Identification of discrete structural domains in the retinoblastoma protein. Amino-terminal domain is required for its oligomerization.

To characterize the protein product of the retinoblastoma tumor suppressor gene biochemically, a recombinant human protein was produced in an Escherichia coli expression system. The full-length protein, p110RB, and an amino-terminal truncated form, p56RB, were expressed and purified to near homogeneity by conventional chromatographic procedures. To probe the structural organization of the retinoblastoma protein the purified proteins were subjected to partial proteolysis by trypsin, chymotrypsin, and subtilisin. Four discrete structural domains were revealed in p110RB by this method. Two of these structural domains, found in both p56RB and p110RB, were mapped to the carboxyl-terminal half of the protein and corresponded to the SV40 large T binding domains defined previously by genetic methods. In addition two distinct domains in the amino-terminal half of the protein were also defined. A potential role for these newly defined amino-terminal domains was uncovered upon analysis of the purified proteins by nondenaturing polyacrylamide gel electrophoresis. p110RB revealed multiple bands by this method, suggesting the formation of oligomeric structures by the protein, while this property was not observed for p56RB. Electron microscopy of p110RB revealed linearly extended, macromolecular structures, further supporting the formation of homologous higher order structures by the full-length retinoblastoma protein. Analysis of the interactions between retinoblastoma protein molecules using the yeast two-hybrid system confirmed that the retinoblastoma protein could self-associate and that this association was mediated by interactions between the amino- and carboxyl-terminal ends of the protein. These observations suggest that the retinoblastoma protein contains multiple structural domains with the amino-terminal domains being required for oligomerization of the full-length protein.

Retinoblastoma protein and the cell cycle.

Deregulation of the cell cycle may contribute one of the primary mechanisms through which cancer arises. Eukaryotic cell division has been found to be a strictly controlled process, involving response to both positive and negative external signals and assessment of the cell's internal state. Several recent discoveries have strengthened and refined the theory that the retinoblastoma protein is involved in the decision between cell division and differentiation, and have begun to provide an outline of the nature of this involvement.

Effect of staurosporine on the phorbol ester-induced activation of the Na+/H+ antiporter in smooth muscle cells.

Evidence is presented suggesting that the Na+/H+ antiporter activity of aortic smooth muscle cells is stimulated by protein kinase C activation. However, once the transporter has been activated, inhibitors of protein kinase C are not effective, supporting a model in which the Na+/H+ antiporter conserves memory of its activation by protein kinase C.

Suramin, an anti-cancer drug, inhibits protein kinase C and induces differentiation in neuroblastoma cell clone NB2A.

Protein kinase C purified from rat brain was found to be inhibited by suramin, a substance used originally in the therapy of antitrypanosomic infections and more recently proposed as antineoplastic agent. The inhibition of suramin was competitive with one of the substrates of the enzyme, ATP with a Ki of 10 microM. At concentrations adequate to inhibit the isolated enzyme, suramin was shown to slow the rate of proliferation of neuroblastoma NB2A cells in vitro and to induce their differentiation as evidenced by typical morphological changes.