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Distinctive regulation of v-Src-associated phosphatidylinositol 3-kinase during PC12 cell differentiation.

ABSTRACT: In chicken embryo fibroblasts, the binding of v-Src to PtdIns 3-kinase requires Src homology domains, SH3, SH2 and the SH1 or kinase domain, which induces the cytoskeletal disruption associated with fibroblast transformation. In the rat phaeochromocytoma PC12 cell line, v-Src has a different effect on the cytoskeleton, inducing neurite extension rather than cytoskeletal disruption. Here we show that v-Src-induced neurite outgrowth is suppressed by the selective PtdIns 3-kinase inhibitor LY294002, suggesting that this effect of v-Src in PC12 cells also requires the activity of the lipid kinase. However, in contrast with chicken embryo fibroblasts, the association of PtdIns 3-kinase with v-Src in PC12 cells is delayed until several hours after activating the v-Src tyrosine kinase. Furthermore the v-Src-associated p85 regulatory subunit of PtdIns 3-kinase is not phosphorylated on tyrosine in PC12 cells and associates only weakly with isolated v-Src homology domains (SH3/SH2) in a Src kinase-independent manner. However, p85 and v-Src both associate with an unidentified protein (of molecular mass approx. 68 kDa; termed p68), which becomes tyrosine phosphorylated concomitantly with the association of both p85 and PtdIns 3-kinase with v-Src in PC12 cells. Thus we conclude that the mode of regulation of v-Src-associated PtdIns 3-kinase is cell-context-dependent and that p68 might act as an adaptor protein to mediate the association of p85 and v-Src in PC12 cells. The different regulation of PtdIns 3-kinase in PC12 and in chicken embryo fibroblasts in response to v-Src activity might reflect the different cytoskeletal rearrangements induced by this oncoprotein in the two cell types.

SUBMITTER: Haefner B

PROVIDER: S-EPMC1218967 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase.

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Phosphatidylinositol 3-kinase modulation of trophoblast cell differentiation

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| S-EPMC7912711 | biostudies-literature

Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation.

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Project description:Shootin1 is a protein involved in neuronal polarization, and has been shown to be a key molecule for the positive/negative feedback loop for axon induction required during neuronal symmetry breaking. To better understand the molecular basis of shootin1 dynamics, we analysed the regulatory pathways and the expressional status of shootin1 gene during NGF-induced neuronal differentiation. We demonstrated that the isoform-1 and isoform-2 of shootin1 is differentially expressed during neuronal differentiation. By blocking individual downstream pathways of NGF signalling, we found that PI3K/Akt pathway plays a major role in the expression of shootin1 isoform-2. Western blot and RT-PCR results showed that the isoform-1 of shootin1 is constitutively expressed, while the isoform-2 is expressed in a manner that is strictly dependent on NGF-stimulation. Isoform-specific RT-PCR results demonstrated that the differential expression of the isoform-1 and isoform-2 of shootin1 is a consequence of alternative splicing of shootin1 pre-mRNA, in response to NGF-signalling. Collectively these findings provide the first information on the molecular mechanisms regulating the expression of shootin1 gene and represent the first example of NGF-induced alternative splicing process that has a regulatory role in neuritogenesis.

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Phosphatidylinositol 4-phosphate 5-kinase ? negatively regulates nerve growth factor-induced neurite outgrowth in PC12 cells.

Project description:Neurite outgrowth, a cell differentiation process involving membrane morphological changes, is critical for neuronal network and development. The membrane lipid, phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), is a key regulator of many important cell surface events of membrane signaling, trafficking and dynamics. This lipid is produced mainly by the type I PI 4-phosphate 5-kinase (PIP5K) family members. In this study, we addressed whether PIP5K?, an isoform of PIP5K, could have a role in neurite outgrowth induced by nerve growth factor (NGF). For this purpose, we knocked down PIP5K? in PC12 rat pheochromocytoma cells by stable expression of PIP5K? microRNA that significantly reduced PIP5K? expression and PIP2 level. Interestingly, NGF-induced neurite outgrowth was more prominent in PIP5K?-knockdown (KD) cells than in control cells. Conversely, add-back of PIP5K? into PIP5K? KD cells abrogated the effect of NGF on neurite outgrowth. NGF treatment activated PI 3-kinase (PI3K)/Akt pathway, which seemed to be associated with reactive oxygen species generation. Similar to the changes in neurite outgrowth, the PI3K/Akt activation by NGF was potentiated by PIP5K? KD, but was attenuated by the reintroduction of PIP5K?. Moreover, exogenously applied PIP2 to PIP5K? KD cells also suppressed Akt activation by NGF. Together, our results suggest that PIP5K? acts as a negative regulator of NGF-induced neurite outgrowth by inhibiting PI3K/Akt signaling pathway in PC12 cells.

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Hypergravity stimulation enhances PC12 neuron-like cell differentiation.

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