Ability of adenovirus 5 E1A proteins to suppress differentiation of BC3H1 myoblasts correlates with their binding to a 300 kDa cellular protein.

Abstract

We have used deletion mutants to define the regions in Ad5 ElA proteins necessary to suppress differentiation of mouse BC3H1 myoblasts. We examined the differentiation of cells infected at a low multiplicity with viruses containing the ElA deletions and constructed so as to produce only the smaller of the two major ElA proteins. Only four of the mutant viruses containing deletions within the N-terminal 69 residues failed to suppress differentiation as judged by changes in morphology and in levels of muscle-specific a-actin mRNA and creatine kinase activity. The results were confirmed by analyses of lines of cells stably transfected with representative ElA mutants. The mouse cellular proteins to which mutant ElA proteins bound were identified by immunoprecipitating ElA proteins specifically from infected BC3H1 cells and by analyzing the precipitates on denaturing gels. Bands of proteins of 300, 130, 107, 105 (the retinoblastoma product), and 60 kDa (cyclin A) were distinguished. Failure to suppress differentiation correlated with loss of binding to the 300-kDa protein but not to any of the others. The regions of ElA defined in this way have been shown to be required for several other activities, including enhancer repression and transformration. One function of the 300-kDa protein appears to be to facilitate the action of transcriptional enhancers of differentiation-specific genes.

Multiple pathways for gene activation in rodent cells by the smaller adenovirus 5 E1A protein and their relevance to growth and transformation

“…bound p107 and pRb less strongly. We found reduced binding ofpRb by dl1143/520 in other cells (Howe et al, 1990;Mymryk et al, 1992), indicating that between residues 35 and 60 in E1A deletions such as dl1143 that are larger than dl1103 and dl1104 affect binding to pRb. These results are summarized in Table 1 and Fig.…”

“…The construction of the mutant Ad5 viruses dl1101/520 to dl1109/520, (Ill 141/520, dI1142/ 520 and dl1143/ 520 that produce only the 243R E1A protein and contain single deletions in exon 1, as welt as viruses containing different pairs of these exon 1 deletions in the dl520 background, have been described (Jelsma et al, 1988(Jelsma et al, , 1989Howe et al, 1990). Ad5 dl1151 lacks residues 151 to 289 of the larger E1A protein and so effectively expresses only the first exon of E1A (Mymryk et al, 1992).…”

“…[Dl1109/520 has given low levels of E1A protein before (Mymryk et al, 1992); we assume it was because the protein was unstable, but we have not studied the question further.] In addition to bands of these proteins, there were bands corresponding to proteins of 400K, 300K, 107K and 100K.…”

“…In the upper part of the figure, the deletions in each of the mutants used in this study are indicated by rectangles, together with the numbers of the first and last residues deleted. Below this, results with these mutants are summarized with rectangles that mark regions required in the cell types indicated, for binding to cellular proteins p400, p300, p107 and pRb (this paper), for general trans-activation (Jelsma et al, 1988), for transformation (Jelsma et al, 1989), for induction of mitosis (Howe & Bayley, 1992), cellular DNA synthesis (Howe et al, 1990) and gene expression by exon 1 (this paper), for the repression of enhancer-driven transcription (Jelsma et al, 1989), and for the suppression of myogenic differentiation (Mymryk et al, 1992). Solid rectangles indicate esential regions.…”

Granulocytic differentiation of myeloid progenitor cells by p130, the retinoblastoma tumor suppressor homologue

“…When the 32Dcl3 cells are treated with G-CSF in the absence of IL-3, they exit from the cell cycle and terminally di erentiate to granulocytes. As are the cases of muscle or neuronal di erentiation (Gu et al, 1993;Mymryk et al, 1992;Kalman et al, 1993;Webster et al, 1988;Boulukos and Zi , 1993;Tiainen et al, 1996), this G-CSF-dependent granulocytic di erentiation is blocked by ectopic expression of the E1A oncoprotein, which binds and functionally inactivates the pRB family of pocket proteins (Whyte et al, 1988;Giordano et al, 1991;Ewen et al, 1991;Cobrinik et al, 1993). The observation indicates a requirement for the biologically active pRB family proteins in the process of granulocytic di erentiation.…”