[Analysis of nuclear protein complexes comprising alpha-actinin-4 by 2D-electrophoresis and mass-spectrometry].
Actin-binding protein alpha-actinin-4 is a member of spectrin super family. It is located in the cytoplasm and in the nucleus. However, nuclear functions of alpha-actinin-4 are still not clear. In this study, we analyzed composition of nuclear protein complexes associated with alpha-actinin-4 in A431 cells. Using 2D electrophoresis, we have determined that about 50 different proteins may be associated with nuclear alpha-actinin-4. Using mass-spectrometry, we analyzed major proteins of these complexes. beta-Actin, alpha- and beta-tubulins, ribonucleoprotein A2/B1, which regulates splicing and is associated with beta-actin, peroxiredoxin-1, which is involved in oxidative stress, and glycolytic enzyme D-3-phosphoglycerate dehydrogenase were identified by MALDI-TOF. Detection of these proteins in nuclear complexes with alpha-actinin-4 may suggest that alpha-actinin-4 is involved in transcription and splicing. Presence of beta-actin in the investigated complexes was confirmed by tandem mass-spectrometry (MALDI-TOF-TOF). Immunoprecipitation of nuclear proteins with antibodies against alpha-tubulin confirmed association of alpha-actinin-4 with alpha-tubulin in the protein complex. Nuclear alpha-actinin-4 constitutes of 105 KDa fullsize isoform and two truncated isoforms of 65 and 75 kDa, whereas only the truncated isoform have been found in nuclear complexes with alpha-tubulin. These data suggest that alpha-actinin-4 is associated with a number of different nuclear protein complexes which may carry out different functions in the cell nucleus.
M G
MG
Khotin
L V
LV
Turoverova
E P
EP
Podol'skaia
I A
IA
Krasnov
A V
AV
Solov'eva
V Iu
VIu
Aksenova
K E
KE
Magnusson
G P
GP
Pinaev
D G
DG
Tentler
D000185:Actinin / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D047468:Immunoprecipitation; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D009687:Nuclear Proteins; D020033:Protein Isoforms; D000185:Actinin