Faculty Bibliography

Using recombinant baculovirus expressed p53 and Raf proteins, we show that activated Raf-1 kinase can phosphorylate mouse p53 in vitro. We also show that co-expression of vRaf and p53 in NIH3T3 fibroblasts, potentiates the ability of p53 to transactivate a minimal promoter with a p53 cognate DNA binding site. A dominant negative mutant of Raf inhibits the transactivation function of p53 in NIH3T3 fibroblasts. Incubation of Raf-1 kinase with a series of p53 derived synthetic peptides maps the Raf-1 phosphorylation sites to the 27 amino terminal residue region of p53 which coincides with the transactivation domain. Phosphorylation occurs on serines which are phosphorylated in vivo. Our results suggest that the transactivation function of p53 can be regulated by a signaling cascade involving Raf

A primary response to many growth factor-induced transmembrane signals is the rapid activation of transcription of the proto-oncogene c-fos and other early-response genes, including the beta-actin gene. The c-raf gene encodes a cytoplasmic serine/threonine kinase, raf-1, whose activity is also responsive to transmembrane signals and which in mutant form can transform cells. Here we show that in transient assays, the v-raf protein, which is a constitutively activated oncogenic counterpart of raf-1, can transactivate transcription from two early-response promoters, including the c-fos promoter from human and murine cells and the human beta-actin gene promoter. Multiple elements of the human fos promoter, including the dyad symmetry element necessary for growth-factor induction, an octanucleotide direct repeat element, and the region spanning the sequence from nucleotides -225 to -99 can all serve as targets for raf induction. The c-myc promoter and two adenovirus-2 early promoters are not induced. These findings indicate that raf kinase, when activated by a transmembrane signal or by mutation of a regulatory domain, can phosphorylate a factor(s) capable of regulating transcription of the c-fos and actin genes. The oncogenic form of raf may transform by constitutively activating early response protooncogenes such as c-fos.

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane

We describe the first isolation of a human creatine kinase M cDNA clone and its mapping of the gene to human chromosome 19. A human creatine kinase M cDNA clone, pJN2CK-M, harboring a 1,160-bp insert, was isolated by colony hybridization with a previously sequenced chicken creatine kinase M cDNA probe. The human cDNA was used as a probe in Southern transfers of TaqI-digested genomic DNA from mouse/human somatic-cell hybrids to localize the human creatine kinase-M gene to chromosome 19. In situ hybridization of the tritiated cDNA probe to metaphase chromosomes of peripheral blood lymphocytes from normal males revealed significant labeling to chromosome 19. These two independent methodologies assign the human creatine kinase-M gene to chromosome 19. Since greater than 69% of the grains of chromosome 19 label band q13, the human creatine kinase-M gene has been mapped to 19q13. On the basis of high-resolution G-banding, the predominant labeling site was 19q13.2-q13.3