Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

Abstract

The v-vis gene encodes p28sis, the transforming protein of simian sarcoma virus. This gene resulted from a fusion of the env gene of simian sarcoma-associated virus and the woolly monkey gene for the B chain of platelet-derived growth factor (PDGF). Previous work has shown that the v-sis gene product undergoes signal sequence cleavage, glycosylation, dimerization, and proteolytic processing to yield a secreted form of the protein. It transport across the endoplasmic reticulum is blocked by the introduction of a charged amino acid residue within the signal sequence, the protein does not dimerize, is not secreted, and is no longer transforming as assayed by focus-forming ability in NIH 3T3 cells. Instead, this mutant protein localizes to the nucleus as demonstrated by both indirect immunofluorescence and cell fractionation. Using a series of deletion mutations, we delimited an amino acid sequence within this protein which is responsible for nuclear localization. This region is completely conserved in the predicted human c-sis protein, although it lies outside of regions required for transformation by the v-sis gene product. This nuclear transport signal is contained within amino acid residues 237 to 255, RVTIRTVRVRRPPKGKHRK. An amino acid sequence containing these residues is capable of directing cytoplasmic v-sis mutant proteins to the nucleus. This sequence is also capable of directing less efficient nuclear transport of a normally cytoplasmic protein, pyruvate kinase. Pulse-chase experiments indicate that the half-lives of nuclear and cytoplasmic v-sis mutant proteins are approximately 35 min. Using the heat-inducible hsp70 promoter from Drosophila melanogaster, we showed that the nuclear v-sis protein accumulates in the nucleus within 30 min of induction. The identification of a nuclear transport signal in the v-sis gene product raises interesting questions regarding the possibility of some function for PDGF or PDGF-related molecules in the nucleus.

A novel mechanism regulating growth factor association with the cell surface: identification of a PDGF retention domain.

“…In the case of growth factors, recent studies have implicated a positively charged, 7-amino-acid stretch in acidic fibroblast growth factor (aFGF) as responsible for targeting to the nucleus and required for mitogenic activity as well (Imamura et al 1990). There are reports that PDGF can achieve a nuclear location (Lee et al 1987;Yeh et al 1987), and there is evidence that the nuclear targeting signal (Lee et al 1987) corresponds to the region identified by us as a membrane retention domain. Our present studies demonstrate that deletion of this putative nuclear targeting sequence from either chain of PDGF had no effect on its mitogenic properties.…”

The alternatively spliced exon of the platelet-derived growth factor A chain encodes a nuclear targeting signal.

“…Additionally, we noted a similarity between the four residues at the C terminus of the long A chain, LKPT, and a sequence in the B chain, LKET. This LKXT sequence is not required for the nuclear targeting of the B chain (15) and is probably not required for the targeting of the A chain.…”

“…Plasmids transfected into CV-1 cells by the calcium phosphate procedure were pSV2CAT (A), pDM117 (B), pSV2DHFR (C), pDM118 (D), pAL79 (E), and pAL125 (F). pSV2CAT (11), pSV2DHFR (20), and pAL79 (containing PK [15]) contain the wild-type genes under the control of the simian virus 40 early promoter. The CAT-NTS and PK-NTS fusions have the sequence PRESGKKRKRKRLKPT appended to the C terminus of each protein, and the predicted molecular mass is increased from 25.6 to 27.6 kilodaltons for CAT and from 21.6 to 23.6 kilodaltons for DHFR.…”

“…coma virus vector, yielding pDM134 (nonsecreted long form) and pDM135 (nonsecreted short form). The plasmids were transfected into the African green monkey kidney cell line CV-1, and 36 to 48 h posttransfection, the cells were assayed by indirect immunofluorescence (15) to determine the location of the protein. The native long form of the A chain showed the characteristic endoplasmic reticulum and Golgi staining patterns of a secreted protein (Fig.…”

“…The 3' end of the long form of the A chain containing the NTS was added directly to the ends of CAT and DHFR genes by using the StuI site (residue 970) in the PDGF A-chain gene, and the fusions were placed under the Rous sarcoma virus promoter, creating pDM117 (CAT-NTS) and pDM118 (DHFR-NTS). The PK-NTS fusion (pAL125) was created by joining the BstXI site (residue 1567) of the PK-coding sequence in pAL79 (15) to the StyI site (residue 973) in exon 6 of the A chain by using EcoRI linkers to maintain the correct reading frame. The hybrid genes were expressed in CV-1 cells, and the transfected cells were examined by indirect immunofluorescence for the location of the hybrid proteins.…”