Abstract
The human neuroblastoma cell line SH-SY5Y can be induced to differentiate into a neuronal sympathetic phenotype after treatment with 16 nM 12-O-tetradecanoyl phorbol 13-acetate (TPA) in serum, or by a combination of the growth factors basic fibroblast growth factor and insulin-like growth factor-I (bFGF/IGF-I). SH-SY5Y cells stably transfected with TrkA, the nerve growth factor (NGF) receptor, differentiate in response to NGF. The function of protein kinase C (PKC) isoforms in these processes was investigated. SH-SY5Y cells were shown to express PKC-alpha, PKC-epsilon and PKC-zeta protein. All three isoforms remained present during TPA- and bFGF/IGF-I-induced differentiation, while a higher TPA concentration (1.6 µM), that does not promote differentiation, down-regulated PKC-alpha completely. The use of the PKC inhibitor GF 109203X demonstrated that TPA-induced differentiation is completely, while growth factor induced differentiation is partially, PKC-dependent. Cells with down-regulated PKC-alpha differentiated in respons to growth factors, implicating a role for PKC-epsilon in this process. Growth cones isolated from differentiated SH-SY5Y cells were enriched in PKC-alpha and PKC-epsilon. Addition of GF 109203X provoked retraction of growth cone filopodia. Together with the finding that cells with down-regulated PKC-alpha form functional growth cones, this result suggests a role for PKC-epsilon in growth cone function.
A yeast two-hybrid assay was performed using the regulatory domain of PKC-epsilon as a bait, in order to isolate PKC-epsilon binding proteins. A clone encoding the ribosomal protein fte-1/S3a was isolated and was shown to interact with the regulatory domains of PKC-alpha and PKC-epsilon in vitro. Fte-1 expression decreased in differentiating SH-SY5Y cells, and overexpression of fte-1 prevented induced differentiation, suggesting a function for fte-1 as a negative regulator of PKC.
The docking protein p130cas was shown to be PKC-dependently tyrosine phosphorylated in differentiating SH-SY5Y cells. The early phase of p130cas phosphorylation (5-30 min) was independent of pp60c-src or other herbimycin A sensitive kinases, while the late phase could be blocked by herbimycin A. p130cas protein and tyrosine phosphorylated p130cas were enriched in growth cones, indicating a function for the protein in this structure.
A yeast two-hybrid assay was performed using the regulatory domain of PKC-epsilon as a bait, in order to isolate PKC-epsilon binding proteins. A clone encoding the ribosomal protein fte-1/S3a was isolated and was shown to interact with the regulatory domains of PKC-alpha and PKC-epsilon in vitro. Fte-1 expression decreased in differentiating SH-SY5Y cells, and overexpression of fte-1 prevented induced differentiation, suggesting a function for fte-1 as a negative regulator of PKC.
The docking protein p130cas was shown to be PKC-dependently tyrosine phosphorylated in differentiating SH-SY5Y cells. The early phase of p130cas phosphorylation (5-30 min) was independent of pp60c-src or other herbimycin A sensitive kinases, while the late phase could be blocked by herbimycin A. p130cas protein and tyrosine phosphorylated p130cas were enriched in growth cones, indicating a function for the protein in this structure.
Original language | English |
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Qualification | Doctor |
Awarding Institution |
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Supervisors/Advisors |
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Award date | 1998 May 20 |
Publisher | |
ISBN (Print) | 91-628-2975-0 |
Publication status | Published - 1998 |
Bibliographical note
Defence detailsDate: 1998-05-20
Time: 10:15
Place: Medelhavet, Wallenberg Laboratory, University Hospital MAS
External reviewer(s)
Name: Jaken, Susan
Title: Dr
Affiliation: [unknown]
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The information about affiliations in this record was updated in December 2015.
The record was previously connected to the following departments: Molecular Medicine (013031200)
Subject classification (UKÄ)
- Cancer and Oncology
Free keywords
- Neurologi
- neurophysiology
- neuropsychology
- Neurology
- p130cas
- PKC
- fte-1
- neuroblastoma
- SH-SY5Y cells
- growth cones
- neuronal differentiation
- neuropsykologi
- neurofysiologi