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

Abstract

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60Jcsrc. The 60-kilodalton (kDa) form appeared similar to pp6Ocsrc from cultured rat fibroblasts or astrocytes.The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60csrc was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60Csrc was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60csl`population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60CSrC and the increase in specific protein kinase activity may be important for neuronal differentiation.Cellular genes homologous to retroviral oncogenes are present in the genomes of all vertebrates. These cellular proto-oncogenes have been highly conserved throughout evolution, suggesting that they are essential to the organism. Although their function remains largely unknown, evidence is accumulating that some may be important for normal cell differentiation or growth.The cellular gene c-src is homologous to the transforming gene of Rous sarcoma virus (59). 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). Evidence is emerging that pp60C-src is the product of a developmentally regulated gene that may participate in cell differentiation. High levels of pp60csrc are expressed in brain and other neural tissues of both chickens and humans during embryogenesis (22,42,45). Expression of pp60-src in the developing chick neural retina (64) and cerebellum (29) coincides with the onset of neuronal differentiation. Post mitotic neurons from the central nervous system of rat embryos express high levels of a structurally distinct, enzymatically activated form of pp60C-src (9). Similarly, embryonal carcinoma cells induced to differentiate into neuronlike cells have elevated levels of a slower-migrating form of pp60c-src (44). There are other nonproliferating cells, for example, platelets (31) and myeloid cells (2, 30), which contain increased pp6Oc-src kinase activity. The presence of high levels of pp6Oc-src protein-tyrosine kinase activity in * Corresponding author. these nondividing cells suggests that pp60csrc may be importan...

Lipid-dependent Recruitment of Neuronal Src to Lipid Rafts in the Brain

“…This is in accordance with previous studies done on O1A1 cells (41) and cultured rat neurons (42) showing that upon differentiation, neurons switch from expressing cSrc to expressing NSrc. However, the NSrc-specific insert in the SH3 domain did not account for the ability of a subpopulation of Src to enter lipid rafts in brain but not fibroblasts.…”

“…Lipid Raft-associated NSrc Is Activated-NSrc is reportedly more catalytically active than cSrc (41,42). Activity is known to be regulated by dephosphorylation of the COOH-terminal regulatory tyrosine, Tyr-529 (Tyr-535 in NSrc), and phosphorylation on the activation loop tyrosine (Tyr-418; Tyr-424 in NSrc) (1,43).…”

“…NSrc differs from the normal splice form (called here cSrc) by 6 amino acids (RKVDVR) that are inserted into the SH3 domain because of an alternative splicing event (39,40). The activity of NSrc is 2-4-fold higher than that of cSrc (41,42). It is thought that these 6 amino acids destabilize the inactive conformation of Src, thereby leading to enhanced activity (43).…”

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

“…Likewise, lysates from thrombin-stimulated platelets showed no enTyr 527. platelets. Tryptic 32P-phosphopeptides were identified by comparison with published patterns (3,10). The identity of the Tyr-416-containing phosphopeptide was confirmed by mixing experiments with in vitro-labeled pp60f-s"c, which is labeled almost exclusively at Tyr-416.…”

“…However, the tissue distribution of pp6Oc-src expression suggests that this is not its function; those cells which express the highest levels of pp60csrc do not divide. For example, postmitotic differentiated or differentiating neurons express high levels of both c-src proteins (1,3,7,8,17,21). Mammalian platelets, terminally differentiated cells with no growth potential, express still higher levels of the smaller c-src protein (9).…”