cDNAs encoding the two putative forms of the DEN4 C, Cap1 and Cap2, were cloned into the pTM-1 expression vector (as reported for project Z01 BF 06001-01 LVVD). Cap1 and Cap2 RNA transcripts were translated in vitro in rabbit reticulocyte lysate, and Cap1 and Cap2 proteins were respectively identified among the products of translation. In addition, higher molecular weight forms of Cap1 and to a lesser extent Cap2 were identifiable and were shown to be ubiquitinated derivatives of the C proteins. Ubiquitin is conjugated to cellular proteins by a covalent linkage to Lys residues in the targeted protein. Ubiquitination of Cap1 was further investigated, since multi-ubiquitination of a Lys residue is known to be linked to ATP-dependent degradation of the substrate, whereas proteins containing mono-ubiquitin- ated Lys residues are stable. Both Cap1 and Cap2 were shown to be stable in vitro and in vivo in LLCMK2 cells. Since Cap1 contains 12 Lys residues in the 99-amino- acid sequence, and since analysis of our data showed that at least 5 ubiquitin moieties could be linked to Cap1, it was possible that Cap1 was not degraded, because no single Lys residue was multi-ubiquitinated. Analysis of PCR-generated mutants in which N-terminal Lys residues in Cap1 (in positions 5,6, and 16) were changed to Arg suggested that Lys-5 and/or Lys-6 were multi-ubiquitinated. To confirm multi-ubiquitination of Lys-5 or -6, RNA transcripts encoding a carboxy-terminally truncated 46-amino-acid Cap1 mutant protein, including only two additional Lys residues downstream from Lys-5,-6, and -16, were analyzed. Truncated Cap1 molecules containing either Lys or Arg at positions 5,6, and 16 appeared to contain multi-ubiquitinated Lys (evidently at the two available positions downstream from Lys-16) and were degraded in vitro, in contrast to results with full-length Cap1. Future plans include proving that in vitro degradation of truncated Cap1 is related to its ubiquitination, showing that truncated Cap1 is degraded in vivo, and explaining the stability of full-length Cap1. It is possible for example that full length Cap1 is stabilized by an association with another protein which is abrogated by its carboxy-terminal truncation.
