Common in vitro substrate specificity and differential Src homology 2 domain accessibility displayed by two members of the Src family of protein-tyrosine kinases, c-Src and Hck.

Hck and Src are members of the Src family of protein- tyrosine kinases that carry out distinct and overlapping functions in vivo (Lowell, C. A., Niwa, M., Soriano, P., and Varmus, H. E. (1996) Blood 87, 1780-1792). In an attempt to understand how Hck and Src can function both independently and in concert, we have compared 1) their in vitro substrate specificity and 2) the accessibility of their Src homology 2 (SH2) domain. Using several synthetic peptides, we have demonstrated that Hck and Src recognize similar structural features in the substrate peptides, suggesting that both kinases have the intrinsic ability to carry out overlapping cellular functions by phosphorylating similar cellular proteins in vivo. Using a phosphotyrosine-containing peptide that has previously been shown to bind the SH2 domain of Src family kinases with high affinity, we found that although Src could bind to the phosphopeptide, Hck showed no interaction. The inability of Hck to bind the phosphopeptide was not a result of a stable intramolecular interaction between its SH2 domain and C-terminal regulatory phosphotyrosine residue (Tyr-520), as most Hck molecules in the purified Hck preparation were not tyrosine-phosphorylated. In contrast to intact Hck, a recombinant truncation analog of Hck was able to bind the phosphopeptide with an affinity similar to that of the Src SH2 domain, suggesting that conformational constraints are imposed on intact Hck that limit accessibility of its SH2 domain to the phosphopeptide. Furthermore, the difference in SH2 domain accessibility is a potential mechanism that enables Src and Hck to perform their respective unique functions by 1) targeting them to different subcellular compartments, whereupon they phosphorylate different cellular proteins, and/or 2) facilitating direct binding to their cellular substrates.

Autophosphorylation induces autoactivation and a decrease in the Src homology 2 domain accessibility of the Lyn protein kinase.

Lyn is a member of the Src family of protein-tyrosine kinases that can readily undergo autophosphorylation in vitro. The site of autophosphorylation is Tyr397 which corresponds to the consensus autophosphorylation site of other Src family tyrosine kinases. The rate of autophosphorylation is concentration-dependent, indicating that the reaction follows an intermolecular mechanism. Autophosphorylation results in a 17-fold increase in protein-tyrosine kinase activity. Kinetic analysis demonstrates that phosphorylation of a substrate peptide by Lyn following autophosphorylation occurs with a 63-fold decrease in Km but no significant change in Vmax, suggesting that autophosphorylation relieves the conformational constraint that prevents binding of the substrate peptide to the active site of the kinase. Using a phosphotyrosine-containing peptide (pYEEI) that has previously been shown to bind to the Src homology 2 (SH2) domain of Src family tyrosine kinases with high affinity, we found that autophosphorylation results in a significant decrease in accessibility of the Lyn SH2 domain, indicating that conformational changes in the protein kinase domain induced by autophosphorylation can be propagated to the SH2 domain. Our study suggests that autophosphorylation plays an important role in regulating Lyn by modulating both its kinase activity and its interaction with other phosphotyrosine-containing molecules.

Effect of extradural analgesia on stress responses to abdominal surgery in infants.

We studied 40 children younger than 4 yr having elective abdominal surgery under general anaesthesia supplemented with either systemic opioids or extradural bupivacaine. Venous blood samples were obtained before tracheal intubation to measure baseline concentrations of adrenaline, noradrenaline, glucose, ACTH and cortisol. Additional samples were obtained 45 min after the start of surgery, at the end of surgery, 1 h and 24 h after the end of surgery. Plasma concentrations of bupivacaine were measured also in the extradural group at each sampling time. Both techniques provided acceptable analgesia, but the perioperative increases in adrenaline, glucose and ACTH were significantly greater in the opioid group. Noradrenaline concentrations decreased to less than baseline values in the extradural group and were significantly less than in the opioid group. The perioperative increase in cortisol was similar in the two groups, despite the differences in ACTH responses. Most responses returned to the baseline values within 24 h. Plasma bupivacaine concentrations remained within safe limits during the study, but systemic concentrations increased in some of the patients during postoperative infusion with 0.125% bupivacaine.

Growth factor-induced phosphorylation of c-ras p21 in normal hemopoietic progenitor cells.

