SPEG Controls Calcium Re-Uptake into the Sarcoplasmic Reticulum Through Regulating SERCA2a by Its Second Kinase-Domain

Rationale: Striated muscle preferentially expressed protein kinase (SPEG) has two kinase-domains and is critical for cardiac development and function. However, it is not clear how these two kinase-domains function to maintain cardiac performance.

Objective: To determine the molecular functions of the two kinase-domains of SPEG. M Methods and Results: A proteomics approach identified sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a (SERCA2a) as a protein interacting with the second kinase-domain but not the first kinase-domain of SPEG. Furthermore, the second kinase-domain of SPEG could phosphorylate Thr484 on SERCA2a, promote its oligomerization and increase calcium re-uptake into the sarcoplasmic/endoplasmic reticulum in culture cells and primary neonatal rat cardiomyocytes. Phosphorylation of SERCA2a by SPEG enhanced its calcium transporting activity without affecting its ATPase activity. Depletion of Speg in neonatal rat cardiomyocytes inhibited SERCA2a Thr484 phosphorylation and SR calcium re-uptake. Moreover, over-expression of SERCA2aThr484Ala mutant protein also slowed SR calcium re-uptake in neonatal rat cardiomyocytes. In contrast, domain-mapping and phosphorylation analysis revealed that the first kinase-domain of SPEG interacted and phosphorylated its recently-identified substrate junctophilin-2 (JPH2). An inducible heart-specific Speg knockout mouse model was generated to further study this SPEG-SERCA2a signal nexus in vivo. Inducible deletion of Speg decreased SERCA2a Thr484 phosphorylation and its oliogomerization in the heart. Importantly, inducible deletion of Speg inhibited SERCA2a calcium transporting activity and impaired calcium re-uptake into the SR in cardiomyocytes, which preceded morphological and functional alterations of the heart and eventually led to heart failure in adult mice.

Conclusions: Our data demonstrate that the two kinase-domains of SPEG may play distinct roles to regulate cardiac function. The second kinase-domain of SPEG is a critical regulator for SERCA2a. Our findings suggest that SPEG may serve as a new target to modulate SERCA2a activation for treatment of heart diseases with impaired calcium homeostasis.