Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells.
The HER2/neu gene encodes a receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor. Overexpression of the receptor in mammary and ovarian carcinoma correlates with poor patient prognosis. To determine how the overexpression of a normal receptor leads to the generation of an oncogenic signal, we compared the patterns of tyrosine phosphorylation in tumor-derived human cell lines expressing high levels of p185HER2/neu. In intact SKBR3 cells, basal phosphorylation of p185HER2/neu was not detected. However, pretreatment of cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, led to the detection of phosphotyrosine on phospholipase C-gamma (PLC-gamma), GTPase-activating protein but not on the RAF-1 kinase. Strikingly, PLC-gamma was detected in a complex which contained multiple tyrosine-phosphorylated polypeptides. This complex was detected only in cytoplasmic fractions and had a distinct composition in different p185HER2/neu-overexpressing cell lines. Although GTPase-activating protein has been found previously in association with proteins of 190 and 62 kDa in fibroblasts, in SKBR3 cells it was found associated with multiple additional tyrosine-phosphorylated polypeptides. These experiments show that SKBR3 cells possess high levels of protein tyrosine phosphatase that can act upon p185HER2/neu. Moreover, they reveal, for the first time, the presence of PLC-gamma and GTPase-activating protein in cytosolic complexes containing a variety of other tyrosine-phosphorylated polypeptides. These observations suggest novel possibilities for the specific definition of receptor-generated signals in tumor cells.
Department of Molecular Biology, Max-Planck-Institut für Biochemie, Martinsried, Germany.
B
B
Jallal
J
J
Schlessinger
A
A
Ullrich
D000595:Amino Acid Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D006801:Humans; D010051:Ovarian Neoplasms / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D018719:Erb-b2 Receptor Tyrosine Kinases; D015398:Signal Transduction; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D020690:GTPase-Activating Proteins; D017027:Protein Tyrosine Phosphatases; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; D018719:Erb-b2 Receptor Tyrosine Kinases; D010738:Type C Phospholipases; D014443:Tyrosine