Abstract
The mechanism of SV40-induced cellular transformation was investigated by two-dimensional gel analysis of 35S- and 32P-labeled proteins of various cells. These included rat and mouse cells, either transformed or abortively infected by SV40 wild type, small t deletion mutants, and a large T temperature-sensitive mutant. Synthesis, turnover, or (de)phosphorylation of multiple protein spots was found to be reproducibly and quantitatively influenced by the transformed and/or infected status. Several of these alterations were attributable to the biological activity of either large T or small t antigen. Most changes in 35S-labeled proteins corresponded to a decreased intensity of the gel spots in transformed cells, while hyperphosphorylated proteins were more common than hypophosphorylated ones. About half of the polypeptide alterations in 35S-and 32P-labeled SV40-transformed rat cells, including a set of 35S-labeled small t-dependent changes were shared by Rous sarcoma virus-transformed cells. In contrast, small t-dependent (de)phosphorylation was rarely detected. Phosphoamino acid analysis of selected phosphoprotein spots of rat cells and alkaline hydrolysis of whole two-dimensional gels did not reveal any evidence for increased tyrosine-specific phosphorylation after SV40-induced transformation. Abortively infected mouse cells showed many protein alterations, also observed in stably transformed cells. However, the latter cells contained additional changes, also affecting several phosphoproteins and possibly related to the establishment of transformation. These findings are discussed in relation to the biological functions, known or presumed, for SV40 large T and small t antigens during transformation.
Highlights
These included rat and mouse cells, either transformed in transformed cells [6], including a 36-kDa protein [7,8,9], a or abortively infected b y SV40 wild type, small t deletion mutants, and a large T temperature-sensitive mutant
There is some evidence that protein phosphorylation is important inSV40-specifictransformation [17].SV40-encoded and SV40-induced tumor antigens were reported to have corresponded to a decreased intensity of the gel spots intrinsic or associated protein kinase activity [18,19,20,21,22,23].A numin transformed cells, while hyperphosphorylated pro- ber of investigators have compared untransformed cell lines teins were morecommon than hypophosphorylated with SV40-transformed derivative cells by two-dimensional ones
Changes unique to transformed FR3T3 cells were not recorded. Changes present in both RSVtransformed and some or all SV40-transformed rat cells were designated as fully transformation-dependent
Summary
CLassification of Changes Detected in Two-dimensional Gel Patterns of Transformed Rat and Transformed o r Infected Mouse Cells-Polypeptide changes were divided into several classes according to the combination of cell lines in which they were detected (Tables I and 11). Spotsfound to remain unaltered upon infection with ts A58 at the nonpermissive temperature but altered at the permissive temperature were designated large T-dependentS.mall t-dependent spots showed an altered intensity afteirnfection with SV40 WT, but infection with the dl 884 mutant had no detectable effect on these spots Each of these groups of alterations can be further subdivided into repressed and induced spots, i.e. spots with intensities that are eithderecreased or enhanced inthe transformed and/or infected cell line. 0, part of the two-dimensional gel pattern of F l l l cells (shown in A ) , containing reference spots (seealso Table 111) Both %- and =P-labeled polypeptides are classified into different groups according to the combination of rat cell l i e s in which they are affected. Changes in spot intensity are designated induced (+) or repressed (-) relative to theintensity of the corresponding spots of F l l l cells. =, no significant alteration was detected in that particular cell line
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