Coexpression of c-kit and stem cell factor in breast cancer results in enhanced sensitivity to members of the EGF family of growth factors.

Kit, a tyrosine kinase growth factor receptor, and its ligand, stem cell factor (SCF), are commonly coexpressed in breast cancer. We have previously shown that MCF7 cells (that naturally express SCF) transfected with a c-kit expression vector exhibit enhanced growth in serum-free medium supplemented with IGF-1. Consequently, we wished to examine the interaction of Kit/SCF with additional growth factors important in the biology of breast cancer. MCF7 transfectants expressing Kit, cultured in serum-free medium supplemented with EGF, displayed more than twice the growth of controls at identical EGF concentrations. Similar responses were seen in the presence of heregulin alpha. The specificity of the Kit-mediated response was illustrated by a reduction in heregulin-stimulated growth in the presence of a monoclonal antibody directed against the Kit receptor. In addition, EGF- and heregulin-stimulated growth of the ZR75-1 cell line that naturally coexpresses Kit and SCF was also inhibited by the Kit blocking antibody. Preliminary investigations into the signal transduction pathways activated by these growth factors revealed that SCF activated both the Ras-MAP kinase and phosphatidyl-inositol-3-kinase (PI3 kinase) pathway. Both EGF and heregulin activated MAPK but to a lesser degree than SCF, and combination of SCF with these growth factors resulted in enhanced MAPK activation. Assessment of PI3K pathway activation using antiphospho-Akt antibodies revealed that EGF was a poor activator of Akt; activation of this pathway was markedly enhanced by the addition of SCF. Heregulin activated Akt and addition of SCF provided no further activation. Taken together these results suggest that coexpression of SCF and Kit may enhance responsiveness to erbB ligands by enhancing activation of the MAPK and PI3K pathways.

Hormonal regulation of cholesterol 7 alpha-hydroxylase mRNA levels and transcriptional activity in primary rat hepatocyte cultures.

In primary cultures of adult rat hepatocytes the level of cholesterol 7 alpha-hydroxylase steady-state mRNA markedly decreased by 72 h. However, the addition of L-thyroxine (T4) and dexamethasone synergistically returned cholesterol 7 alpha-hydroxylase steady-state mRNA levels near to that of cholestyramine-fed animals. The maximal responses to T4 and dexamethasone in serum-free medium were at 1.0 and 0.1 microM, respectively. The addition of T4 in combination with dexamethasone resulted in an 11-fold increase in transcriptional activity of the cholesterol 7 alpha-hydroxylase gene as compared to no addition controls. The specific activities of cholesterol 7 alpha-hydroxylase in microsomes prepared from cultures treated with dexamethasone and T4 were 1.56 +/- 1.17 nmol/h/mg protein which is similar to that of intact liver (1.70 +/- 0.062 nmol/h/mg protein), but lower than cholestyramine-fed animals. Cholesterol 7 alpha-hydroxylase activity was not detectable (less than 0.020 nmol/h/mg protein) at 72 h in cultures without the addition of both dexamethasone and T4. In the presence of optimal concentrations of dexamethasone and T4, glucagon (0.2 microM), or dibutyryl cAMP (50 microM) decreased (90%) cholesterol 7 alpha-hydroxylase mRNA within 6 h. Transcriptional activity decreased (62%) in 6 h following the addition of glucagon (0.2 microM) to the culture medium. The results reported in this paper suggest an important role for multiple hormones in the regulation of cholesterol 7 alpha-hydroxylase in the liver.

Electroconvulsive treatment induces a rapid and transient increase in tyrosine phosphorylation of a 40-kilodalton protein associated with microtubule-associated protein 2 kinase activity.

Recent studies have identified protein tyrosine phosphorylation as a major intracellular signaling pathway. However, little is known about regulation of this signaling pathway in neuronal systems. To help identify changes in levels of protein tyrosine phosphorylation in brain, we have utilized specific anti-phosphotyrosine antibodies to detect phosphotyrosine-containing proteins by immunoblotting techniques. We have found that electroconvulsive treatment induces a selective increase in tyrosine phosphorylation of a soluble 40-kDa protein. The rise is rapid and transient, reaching maximal levels at 1-2 min and returning to basal levels by 8 min. The phosphotyrosine-containing 40-kDa protein is most prominent in hippocampus, smaller in neocortex, and not detected in brainstem or cerebellum. A phosphotyrosine-containing 42-kDa protein present in several cell types has recently been identified as a serine/threonine phosphotransferase, referred to as microtubule-associated protein 2 kinase. Comparison of the levels of tyrosine phosphorylation of the 40-kDa protein and microtubule-associated protein 2 kinase activity during column chromatography of hippocampal extracts demonstrates that the phosphotyrosine-containing 40-kDa protein and microtubule-associated protein 2 co-purify. Moreover, the tyrosine phosphorylation of the 40-kDa protein and microtubule-associated protein 2 kinase activity are increased to a similar extent following electroconvulsive treatment. These findings suggest that the phosphotyrosine-containing 40-kDa protein identified in brain is closely related to microtubule-associated protein 2 kinase.

