Proteomic-based analysis of nuclear signaling: PLCbeta1 affects the expression of the splicing factor SRp20 in Friend erythroleukemia cells.
An extensive body of evidence links inositide-specific phospholipase C (PLC) to the nucleus and the main isoform located in the nucleus is PLCbeta(1). Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend erythroleukemia cells differentiation and to induce cell cycle progression targeting cyclin D3. The aim of this study was to identify new proteins regulated by PLCbeta(1) overexpression, given the role exerted by its signaling in the nucleus during cell growth and differentiation. To identify novel downstream effectors of nuclear PLCbeta(1)-dependent signaling in Friend erythroleukemia cells, we performed the high-resolution 2-DE-based proteomic analysis. Using a proteomic approach we found that SRp20, a member of the highly conserved SR family of splicing regulators, was down-regulated in cells overexpressing nuclear PLCbeta(1) as compared with wild-type cells. Reduction in SRp20 was confirmed by 2-D Western blotting. Moreover, we have shown that nuclear PLCbeta(1) is bound to the SRp20 splicing factor. Indeed, by immunoprecipitation and subcellular fractioning, we have demonstrated that endogenous PLCbeta(1) and SRp20 physically interact in the nucleus. Here we show the existence of a PLCbeta(1)-specific target, the splicing factor SRp20, whose expression is specifically down-regulated by the nuclear signaling evoked by PLCbeta(1).
Bavelloni
Alberto
A
Laboratory of Cell Biology and Electron Microscopy, IOR, Bologna Italy.
Faenza
Irene
I
Cioffi
Gabriella
G
Piazzi
Manuela
M
Parisi
Daniela
D
Matic
Ivan
I
Maraldi
Nadir M
NM
Cocco
Lucio
L
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D016650:Fluorescein-5-isothiocyanate; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D015972:Gene Expression Regulation, Neoplastic; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008856:Microscopy, Fluorescence; D010449:Peptide Mapping; D054799:Phospholipase C beta; D011233:Precipitin Tests; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D007527:Isoenzymes; D016601:RNA-Binding Proteins; C106161:Srsf3 protein, mouse; D000068103:Serine-Arginine Splicing Factors; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D016650:Fluorescein-5-isothiocyanate