Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts.
Oncogenic Ras signaling has been long known to play an important role in tumorigenesis and human cancer. In this report, we have used the sensitive 2-D-DIGE coupled to MS for the identification of proteins differentially expressed at the cell membrane level between oncogenic H-RasV12-transformed wild-type and p38alpha-deficient mouse embryo fibroblasts (MEFs). Following trifluoroethanol solubilization, 76 proteins were found to be differentially regulated. After PMF, 63 spots containing 42 different proteins were unequivocally identified by MALDI-TOF MS coupled with database interrogation. As expected, many of them were membrane proteins. Six proteins were selected for further validation studies based on their potential functional link with malignant transformation and signal transduction. These were prohibitin (PHB), protein disulfide isomerase 3 (PDIA3), focal adhesion kinase 2 (FAK2), c-GMP dependent protein kinase 2 (KGP2), NADH-ubiquinone oxidoreductase 30 kDa subunit (NUGM) and translationally controlled tumor protein (TCTP). All these proteins were up-regulated in the membranes of H-RasV12-transformed p38alpha-/-cells, except for prohibitin, which was down-regulated. An excellent correlation was found between DIGE results and Western blot studies, indicating the reliability of the 2-D-DIGE analysis. The available evidence about the putative function of the identified proteins supports the emerging role of p38alpha as a negative regulator of tumorigenesis. Further studies are in progress to elucidate the implications of these findings in the regulation of H-Ras-induced transformation by p38alpha signaling.
Protein Technology Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain.
Patricia
P
Alfonso
Ignacio
I
Dolado
Aneta
A
Swat
Antonio
A
Núñez
Ana
A
Cuadrado
Angel R
AR
Nebreda
J Ignacio
JI
Casal
D000818:Animals; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology* / Q000473:pathology; D018014:Gene Transfer Techniques; D011905:Genes, ras; D006801:Humans; D051379:Mice; D048308:Mitogen-Activated Protein Kinase 14 / Q000502:physiology*; D000091402:Prohibitins; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D013347:Subcellular Fractions; D000090324:Tumor Protein, Translationally-Controlled 1
C000717328:PHB protein, human; D000091402:Prohibitins; D020543:Proteome; C000715647:TPT1 protein, human; C000715648:Tpt1 protein, mouse; D000090324:Tumor Protein, Translationally-Controlled 1; D048308:Mitogen-Activated Protein Kinase 14