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

Abstract

ABSTRACTp36 is a major substrate of both viral and growth factor-receptor-associated tyrosine protein kinases. p36 can be isolated as a complex consisting of a subunit of Mr 36,000 (p36) and a subunit ofMr 10,000 (p10), and it represents an abundant cellular protein. We have isolated the p36-plO complex from bovine intestinal epithelium and analyzed the amino terminus of both subunits. Sequence analysis of the first 56 amino acids of p1O demonstrates a striking sequence homology (48% identically placed residues) with the Mr 10,000 calcium-binding proteins from bovine brain, termed S-100. Intestinal p36 could be effectively labeled on a single tyrosine in vitro with immunoprecipitated pp60V1 and [-32P]ATP. Mild proteolysis of p36 with chymotrypsin resulted in the cleavage into large (Mr,33,000) and small domains (Mr, 3000), with the latter representing the phosphorylated amino terminus. Although the amino terminus is apparently blocked, sequence analysis of a secondary tryptic peptide of the Mr 3000 fragment as well as the amino-terminal sequence of the Mr 33,000 domain and overlapping peptides clearly established the site of tyrosine phosphbrylation.A number of oncogenic viruses and growth factor receptors are known to encode or be associated with a tyrosine protein kinase activity (1)(2)(3)(4)(5). Since this finding, there has been a search for relevant substrates whose phosphorylation could cause the altered phenotype of transformed cultured cells. Known substrates for tyrosine kinases include glycolytic enzymes (6) as well as the cytoskeletal proteins vinculin (7) and the Mr 36,000 polypeptide (p36). p36 was first identified as a major substrate phosphorylated on tyrosine in Rous sarcoma virus/chicken embryo fibroblast cells (8, 9) as well as being phosphorylated by other transforming proteins and in response to growth factors (10-12).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 (13-16), 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). Intestinal p36 was shown to be comprised of polypeptides of Mr 36,000 and 10,000 as a native complex of Mr 85,000 (23) that can interact with both actin and spectrin (20). Similar subunits of p36 have been identified in fibroblasts in addition to monomeric p36 (24). Antibodies to fibroblast p36 were shown to react with intestinal p36 and antibodies to intestinal p36 decorated a reticular network in cultured cells and would immunoprecipitate a protein of Mr 36,000 that was phosphorylated on tyrosine in Rous sarcoma virus-transformed cells (20). These properties suggest a function for p36 in membrane structure. We now report the partial sequence of...

Elevated expression of Annexin II (Lipocortin II, p36) in a multidrug resistant small cell lung cancer cell line

“…The NH2-terminal tails of the annexins are quite variable in sequence and length. In p36 (annexin II), this region contains both serine and tyrosine phosphorylation sites (Glenney & Tack, 1985;Gerke, 1989) and p36 has been shown to be a substrate for pp6Ov-src (Huang et al, 1986;Erikson et al, 1984;Saris et al, 1986) and protein kinase C (Gould et al, 1986;Barnes et al, 1991).…”

“…p36 exists as either a monomer or as a heterotetramer with an 11 kDa protein, pll, forming a (p36)2(p1l)2 complex (Erikson et al, 1984;Glenney & Tack, 1985;Johnsson et al, 1988;Glenney et al, 1986;Zokas & Glenney, 1987). In the latter form, it may be a structural component of the cytoskeletal framework.…”

The C Terminus of Annexin II Mediates Binding to F-actin

“…The similarity in function between the ERM proteins and AIIt, and the presence of the F-actin binding domain within the C terminus of the ERM proteins, led us to suspect that the C-terminal region of the annexin II subunit of AIIt might be involved in F-actin binding. It is known that the N terminus of annexin II is responsible for binding of p11 (23,46,47), however, a role has not been attributed to the C-terminal region of the protein. Therefore, we have used site-directed mutagenesis to truncate the C terminus of annexin II to assess its possible function in F-actin binding.…”

Phosphorylation by Protein Kinase C of Annexin 2 in Chromaffin Cells Stimulated by Nicotine

“…growth factor and platelet-derived growth factor, annexin 2 is phosphorylated on a tyrosine as well as on a serine (Isacke et a!., 1986). The serine residue was identified as Ser25 within the amino-terminal region (Glenney and Tack, 1985). Alternatively, in vitro phosphorylations of annexin 2 with calmodulin-dependent protein kinase, cyclic AMP-dependent protein kinase, and protein kinase C indicated that the monomeric heavy chain is a much better substrate than the complex (Johnsson et al, 1986); these studies showed also the presence of a second site phosphorylated by protein kinase C, probably Ser°.…”