SEVERAL SMALL GTP-BINDING PROTEINS ARE STRONGLY DOWN-REGULATED IN SIMIAN-VIRUS-40 (SV40) TRANSFORMED HUMAN KERATINOCYTES AND MAY BE REQUIRED FOR THE MAINTENANCE OF THE NORMAL PHENOTYPE

High resolution two-dimensional (2-D) gel electrophoresis in combination with the blot overlay nucleotide binding assay was used to reveal low molecular weight GTP-binding proteins expressed by primary cultured, normal human keratinocytes. Forty one small GTP-binding proteins (30 isoelectric focusing, IEF; and 11 nonequilibrium pH gradient electrophoresis, NEPHGE) ranging in molecular weights from 18000 to 30000 and isoelectric points from 4.4 to 8.0 were detected and mapped in the master human keratinocyte database. Four GTP-binding proteins were identified by 2-D gel immunoblotting and these correspond to rap1 and 2 and two forms of rab6. rasProteins are most likely present in the [alpha(32)P]GTP 2-D gel blots but their levels may be too low to be detected by immunoblotting. Quantitative changes in the relative expression levels of [alpha(32)P]GTP-binding proteins in normal proliferating and simian virus 40 (SV40) transformed human keratinocytes (K 14) were determined by scintillation counting of the radioactive spots excised from the nitrocellulose blots. The results showed that thirteen of these proteins were not expressed in transformed K14 keratinocytes, implying that they may play a role in the maintenance of the normal cell phenotype.