The p36 substrate of tyrosine-specific protein kinases co-localizes with non-erythrocyte alpha-spectrin antigen, p230, in surface lamina of cultured fibroblasts.

Abstract

Biochemical and immunofluorescence studies have demonstrated that p36, a major substrate for the tyrosinespecific protein kinases induced by several sarcoma viruses and epidermal growth factor, is associated with plasma membranes and detergent-resistant cytoskeletal structures of cultured cells. We have used here polyclonal antisera and monoclonal antibodies in indirect immunofluorescence microscopy to study the subcellular location of p36 and the p230, which is a subplasmalemmal polypeptide showing immunologic cross-reactivity with erythrocyte a-spectrin. Both p36 and p230 showed a diffuse distribution in fixed and permeabilized cells and were localized in a surface lamina-like network in Triton-extracted cells. In double-staining experiments, an extensive co-distribution between these proteins was seen in detergent-treated cultured fibroblasts. These results, together with our previous work, suggest that the p36 protein is an integral part of the detergent-resistant proteinaceous network at the cytoplasmic face of the plasma membrane.

Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100.

“…The sequence obtained for p10 confirmed and extended by eight residues that previously derived for the p36-plO complex. The p1O sequence (positions [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] was then used in a computer search of known amino acid sequences (Protein Identification Resource Databank, Na-…”

“…The phosphorylation assays were performed with 250 pug of p36-plO complex per mi/10 mM Tris HCl/0.05 M KC1/5 mM MgCl2/1 mM CaCl/0.5 mM dithiothreitol, pH 6.8, containing [10][11][12][13][14][15][16][17][18][19][20] ACi of [y-32P]ATP (1 Ci = 37 GBq) (Amersham). Under these conditions, incorporation was <1% (mol/mol) but it could be increased with additional pp6(V-src.…”

“…Immunofluorescence microscopy studies have shown that antibodies directed against p36 decorate a reticular network just beneath the plasma membrane in fibroblasts, a distribution similar to that of nonerythroid spectrin (13)(14)(15)(16), a membrane structural element having a widespread tissue distribution (15)(16)(17)(18)(19). Additional localization experiments on tissue sections have shown that p36 is also present in the terminal web region of intestinal epithelial cells (20)(21)(22), and recently p36 has been isolated from this source (20).…”

Tumor promoter induces reorganization of actin filaments and calspectin (fodrin or nonerythroid spectrin) in 3T3 cells.

“…In fact, biochemical and immunocytochemical studies support the idea that it mainly locates at the inner surface of the plasma membrane (5)(6)(7)(8)(9). In cultured cells, it remains on the cytoskeletal meshwork even after cells are permeabilized with detergent (10,11). Like erythrocyte spectrin, it also binds to globular (G) and filamentous (F) forms of actin, resulting in an increased viscosity of actin filaments in vitro (12,13).…”

Changes in the distribution of the 34-kdalton tyrosine kinase substrate during differentiation and maturation of chicken tissues.

“…In some cases, however, stimulation of fibroblasts with epidermal or platelet-derived growth factor (18-20) is not accompanied by increased 34-kD protein phosphorylation . In addition, phosphorylation of this protein does not appear to be required for transformation of some cells by tyrosine kinase encoding retroviruses; several cell lines transformed by Abelson murine leukemia virus do not contain detectable amounts of the 34-kD protein (21).Recently, the 34-kD tyrosine kinase substrate has been localized at the inner surface of the plasma membrane by immunofluorescence staining (22-24) and subcellular fractionation studies (22,(24)(25)(26)(27) and was observed by immunofluorescence staining to have a distribution in chick or human embryo fibroblasts similar to that of the cytoskeletal associated protein a-spectrin (22,28). However, the role of the 34-kD protein in cellular transformation by retroviruses as well 473 on May 7, 2018 jcb.rupress.org Downloaded from…”