Abstract 1480: A Cullin 4a E3 Ligase Complex Mediates Rapid Degradation of the Calcineurin Regulatory Protein MCIP1.4 In Cardiac Myocytes.
Background: Activation of the protein phosphatase calcineurin is a fundamental signaling event promoting hypertrophic growth and pathological remodeling of the heart. The modulatory calcineurin-interacting protein 1 (MCIP1) is an endogenous feedback inhibitor of calcineurin. We have previously shown that increasing the level of MCIP1 protein protects the heart from unrestrained activation of calcineurin, suggesting that strategies to increase MCIP1 protein in the heart may be a viable therapeutic approach. To this end we have undertaken genetic and biochemical studies to decipher mechanisms that regulate degradation of MCIP1 proteins.
Methods and Results: There are two major isoforms of MCIP1 (MCIP1.1 and MCIP1.4). MCIP1.1 levels are extremely stable with a half-life of over 8 hours in cultured cardiomyocytes. In contrast, MCIP1.4 levels are very low in an unstressed heart but increase precipitously in response to stress and calcineurin activation. Unlike MCIP1.1, the MCIP1.4 protein is extremely unstable with a half-life of less than 5 minutes. Mutation of a highly conserved serine-proline (SP) repeat common to all MCIP proteins increased the stability of either MCIP1.1 or MCIP1.4 suggesting that it may act as a phosphodegron targeting all MCIP proteins for degradation. However, because both proteins contain this motif, some other pathway must be responsible for the rapid turnover of MCIP1.4. To identify the specific E3 ligase complex that targets MCIP1.4 for rapid degradation we co-expressed MCIP1.4 with a series of dominant-negative cullins (dnCUL1–5) and found that dnCUL4A increased the stability of MCIP1.4 in a dose dependent manner but had no measurable effect on MCIP1.1. CUL4A E3 ligase complexes control the proteolysis of proteins involved in DNA-damage and cell cycle control including DDB2, CDTD1, p27, cyclin E, and c-jun. We verified that CUL4A and MCIP1.4 form a complex in vivo using co-immunoprecipitation. Furthermore we found that CUL4A-mediated degradation is independent of the SP-repeat.
Conclusions: A CUL4A E3 ligase complex important in DNA-damage and cell cycle control mediates the rapid turnover of MCIP1.4. These findings identify a new pathway through which it may be possible to regulate calcineurin activity in the heart.