Transformation of BALB3T3 cells caused by over-expression of rat CD98 heavy chain (HC) requires its association with light chain: mis-sense mutation in a cysteine residue of CD98HC eliminates its transforming activity.
CD98 is a 125-kDa glycoprotein (GP125) consisting of an 85-kDa heavy chain (HC) and a 40-kDa light chain (LC), and is highly expressed on the cell surface of activated lymphocytes and various tumor cells. In addition to the regulatory role of CD98HC in L-, y(+)L- and Xc-amino-acid transport systems, which are principally mediated by CD98LC, we have reported transforming activity of human CD98HC. In this study, we established and analyzed BALB3T3 clones transfected with cDNAs encoding wild-type and mutated rat CD98HC proteins designated as BrH/Wild, C103S, C325S and 103/325, in which 103 and/or 325 cysteine were intact or replaced with serine. Flow cytometry with anti-rat CD98HC MAb B3 revealed that wild-type and mutated CD98HC transfectants expressed almost the same amounts of rat CD98HC proteins on the cell surface. Immunoprecipitation with B3 revealed that exogenous rat CD98HC proteins were associated with endogenous mouse CD98LC by a disulfide bond in BrH/Wild and C325S, but not in C103S and 103/325 transfectants. These transfectants showed similar doubling times and leucine and arginine transport activities, as compared with BALB3T3 and control transfectants in monolayer culture. Wild-type and C325S transfectants, however, formed much larger anchorage-independent colonies than C103S, 103/325 and control transfectants in soft agar. In addition, wild-type and C325S transfectants showed tumorigenicity in nude mice, although C103S, 103/325 and control transfectants did not. These findings indicate that over-expression of CD98HC and its disulfide-linkage with CD98LC at the cell surface result in malignant transformation of murine fibroblasts.
Shishido
T
T
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Uno
S
S
Kamohara
M
M
Tsuneoka-Suzuki
T
T
Hashimoto
Y
Y
Enomoto
T
T
Masuko
T
T
D019943:Amino Acid Substitution; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003260:Contact Inhibition; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D005347:Fibroblasts / Q000473:pathology* / Q000637:transplantation; D027261:Fusion Regulatory Protein-1; D015972:Gene Expression Regulation, Neoplastic; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D020125:Mutation, Missense; D009368:Neoplasm Transplantation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014162:Transfection; D014410:Tumor Stem Cell Assay
D015703:Antigens, CD; D002352:Carrier Proteins; D018076:DNA, Complementary; D027261:Fusion Regulatory Protein-1; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D003553:Cystine; D003545:Cysteine