Src family protein tyrosine kinases are activated following engagement of many different classes of cellular receptors and participate in signaling pathways that control a diverse spectrum of receptor-induced biological activities. While several of these kinases have evolved to play distinct roles in specific receptor pathways, there is considerable redundancy in the functions of these kinases, both with respect to the receptor pathways that activate these kinases and the downstream effectors that mediate their biological activities. This chapter reviews the evidence implicating Src family kinases in specific receptor pathways and describes the mechanisms leading to their activation, the targets that interact with these kinases, and the biological events that they regulate.

Cellular functions regulated by src family kinases

Background Transforming growth factor (TGF)-β1 is a profibrogenetic cytokine that has been implicated in the development of fibrosis in transplanted tissues. In this study, we have analyzed the genetic regulation of TGF-β1 production in lung transplant recipients. Method. A polymerase chain reaction-single-stranded conformational polymorphism technique was used to detect polymorphisms in the TGF-β1 gene from genomic DNA. Polymorphisms were shown to correlate with in vitro TGF-β1 production by stimulated lymphocytes. A single-specific oligonucleotide probe hybridization method was devised to screen for these polymorphisms in lung transplant groups and controls. Results. We have identified five polymorphisms in the TGF-β1 gene: two in the promoter region at positions -800 and -509, one at position +72 in a nontranslated region, and two in the signal sequence at positions +869 and +915. The polymorphism at position +915 in the signal sequence, which changes codon 25 (arginine→proline), is associated with interindividual variation in levels of TGF-β1 production. Stimulated lymphocytes of homozygous genotype (arginine/arginine) from control individuals produced significantly more TGF-β1 in vitro (10037±745 pg/ml) compared with heterozygous (arginine/proline) individuals (6729±883 pg/ml; P<0.02). In patients requiring lung transplantation for a fibrotic lung condition, there was an increase in the frequency of the high-producer TGF-β1 allele (arginine). This allele was significantly associated with pretransplant fibrotic pathology (P<0.02) (n=45) when compared with controls (n=107) and with pretransplant nonfibrotic pathology (P<0.004) (n=50). This allele was also associated with allograft fibrosis in transbronchial biopsies when compared with controls (P<0.03) and with nonallograft fibrosis (P<0.01). Conclusion. The production of TGF-β1 is under genetic control, and this in turn influences the development of lung fibrosis. Hence, the TGF-β1 genotype has prognostic significance in transplant recipients.

Genotypic variation in the transforming growth factor-beta1 gene: association with transforming growth factor-beta1 production, fibrotic lung disease, and graft fibrosis after lung transplantation

Several lines of evidence have suggested that ganglioside GM1 stimulates neuronal sprouting and enhances the action of nerve growth factor (NGF), but its precise mechanism is yet to be elucidated. We report here that GM1 directly and tightly associates with Trk, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances neurite outgrowth and neurofilament expression in rat PC12 cells elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. The potentiation of NGF activity by GM1 appears to involve tyrosine-autophosphorylation of Trk, which contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain. In the presence of GM1 in culture medium, there is a > 3-fold increase in NGF-induced autophosphorylation of Trk as compared with NGF alone. We also found that GM1 could directly enhance NGF-activated autophosphorylation of immunoprecipitated Trk in vitro. Monosialoganglioside GM1, but not polysialogangliosides, is tightly associated with immunoprecipitated Trk. Furthermore, such tight association of GM1 with Trk appears to be specific, since a similar association was not observed with other growth factor receptors, such as low-affinity NGF receptor (p75NGR) and epidermal growth factor receptor (EGFR). Thus, these results strongly suggest that GM1 functions as a specific endogenous activator of NGF receptor function, and these enhanced effects appear to be due, at least in part, to tight association of GM1 with Trk.

https://www.pnas.org/content/pnas/92/11/5087.full.pdf

Ganglioside GM1 binds to the Trk protein and regulates receptor function

The Genomic Action Potential

A gene for neuronal plasticity in the mammalian brain: Zif268/Egr-1/NGFI-A/Krox-24/TIS8/ZENK?

Nerve growth factor induces transcription of transforming growth factor-beta 1 through a specific promoter element in PC12 cells.

Nerve growth factor, a differentiating agent, and epidermal growth factor, a mitogen, increase the activities of different S6 kinases in PC12 cells.

The PC12 rat pheochromocytoma cell line is used extensively as a model to study neuronal differentiation. These cells resemble adrenal chromaffin cells, differentiating both morphologically and biochemically when cultured in the presence of dexamethasone, but develop a sympathetic neuron-like phenotype when cultured in the presence of nerve growth factor. Expression of the protein product of the v-src oncogene in PC12 cells also induces neurite outgrowth similar to that resulting from nerve growth factor treatment (Alema et al: Nature 316:557-559, 1985). It is thus possible that c-src or a src-like tyrosine kinase participates in the signal transduction pathway by which nerve growth factor acts on PC12 cells. In this study a temperature-sensitive v-src gene has been introduced into PC12 cells. When cultures of these src-transformed cells are switched from the nonpermissive (40 degrees C) to the permissive (37 degrees C) temperature they elaborate neurites. The differentiation induced by src has been compared with that induced by nerve growth factor by determining whether src-transformed PC12 cells at 37 degrees C exhibit the same biochemical alterations as those induced in PC12 cells treated with nerve growth factor. Neurite extension at 37 degrees C in v-src-transformed cells, like NGF-induced differentiation, is accompanied by an increase in the nerve growth factor-inducible large external (NILE) protein. However, neurite extension in v-src-transformed cells is not blocked by the protein kinase inhibitor K-252a, which completely blocks NGF-induced neurite extension. Likewise, EGF receptor down-regulation and the development of saxitoxin and tetanus toxin binding sites are either much reduced or completely absent in src-differentiated compared with NGF-differentiated PC12 cells.

Differentiation of PC12 cells with v‐src: Comparison with nerve growth factor

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.

Brian B. Rudkin

Papers

Nerve growth factor induces transcription of transforming growth factor-beta 1 through a specific promoter element in PC12 cells.

Nerve growth factor, a differentiating agent, and epidermal growth factor, a mitogen, increase the activities of different S6 kinases in PC12 cells.

Differentiation of PC12 cells with v‐src: Comparison with nerve growth factor

Differential Responses of the Phosphorylation of Ribosomal Protein S6 to Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells