Introduction Phospholipase Cb1 (PLC-β1) is a key player in the regulation of nuclear inositol lipid
signaling and of a wide range of cellular functions, such as proliferation and differentiation (1,2,3).
PLCb1 signaling depends on the cleavage of phosphatidylinositol 4,5-bisphosphate and the
formation of the second messengers diacylglycerol and Inositol tris-phosphate which activate
canonical protein kinase C (cPKC) isoforms. Here we describe a proteomic approach to find out a
potential effector of nuclear PLC-b1 dependent signaling during insulin stimulated myogenic
differentiation.
Methods Nuclear lysates obtained from insulin induced C2C12 myoblasts were
immunoprecipitated with anti-phospho-substrate cPKC antibody. Proteins, stained with Comassie
blue, were excised, digested and subsequently analysed in LC-MS/MS. For peptide sequence
searching, the mass spectra were processed and analyzed using the Mascot MS/MS ion search
program with the NCBI database. Western blotting, GST-pull down and co-immunoprecipitation
were performed to study the interaction between eEF1A2 and cPKCs. Site direct mutagenesis was
performed to confirm the phosphorylated motif recognized by the antibody. Immunofluorescence
analysis, GFP-tagged eEF1A2 vector and subcellular fractionation were performed to study nuclear
localization and relative distribution of eEF1A2.
Results We have previously shown that PLC-β1 is greatly increased at the nuclear level during
insulin-induced myoblasts differentiation and that this nuclear localization is essential for induction
of differentiation. Thus, nuclear proteins of insulin stimulated C2C12 myoblasts, were
immunoprecipitated with an anti-phospho-substrate cPKC antibody. After Electrophoretic gel
separation of proteins immunoprecipitated, several molecules were identified by LC-MS/MS.
Among these most relevant and unexpected was eukaryotic elongation factor 1 alpha 2 (eEF1A2).
We found that eEF1A2 is phosphorylated by PKCb1 and that these two molecules coimmunolocalized
at the nucleolar level. eEF1A2 could be phosphorylated in many sites among
which both threonine and serine residues. By site direct mutagenesis we demonstrated that it is the
serine residue of the motif recognized by the antibody that is specifically phosphorylated by
PKCb1. The silencing of PLCb1 gives rise to a reduction of expression and phosphorylation levels
of eEF1A2 indicating this molecule as a target of nuclear PLCb1 regulatory network during
myoblasts differentiation.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:2226 |
| Date | 19 January 2009 |
| Creators | Piazzi, Manuela <1980> |
| Contributors | Cocco, Lucio Ildebrando |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | Italian |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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