Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling.
Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades.
Kim
Euikyung
E
Institue of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, Korea.
Lee
Seunghwan
S
Mian
Md Firoz
MF
Yun
Sang Uk
SU
Song
Minseok
M
Yi
Kye-Sook
KS
Ryu
Sung Ho
SH
Suh
Pann-Ghill
PG
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002645:Chickens; D004815:Epidermal Growth Factor / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism; D020394:Vault Ribonucleoprotein Particles / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases; D000099187:Major Vault Protein
D000081247:CSK Tyrosine-Protein Kinase; D004815:Epidermal Growth Factor; D048049:Extracellular Signal-Regulated MAP Kinases; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D020394:Vault Ribonucleoprotein Particles; D019061:src-Family Kinases; D000099187:Major Vault Protein; C000631346:CSK protein, human