Phosphorylation-Regulated Dynamic Phase Separation of HIP-55 Protects Against Heart Failure.

BACKGROUND: Heart failure (HF), which is the terminal stage of many cardiovascular diseases, is associated with low survival rates and a severe financial burden. The mechanisms, especially the molecular mechanism combined with new theories, underlying the pathogenesis of HF remain elusive. We demonstrate that phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 (hematopoietic progenitor kinase 1-interacting protein of 55 kDa) protects against HF. METHODS: Fluorescence recovery after photobleaching assay, differential interference contrast analysis, pull-down assay, immunofluorescence, and immunohistochemical analysis were used to investigate the liquid-liquid phase separation capacity of HIP-55 and its dynamic regulation in vivo and in vitro. Mice with genetic deletion of HIP-55 and mice with cardiac-specific overexpression of HIP-55 were used to examine the role of HIP-55 on ß-adrenergic receptor hyperactivation-induced HF. Mutation analysis and mice with specific phospho-resistant site mutagenesis were used to identify the role of phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 in HF. RESULTS: Genetic deletion of HIP-55 aggravated HF, whereas cardiac-specific overexpression of HIP-55 significantly alleviated HF in vivo. HIP-55 possesses a strong capacity for phase separation. Phase separation of HIP-55 is dynamically regulated by AKT-mediated phosphorylation at S269 and T291 sites, failure of which leads to impairment of HIP-55 dynamic phase separation by formation of abnormal aggregation. Prolonged sympathetic hyperactivation stress induced decreased phosphorylation of HIP-55 S269 and T291, dysregulated phase separation, and subsequent aggregate formation of HIP55. Moreover, we demonstrated the important role of dynamic phase separation of HIP-55 in inhibiting hyperactivation of the ß-adrenergic receptor-mediated P38/MAPK (mitogen-activated protein kinase) signaling pathway. A phosphorylation-deficient HIP-55 mutation, which undergoes massive phase separation and forms insoluble aggregates, loses the protective activity against HF. CONCLUSIONS: Our work reveals that the phosphorylation-regulated dynamic phase separation of HIP-55 protects against sympathetic/adrenergic system-mediated heart failure.

Src mediates ß-adrenergic receptor induced YAP tyrosine phosphorylation.

The Hippo pathway is a newly identified pathway and evolutionarily conserved from flies to humans mainly regulating cell proliferation. Transcriptional co-activator Yes-associated protein (YAP) functions as a major downstream effector and key node of the Hippo pathway. Phosphorylation of YAP is critical to regulate YAP activity and its corresponding functions. ß-adrenergic receptor (ß-AR), a typical G protein coupled receptor (GPCR), mediates proliferation in various cell types and regulates multiple physical and pathological processes. However, the role of ß-AR in regulating YAP remains elusive. Here, we report that ß-AR can obviously stimulate YAP tyrosine phosphorylation. The mechanism is that ß-AR stimulation results in tyrosine kinase Src activation and Src phosphorylates YAP tyrosine at Y357. Further studies demonstrate that inhibition of Src kinase activity can obviously alleviate ß-AR induced YAP tyrosine phosphorylation and cell proliferation. We conclude that ß-AR can induce YAP tyrosine phosphorylation and also establish the Src/YAP pathway as a critical signaling branch downstream of GPCR.