Tyrosine phosphorylation at the membrane-microfilament interface: a p185neu-associated signal transduction particle containing Src, Abl and phosphorylated p58, a membrane- and microfilament-associated retroviral gag-like protein.
Microfilaments are associated with the microvillar membrane in the 13762 ascites rat mammary carcinoma cells by stable interaction with a large, multimeric signal transduction particle (STP) containing the (proto)oncogene receptor p185(neu). In vitro kinase assays on isolated microvilli and microvillar fractions enriched in the putative signal transduction particle showed a high specific activity of tyrosine kinase activity compared to that of membranes from EGF receptor-overexpressing A431 cells maximally activated by EGF. Assays of velocity sedimentation fractions from microvillar lysates in the presence and absence of the exogenous tyrosine kinase substrate poly-glu-tyr polypeptide (poly-E(4)Y) suggested association of the tyrosine kinase activity with STP-enriched microvillar fractions. The particulate fractions also contained discrete endogenous tyrosine-phosphorylated proteins, including prominent bands of approximately 42 and 58 kDa. Addition of ATP to these fractions resulted in a rapid increase in tyrosine phosphorylation of these and several other proteins, as detected by anti-phosphotyrosine blots. Immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody of SDS-solubilized ascites cells and microfilament core fractions showed nine major bands; the electrophoretic mobilities of most of these in the cell immunoprecipitate and microfilament core were the same. In vivo and in situ phosphorylation, phosphoamino acid analysis, immunoprecipitation, 2-dimensional isoelectric focusing/SDS PAGE and immunoblot analysis showed that one of the prominent substrates is p58(gag), a retroviral Gag-like cytoplasmic STP component implicated in stabilizing microfilament-membrane interactions. Immunoblotting identified two peripheral membrane tyrosine kinases, p6O(src) and p120(abl), stably associated with the p185(neu)-containing signal transduction particle. These results provide further evidence for the constitutive activation of this aggressive mammary tumor and suggest a rote for phosphorylation of p58(gag) in organization of the STP at the membrane-microfilament interface in these cells.
Juang
S H
SH
Department of Biochemistry, University of Miami School of Medicine, Florida 33101, USA.
Carvajal
M E
ME
Whitney
M
M
Liu
Y
Y
Carraway
C A
CA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D015674:Mammary Neoplasms, Animal / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D010590:Phalloidine / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D066246:ErbB Receptors; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010590:Phalloidine; D011505:Protein-Tyrosine Kinases; D018719:Erb-b2 Receptor Tyrosine Kinases; C500813:membrane and microfilament-associated protein p58, rat