The p36 substrate of pp60src kinase is located at the cytoplasmic surface of the plasma membrane of fibroblasts; an immunoelectron microscopic analysis.
p36, a member of the family of Ca2+/lipid-binding proteins, is a major cellular substrate for the tyrosine kinase encoded by the src oncogene. It occurs in two distinct physical states, as either a monomer or a heterotetramer (protein I), which comprises two copies each of p36 and a p11 polypeptide. Immunofluorescence microscopy and cell fractionation studies suggest that p36 and p11 are located underneath the plasma membrane. To investigate whether p36 is indeed associated with the plasma membrane, we have examined its cellular distribution at the electron microscopic level with gold-labeled antibodies. In human fibroblasts, p36 is clearly associated with the cytoplasmic side of the plasma membrane and shows a uniform and regular distribution. Decoration with monoclonal antibodies against p11 reveals the same distribution, suggesting that the p36(2)p11(2) complex (protein I) occurs in the cell in a strict association with the plasma membrane. Titration experiments show that this association is Ca2+ dependent and still occurs at physiological Ca2+ concentrations (10(-7) M). Fodrin, a non-erythroid spectrin, known to bind p36 in vitro, shows a very similar distribution on the cytoplasmic side of the plasma membrane. The results suggest that in a resting and unstimulated cell p36 and p11 reside as a complex bound to the inner side of the plasma membrane.
Semich
R
R
Institute for Arteriosclerosis Research, University of Münster, Federal Republic of Germany.
Gerke
V
V
Robenek
H
H
Weber
K
K
D000911:Antibodies, Monoclonal; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D002462:Cell Membrane / Q000032:analysis*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000032:analysis*; D005347:Fibroblasts; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D008854:Microscopy, Electron; D010743:Phospholipids / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D010743:Phospholipids; D011505:Protein-Tyrosine Kinases; D002118:Calcium