Normal murine hemopoietic progenitor cells (colony-forming cells, CFC), representing 0.2% of the bone marrow cell population, were purified to homogeneity by fluorescence-activated cell sorting. CFC require the presence of the murine hemopoietic regulator, granulocyte-macrophage-colony stimulating factor (GM-CSF) for survival, proliferation, and differentiation along the myeloid pathway. An analysis of protein phosphorylation in GM-CSF-stimulated CFC over a 20-hr period demonstrated three phosphoproteins of approximate MW 21 kd and pI 6.2, 5.7 and 5.2 p21-6.2 persisted for 14 hr, while p21-5.7 and p21-5.2 were only detected during the first 5 hr of the analysis. The phosphate turnover time in all three p21 proteins was less than 3 hr and p21-5.2 contains an alkaline-resistant phosphorylation site. Low levels of p21-6.2 were also detected in unstimulated CFC. The observation of these phosphoproteins led us to investigate c-ras p21 in CFC. Immune precipitation with the anti-Ha/Ki-ras p21 monoclonal antibody (Y13-259) showed that expression of c-ras p21 in CFC was independent of GM-CSF stimulation, but that phosphorylated c-ras p21 was present only after GM-CSF stimulation. CFC contained one-tenth of the amount of phosphorylated c-ras p21 per cell compared with v-Ha-ras-transformed fibroblasts. It is possible that the phosphorylation of c-ras p21 in CFC has a significant role in the growth factor-directed molecular cascade responsible for normal hemopoietic development.

Epitope diversity of monoclonal antibodies revealed by cross-species reactivity.

The epitope specificity of two monoclonal antibodies (MAb) which have the same functional activity has been studied. These two independently raised rat IgG2b MAb, NIMP-R10 and M1/70 (Springer et al., 1979), blocked the complement (C) receptor on mouse macrophages. Both MAb showed essentially the same binding pattern with mouse cells, binding to the same extent mouse eosinophils, macrophages, neutrophils, a small proportion of spleen and bone marrow cells, but not thymocytes. That both MAb were apparently recognizing the same epitope was suggested from experiments in which MAb M1/70 inhibited the binding of MAb NIMP-R10. In addition, both MAb showed identity at the molecular level, precipitating the same molecules from the surface of mouse cells. However, NIMP-R10 and M1/70 could be shown to recognize different epitopes when they were tested on human cells. Thus, NIMP-R10 was found to bind to neutrophils and to large granular lymphocytes with natural killer cell activity but not to eosinophils or monocytes, while M1/70 bound to all of these cell types. It is suggested that inter-species testing may have general application in the analysis of antibody specificity.

A synthetic peptide containing the autophosphorylation site of the transforming protein of Harvey sarcoma virus is phosphorylated by the EGF-stimulated tyrosine kinase.

The transforming proteins (p21) of Harvey and Kirsten sarcoma viruses threonine kinase activity, which phosphorylates threonine 59 of the p21 proteins themselves. A tridecapeptide: Arg-Arg-Leu56-Asp-Thr-Thr59-Gly-Gln-Glu-Tyr-Ser-Ala66 containing residues 56-66 of p21 is phosphorylated solely on tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Km-Values of 240 and 80 microM and Vmax values of 1.7 and 0.1 nmol.min-1.mg-1 were obtained in the presence and absence of EGF, respectively.

Granulocyte macrophage-colony stimulating factor stimulates the synthesis of membrane and nuclear proteins in murine neutrophils.

The effect of granulocyte macrophage-colony stimulating factor (GM-CSF), a well-characterized hemopoietic regulator, on protein synthesis in murine bone marrow neutrophils is described. Bone marrow neutrophils in excess of 95% purity were obtained by fluorescence-activated cell sorting. While GM-CSF did not appear to slow the rate of dying of peritoneal exudate neutorphils or thymus cells, the viability of bone marrow neutrophils after 17 hr was enhanced (40%) by GM-CSF. GM-CFS had no effect on total 35S-methionine incorporation by thymocytes or peritoneal exudate neutrophils over a 17-hr incubation period; however, bone marrow neutrophils showed increased incorporation (approximately 10%) at all times between 5-17 hr. As viability and 35S-methionine incorporation of bone marrow neutrophils at 5 hr were minimally affected by GM-CSF, this time point was chosen to study the effect of GM-CSF on the synthesis of particular proteins. Two-dimensional polyacrylamide gels of 35S-methionine-labelled lysates were prepared from whole cells, isolated nuclei, and membranes. Quantitative analysis of the fluorograms obtained from the two-dimensional electropherograms by a computer-linked optical data digitiser indicated that out of a total of 180 proteins, the amount of label contained in 11 proteins was significantly higher in the presence of GM-CSF, while three proteins, apparently of cytoplasmic origin, contained less label than control cells. Eight of these proteins were identified as nuclear, and one was membrane derived. Attempts have been made to identify some of the inducible proteins and to correlate results with other studies of normal hemopoietic and leukemic cells. The significance and multiple functions of GM-CSF in hemopoiesis are discussed.