Cellular src gene product detected in the freshwater sponge Spongilla lacustris.

Abstract

Serum from Rous sarcoma virus tumor-bearing rabbits immunoprecipitated from extracts of the freshwater sponge Spongilla lacustris a tyrosine-specific protein kinase with characteristics similar to the chicken pp60c-src kinase activity. An immune competition assay confirmed the relationship between the protein from sponges and viral pp6O-sr'.The transforming gene of Rous sarcoma virus (RSV), vsrc, codes for a 60,000-dalton phosphoprotein, pp6O-src (1), which functions as a tyrosine-specific protein kinase (2, 6). The corresponding normal cellular counterpart, c-src, also codes for a protein, pp60c-rs, which shares structural and functional homology with viral pp60-src. Sequences homologous to v-src are present in the genomes of all vertebrates tested so far (7, 9, 13-15) and have been detected in the genus Drosophila (12). In this communication, we demonstrate the presence of a protein immunologically and functionally related to pp6Ov-src in the freshwater sponge Spongilla lacustris. Sponge cell extracts were tested for a tyrosine-specific kinase activity similar to the avian pp60sr-associated kinase activity. It was shown that the immunoglobulin G heavy chain of tumor-bearing rabbit (TBR) serum could be phosphorylated upon reaction (11) with a sponge cell extract, whereas the reaction with normal rabbit serum was negative (Fig. la). That the 32P-labeled 53,000 (53K) band was indeed the heavy chain of immunoglobulin G was established by running parallel portions of each sample with and without mercaptoethanol, so that under nonreducing conditions, a high-molecular-weight band of 150K could be observed (Fig. la). To prove that the kinase reaction observed actually utilized the same antibodies in TBR serum as those which took part in the reaction with pp60v-src an assay was performed in which a limiting amount of serum was reacted with heat-inactivated (50°C for 40 min) sponge extracts (ca. 0.2 mg of protein) before the addition of RSVinfected chicken cell lysates, followed by an in vitro kinase reaction (10). Preincubation with the heat-inactivated sponge extracts greatly reduced the pp6O-src kinase reaction (Fig. lb). Finally, the 53K product of the in vitro protein kinase reaction seen in Fig. la was cut out of the gel and eluted, and phosphoamino acids were analyzed by the method of Hunter and Sefton (6). The phosphorylated immunoglobulin G heavy chain is exclusively labeled in phosphotyrosine (Fig. lc).Immunoprecipitations with increasing amounts of TBR serum and a constant amount of sponge extract resulted in a maximum kinase activity with 5 ,lI of serum, whereas after the time dependence of the phosphorylation of heavy chain a * Corresponding author. maximum incorporation was revealed between 4 and 5 min ( Fig. ld and e).With respect to all reaction characteristics, the protein kinase activity as detected in sponge extracts could not be distinguished from the activity associated with the chicken cell pp60csrc (A. Barnekow and H. Bauer, Biochim. Biophys. Acta, in press). Thus, it was reasonable to expect ho...

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells.

“…The possibility also must be considered that the localization of an overexpressed chicken pp60 .... in mouse cells does not reflect the true location of the endogeneous mouse pp60 .... . This, however, is improbable since the cellular src gene has been shown to be structurally highly conserved during evolution from sponges to mammalian cells (Barnekow and Schartl, 1984) and, since a second mAb, GDll, that has been shown to be directed against a highly conserved epitope of the molecule (Parsons et al, 1984(Parsons et al, , 1986 give pp60 ~-~' patterns quite similar to the mAb 327. pp60 ~-~ also accumulates, often but not always, in the long cellular processes that develop when the cells are plated at low density. The formation of such processes, however, is inhibited when the cells are plated at high density or when they are plated at low density on a confluent layer of irradiated NIH 3T3 cells.…”

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

“…It is highly conserved phylogenetically, being present in the genome of such widely divergent species as humans, Drosophila melanogaster (35,61,62), and the freshwater sponge Spongilla lacustris (3,55). The c-src gene encodes a 60-kilodalton (kDa) membraneassociated phosphoprotein, pp6Oc-src, which is a proteintyrosine kinase (14-17, 24, 36, 38, 49, 52, 59).…”

The Genes That Carcinogens Act Upon

“…21) [16,53], and intensive efforts are being made in many laboratories to determine whether a cellular oncogene, such as c-src, is capable of mediating neoplastic transformation like its viral counterpart [3,[54][55][56][57][58][59]. If this should be proven we suggest that all individuals of all metazoa are endowed with tbe capacity to develop neoplasia.…”