stretch receptors. 5. Cold exposure and voluntary apnoea applied simultaneously caused a summative effect but when tested with face immersion in water there was a synergistic response greater than the sum of individual responses. 6. The results confirm the bradycardic effect of apnoea and cold exposure, whereas immersion in thermoneutral water had little effect, a finding which has been disputed in the literature.

Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts.

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.

The product of the cellular crk gene consists primarily of SH2 and SH3 regions.

We have cloned and sequenced a complementary DNA encoding the cellular homologue of the transforming oncogene v-crk of avian sarcoma virus CT10. This complementary DNA contains an open reading frame of 915 base pairs that encodes a polypeptide of 305 amino acids. The first 205 amino acids of this c-Crk protein were identical to those of the CT10 encoded v-Crk protein, with the exception of 5 amino acids. Like v-Crk, this portion of c-Crk contained one each of the Src homology domains SH2, SH2', and SH3. The 100 carboxy-terminal amino acids of c-Crk protein, which are not coded for in the CT10 viral genome, contain another SH3 region. We found limited sequence homology between c-crk and the avian retrovirus genome, which explains recombination events in the transduction of this protooncogene. Using a polyclonal antiserum made against bacterially expressed v-crk, we identified a 35-kilodalton protein in normal chicken embryo fibroblasts and in all embryonic chicken tissues examined. This 35-kilodalton protein was indistinguishable from a polypeptide made by in vitro translation of c-crk complementary DNA.

Biological and biochemical activity of v-Crk chimeras containing the SH2/SH3 regions of phosphatidylinositol-specific phospholipase C-gamma and Src.

The chicken CT10 virus oncogene product, P47gag-crk, contains SH2/SH3 domains that have been identified as conserved domains among proteins involved in signal transduction. We studied the functional similarity of the SH2/SH3 domains by replacing those of v-Crk with those of phosphatidylinositol-specific phospholipase C-gamma, v-Src, or c-Src. The transforming activity of v-Crk was partially retained in a mutant with a v-Src SH3 domain but not in the other mutants with heterologous SH2/SH3 domains. Mutant viruses with Crk-SH2/SH2' domains induced tyrosine phosphorylation of cellular proteins, but mutants with phosphatidylinositol-specific phospholipase C-gamma or Src SH2/SH2' domains did not. However, the mutant proteins with heterologous SH2/SH2' regions were able to weakly associate with some phosphotyrosine-containing proteins in vitro. These results indicate that in the context of the P47gag-crk structure, the requirement of Crk-SH2/SH3 is more stringent for its activity to induce cell transformation than to cause phosphorylation of cellular proteins. The substitution with heterologous sequences least perturbs the capacity to bind phosphotyrosine-containing proteins. In each case, the SH3 domain is more flexible to substitution than is the SH2 domain.

Generation of Met-enkephalin Arg6Phe7 immunoreactivity by proteolytic cleavage of mammalian plasma precursors by pepsin.

A region-specific antiserum raised against the C-terminal heptapeptide of proenkephalin A (Met-enk Arg6Phe7) was used in RIA studies to show that rat, human, and ovine plasma contain substrates (mol wt, 68K) that yield nanomolar amounts of Met-enk Arg6Phe7 (ME-RF) after treatment with pepsin under acid conditions. This ovine plasma-derived immunoreactivity diluted in parallel to the ME-RF standard in RIA and chromatographed as two low mol wt species (approximately 1K) which were less hydrophobic than the standard on size exclusion and reverse phase chromatography. The pepsin-generated material displaced [3H]naloxone from rat brain binding sites; its potency was about 1000-fold that of ME-RF, assuming near 100% cross-reactivity with the antiserum. Taken together these observations suggest that the pepsin-generated material is of similar mol wt and amino acid sequence to ME-RF, but differs with respect to opiate-binding efficacy, and that the plasma precursor is distinct from proenkephalin in both size and processing sites.

Beta endorphin and dynorphin levels in rat pituitary and hypothalamus: age studies.

The content of immunoreactive (ir)-beta-endorphin, ir-dynorphin 1-17, and ir-dynorphin 1-8 was determined in hypothalamus, anterior pituitary, and neurointermediate lobe of rats 3-24 months of age. In the anterior pituitary ir-beta-endorphin showed a progressive rise with age (0.5 +/- 0.06 ng/tissue at 3 months to 1.02 +/- 0.23 at 24 months); a similar change was seen in ir-adrenocorticotropic hormone content of the same tissues. No age-related change in neurointermediate lobe ir-endorphin content was observed. In the hypothalamus, the ir-beta-endorphin content fell progressively from 3 to 18 months, but was restored at 24 months to levels indistinguishable from those at 3 months (16.2 +/- 4.3 ng/tissue compared with 11.3 +/- 4.0 at 24 months). The ir-dynorphin content did not change progressively over the age span examined, except in the case of anterior pituitary content of ir-dynorphin 1-17 which fell progressively between 3 and 18 months (from 1.65 +/- 0.15 ng/tissue at 3 months to 0.73 +/- 0.12). Radioimmunoassay following high-performance liquid chromatography analysis of extracts of the tissues showed little variation of the immunoreactive forms with age, with two notable exceptions. In the hypothalamic extracts from 24-month-old rats the ratio of the nonacetylated ir-endorphin to the acetylated ir-endorphin was lower than in equivalent extracts from 3-month-old rats. In anterior pituitary extracts from 3-month-old rats, ir-dynorphin 1-17 appeared as two peaks (putative 6K and 4K species) of approximately equal size.(ABSTRACT TRUNCATED AT 250 WORDS)