The src-family protein-tyrosine kinase p59hck iS mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hCk in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40 % is distributed equally between non-granular membranes and the cytosol.

The release of endogenous noradrenaline and its deaminated metabolite dihydroxyphenylglycol in the myocardium have been studied in the isolated perfused heart of the rat subjected to three models of energy depletion: ischemia, anoxia, and cyanide intoxication. Anoxia and cyanide intoxication were combined with substrate deficiency at constant perfusion flow. All three energy-depleting procedures caused a similar overflow of noradrenaline which, following a constant delay of 10 minutes without increased release, amounted to more than 25% of total heart content within 40 minutes. This noradrenaline overflow was not diminished in the absence of extracellular calcium and was inhibited by the uptake1 blocker desipramine in all three experimental models, indicating a common and nonexocytotic release mechanism. In the presence of glucose, neither anoxia nor cyanide intoxication resulted in a measurable noradrenaline overflow. Conversely, blockade of glycolysis or glucose depletion prior to ischemia or cyanide poisoning accelerated the noradrenaline overflow, demonstrating a key role of the sympathetic nerve cells' energy status in causing nonexocytotic catecholamine release. Blockade of energy metabolism in the presence of oxygen (cyanide model) resulted in the overflow of high amounts of dihydroxyphenylglycol that was not inhibited by uptake1 blockade. The release of the lipophilic dihydroxyphenylglycol by diffusion reflects deamination of axoplasmic noradrenaline by monoamine oxidase. Since saturation of the enzyme could be excluded in this model dihydroxyphenylglycol release can be taken as a mirror of cytoplasmic noradrenaline concentration. The results obtained by these studies indicate that nonexocytotic catecholamine release is a two-step process induced by energy deficiency in the sympathetic varicosity. In a first step, noradrenaline is lost from storage vesicles, resulting in increasing axoplasmic concentrations. The second step is the rate-limiting transport of intracellular noradrenaline across the cell membrane by the uptake1 carrier that has reversed its normal net transport direction.

The mechanism of action of prolactin (PRL) was studied in murine lymphoid BAF‐3 cells transfected with either the long form of the PRL receptor (PRL‐R), or a chimeric receptor consisting of the extracellular domain of the PRL‐R and the transmembrane and intracellular domain of the erythropoietin receptor (PRL/EPO‐R). PRL sustained normal and long‐term proliferation of BAF‐3 cells expressing either the PRL‐R or the hybrid PRL/EPO‐R. Upon [125I]PRL cross‐linking, both types of BAF‐3 transfectants were shown to express two [125I]PRL cross‐linked species differing in size by 20 kDa. These cross‐linked complexes, after denaturation, were recognized by antibody against the PRL‐R, indicating that they contain the transfected receptor. PRL induced rapid and transient tyrosine phosphorylation of both the PRL‐R and the PRL/EPO‐R in BAF‐3 transfectants. Furthermore, PRL induced rapid tyrosine phosphorylation of the Janus kinase 2 (JAK2) which was already physically associated with the PRL‐R or the PRL/EPO‐R in the absence of ligand. JAK1 was also associated with PRL‐R and PRL/EPO‐R in the absence of ligand. However, only in BAF‐3 cells expressing the PRL‐R does PRL induce rapid and transient tyrosine phosphorylation of JAK1. These results demonstrate that JAK protein tyrosine kinases couple PRL binding to tyrosine phosphorylation and proliferation.

The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and vav is a complicated and interesting molecule because of a number of structural features, several of which have suggested a role for Vav in cell signaling (51). Vav is expressed solely in hematopoietic cells, and its temporal pattern of expression during development precedes and coincides with the onset of hematopoiesis (67). Multiple types of evidence show that Vav is involved in signal transduction. We and others have shown that in T cells, Vav is tyrosine phosphorylated upon activation through the CD2 or the CD3 receptor (8, 44, 52), and it has been suggested that Zap-70 could be responsible for Vav tyrosine phosphorylation (38). Vav is also tyrosine phosphorylated upon activation of B and mast cells through the immunoglobulin M (IgM) antigen receptor (7) and the IgE high-affinity FceRI receptor (44), respectively. Furthermore, Vav is tyrosine phosphorylated upon activation of c-Kit by the steel factor (1) and Flk-2 (16) tyrosine protein kinase receptors, by erythropoietin (Epo) stimulation of the Epo receptor (46), and by interleukin-2, interleukin-3, and alpha interferon treatment of hematopoietic cells (16,20,50). Recently, the generation of mice deficient in vav expression in their lymphoid cells has pointed to the essential role of Vav in antigen receptor-induced proliferation of T and B cells. However, these studies also showed that there are Vav-independent signaling pathways involved in the proliferation of both kinds of cells (24,61,66).Vav has two Src homology 3 (SH3) domains flanking an SH2 domain and a proline-rich region in the amino-terminal SH3 (N-SH3) domain (8, 44). It also contains a cysteine-rich region that displays strong similarity to the zinc butterfly domains of protein kinase C isoforms, a pleckstrin homology region, and a Dbl-like domain with homology to a guanine nucleotide release factor. Therefore, it was postulated that Vav may participate in the activation of small GTP-binding proteins. Vav also has an acidic domain, a leucine zipper motif, a helix-loop-helix domain, and nuclear localization signals, suggesting that Vav could play a role as a nuclear factor (5,14,36,47).The function of Vav in cell signaling is not clear, and little is known about the proteins implicated in these pathways. The proline-rich region of Vav, ...