The expression and function of protein kinase C isoforms in differentiating neuroblastoma cells

Sofia Fagerström

Research output: ThesisDoctoral Thesis (compilation)

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.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Department of Translational Medicine
Supervisors/Advisors
  • [unknown], [unknown], Supervisor, External person
Award date1998 May 20
Publisher
ISBN (Print)91-628-2975-0
Publication statusPublished - 1998

Bibliographical note

Defence details

Date: 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

Keywords

  • Neurologi
  • neurophysiology
  • neuropsychology
  • Neurology
  • p130cas
  • PKC
  • fte-1
  • neuroblastoma
  • SH-SY5Y cells
  • growth cones
  • neuronal differentiation
  • neuropsykologi
  • neurofysiologi

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