A 42-kD tyrosine kinase substrate linked to chromaffin cell secretion exhibits an associated MAP kinase activity and is highly related to a 42-kD mitogen-stimulated protein in fibroblasts.

The localization of the protein tyrosine kinase pp60c-src to the plasma membrane and to the membrane of secretory vesicles in neurally derived bovine chromaffin cells has suggested that tyrosine phosphorylations may be associated with the process of secretion. In the present study we have identified two cytosolic proteins of approximately 42 and 45 kD that become phosphorylated on tyrosine in response to secretagogue treatment. Phosphorylation of these proteins reached a maximum (3 min after stimulation) before maximum catecholamine release was observed (5-10 min after stimulation). Both secretion and tyrosine phosphorylation of p42 and p45 required extracellular Ca2+. Tyrosine-phosphorylated proteins of similar Mr have previously been identified in 3T3-L1 adipocytes stimulated with insulin (MAP kinase; Ray, L. B., and T. W. Sturgill. 1987. Proc. Natl. Acad. Sci. USA. 84:1502-1506) and in avian and rodent fibroblasts stimulated with a variety of mitogenic agents (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273; Nakamura, K. D., R. Martinez, and M. J. Weber. 1983. Mol. Cell. Biol. 3:380-390). Comparisons of the secretion-associated 42-kD protein of chromaffin cells with the 42-kD protein of Swiss 3T3 fibroblasts and 3T3-L1 adipocytes provide evidence that these three proteins are highly related. This evidence includes comigration during one-dimensional SDS-PAGE, cochromatography using ion exchange and hydrophobic matrices, similar isoelectric points, identical cyanogen-bromide peptide maps, and cochromatography of MAP kinase activity with the tyrosine-phosphorylated form of pp42. This protein(s), which appears to be activated in a variety of cell types, may serve a common function, perhaps in signal transduction involving a cascade of kinases.

Modulation of pp60c-src tyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells.

High levels of the proto-oncogene product, pp60c-src, have been found in developing and adult neural tissues as well as in certain fully mature cells of the hematopoietic lineage, e.g., platelets and myelomonocytes. Adrenal medullary chromaffin cells exhibit characteristics of both types of cells, i.e., they are derived from the neural crest and carry out exocytosis in response to specific stimuli. Earlier studies have shown that pp60c-src localizes not only to the plasma membrane of chromaffin cells but also to the membranes of chromaffin granules, the secretory vesicles of these cells that store catecholamines and other secretory products. To investigate the possible involvement of pp60c-src in exocytosis, cultured bovine chromaffin cells were analyzed for changes in c-src tyrosine kinase activity in response to stimulation by several secretagogues. Results of in-vitro immune complex kinase assays showed that pp60c-src, derived from cells that had been stimulated for various lengths of time, exhibited decreased auto- and transphosphorylating activities as compared to pp60c-src immunoprecipitated from control cells. The greatest reduction in activity was observed 10 min post-stimulation, while normal levels were regained 2-6 hr after secretagogue treatment. Western immunoblot analysis of the immunoprecipitated pp60c-src revealed that approximately 50% less c-src protein was present in immune complexes prepared 10 min after stimulation as compared to those prepared from mock-stimulated controls, resulting in a specific autophosphorylating activity that was 42-47% of control and little or no reduction in the transphosphorylating specific activity. In experiments in which the rate of secretion of [3H]-norepinephrine from cells preloaded with this compound was compared to the rate of modulation of pp60c-src activity, 50% of the maximal reduction in pp60c-src activity occurred within 2-4 min while 50% maximal release of [3H]-norepinephrine occurred within 1-3 min. Taken together, these results suggest that pp60c-src may play some role (direct or indirect) in the exocytotic process.