Myocardin sumoylation transactivates cardiogenic genes in pluripotent 10T1/2 fibroblasts

Myocardin, a serum response factor (SRF)-dependent cofactor, is a potent activator of smooth muscle gene activity but a poor activator of cardiogenic genes in pluripotent 10T1/2 fibroblasts. Posttranslational modification of GATA4, another myocardin cofactor, by sumoylation strongly activated cardiogenic gene activity. Here, we found that myocardin's activity was strongly enhanced by SUMO-1 via modification of a lysine residue primarily located at position 445 and that the conversion of this residue to arginine (K445R) impaired myocardin transactivation. PIAS1 was involved in governing myocardin activity via its E3 ligase activity that stimulated myocardin sumoylation on an atypical sumoylation site(s) and by its physical association with myocardin. Myocardin initiated the expression of cardiac muscle-specified genes, such as those encoding cardiac alpha-actin and alpha-myosin heavy chain, in an SRF-dependent manner in 10T1/2 fibroblasts, but only in the presence of coexpressed SUMO-1/PIAS1. Thus, SUMO modification acted as a molecular switch to promote myocardin's role in cardiogenic gene expression.     

Sumoylation of Mdm2 by protein inhibitor of activated STAT (PIAS) and RanBP2 enzymes

Mdm2, a ubiquitin ligase that acts on p53, is regulated by sumoylation. In the current study, we identify the enzymes responsible for the sumoylation of Mdm2. When mammalian cells are co-transfected with cDNAs encoding Mdm2 and PIAS1 or PIASxbeta (protein inhibitor of activated STAT) as sumoylation enzymes, Mdm2 is highly sumoylated. Mdm2 is also sumoylated in an in vitro system containing PIASxbeta, PIAS1, and RanBP2. When several lysine residues of Mdm2 were sequentially mutated to arginine, the K182R mutant was not sumoylated in intact cells; however, in the in vitro system this mutant was sumoylated by PIAS1, PIASxbeta, and RanBP2 as efficiently as the wild-type Mdm2 protein. Lysine residues 182 and 185 map within the nuclear localization signal of Mdm2. A K185R mutant of Mdm2 is sumoylated in intact cells, whereas a K182R protein is not. Only a Mdm2 protein bearing the K182R mutation is localized exclusively in the cytoplasm. Because RanBP2 is a nuclear pore protein and PIAS proteins are localized within the nucleus, our data suggest that Mdm2 is sumoylated during nuclear translocation by RanBP2 and then further sumoylated once in the nucleus by PIASxbeta and PIAS1.