In vitro sumoylation and ubiquitination of annexin I and helicase activity

Annexin I, a protein previously termed as lipomodulin or lipocortin I, is a member of the family that bind to phospholipids in a Ca2+ dependent manner. This protein is a major substrate of the oncogenic tyrosine kinases such as c‐met and c‐src, thus being proposed to be involved in cell proliferation and differentiation. While annexin I in the nuclei is thought to be involved in cell transformation and/or proliferation, the nuclear translocation of annexin I is facilitated by oxidative stress and possibly by phosphorylation (and Ca2+ signal). Since annexin I contains the consensus motif, ψKxE, for sumoylation, we tested if annexin I can be sumoylated by Ubc 9 in vitro. We found that purified bovine annexin I is indeed sumoylated by Ubc 9, and that E3 of the sumoylation system is not required. While nonmodified annexin I has no helicase activity, the sumoylation unmasked a helicase activity. On the other hand, the mono‐ubiquitination of annexin I was found to be catalyzed by Ubc H2a and H2b (hHR 6). This reaction required E3 of the ubiquitination system in the cytosolic fraction of HeLa cells. Difference in properties of their helicase activities will be discussed. These observations suggest that annexin I may play an important role in DNA damage bypass and mutagenesis mediated through modifications with ubiquitin and sumo. (Supported in parts by a grant from the Susan G. Komen Breast Cancer Foundation BCTR0600711)