Elevated blood ATP and increased red blood cell (RBC) ATP transport is associated with cystic fibrosis (CF). In this report, we demonstrate the presence of the wild-type and the DeltaF508 mutant form of the CF transmembrane conductance regulator protein in RBC membranes and its putative interaction with ecto-apyrase, an ATP hydrolyzing enzyme also present in the RBC membrane. RBC membranes of control and DeltaF508 individuals and of wild-type and CF transmembrane conductance regulator-knockout mice were examined by immunoblot using several antibodies directed against different epitopes of this protein. These experiments indicated that human RBC membranes contain comparable amounts of the wild-type CF transmembrane conductance regulator protein and the DeltaF508 mutant form of the protein, respectively. CF transmembrane conductance regulator protein was also detected in wild-type mouse RBC membranes but not in the gene knockout mouse RBC membranes. Antibodies directed against ecto-apyrase co-immunoprecipitated CF transmembrane conductance regulator protein of human RBC membranes indicating a physical interaction between these two membrane proteins consistent with ATP transport and extracellular hydrolysis. We conclude that RBCs are a significant repository of CF transmembrane conductance regulator protein and should provide a novel system for evaluating its expression and function.

A single phosphorylation event at T-antigen residue Thr124 regulates initiation of simian virus 40 DNA replication. To explore this regulatory process, a series of peptides were synthesized, centered on Thr124. These peptides contain a nuclear localization signal (NLS) and a recognition site for cyclin/Cdk kinases. When unphosphorylated, the "CDK/NLS" peptides inhibit T-antigen assembly and bind non-sequence specifically to DNA. However, these activities are greatly reduced upon phosphorylation of Thr124. Similar results were obtained by using peptides derived from the CDK/NLS region of bovine papillomavirus E1. Related studies indicate that residues in the NLS bind to DNA, whereas those in the CDK motif regulate binding. These findings are discussed in terms of the control of T-antigen double hexamer assembly and initiation of viral replication.Cell cycle-dependent phosphorylation events regulate the initiation of DNA synthesis in eukaryotes (18,33,50). At the molecular level, however, relatively little is known about these processes. Therefore, model systems, such as that based on simian virus 40 (SV40), are being used to gain insights into phosphorylation-dependent regulation of DNA replication. Initiation of SV40 DNA replication depends upon the binding of the virally encoded T-antigen (T-ag) to the SV40 origin (reviewed in references 3, 17, 18, and 58). Upon binding to the origin, T-ag monomers assemble sequentially into hexamers and then double hexamers (13,15,42,(66)(67)(68). When assembled into a double hexamer, T-ag becomes a DNA helicase that is able to unwind the SV40 origin (4,14,25,59,62,71).In the regulation of SV40 replication, phosphorylation of T-ag on Thr124 is the sole posttranslational modification required for initiation of replication (44,57). Formation of the first hexamer is independent of the phosphorylation status of Thr124; therefore, the phosphorylation of Thr124 is required at a point subsequent to initial hexamer formation (1,45,47,55). Consistent with these studies, on subfragments of the core origin that support double-hexamer formation (35, 61), phosphorylation of Thr124 selectively promotes the formation of the second hexamer (1). The region of T-ag flanking Thr124 contains additional residues that play regulatory roles during SV40 DNA replication. For instance, T-ag residues 126 to 132 contain the nuclear localization signal (NLS) used for nuclear translocation (reviewed in references 22 and 27). Furthermore, the T-ag NLS is flanked by residues that serve as the recognition site for cyclin/Cdk kinases (16, 48), an arrangement found in many other proteins (reviewed in references 26 and 28)To further explore the regulation of T-ag assembly and initiation of viral replication, a series of peptides derived from the CDK/NLS region of T-ag, centered on Thr124, were synthesized. Initial studies revealed that certain of these peptides bind to DNA in a phosphate-dependent manner and interfered with the formation of T-ag hexamers and double hexamers. To establish whether these observatio...