Effects of phosphorylation and mutation R145G on human cardiac troponin I function.

We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.

Cloning of adenovirus type 12 E1 genes into a retroviral vector and their differential splicing in mouse cells.

Early region 1 (E1) of adenovirus type 12 (Ad12) genome is able to transform nonpermissive primary rodent cells in vivo and in vitro. To analyse the role of the E1a gene products alone or in connection with the 58-kDa protein encoded by E1b during oncogenic transformation, we have cloned genomic fragments of both subregions into the retroviral vector, pZIP-NeoSV(X)1. Both constructs are expressed in mouse 3T3 cells, but, in contrast to E1b, the amount of genomic retroviral RNA carrying E1a-specific sequences was low in transfected psi2 cells and not detectable in infected NIH3T3 cells. Nevertheless, we could demonstrate the integration of the complete E1a-carrying provirus into the NIH3T3 genome. However, after infection of primary mouse embryo fibroblasts, high retrovirus-mediated expression of E1a leads to the immortalization of these cells. In the derived cell line, only the 13S transcript and the unspliced form of E1a RNA could be demonstrated, but not the 12S transcript. These results demonstrate that the ratio of genomic vs. subgenomic retroviral RNAs of Ad12 E1-carrying vectors is dependent on the cloned insert and the cell system used.