Zipper-Interacting Protein Kinase Phosphorylates Cardiac Myosin

Zipper-interacting protein kinase (ZIPK) or DAPK3, is a member of the death-associated-protein kinase family associated with apoptosis in nonmuscle cells where it phosphorylates myosin regulatory light chain (RLC) to promote membrane blebbing. ZIPK mRNA is abundant in muscles, prompting our investigation of its role in the heart. A substrate search on mouse heart homogenates led to the discovery that cardiac RLC is a substrate for ZIPK. Enzyme kinetic studies revealed that both smooth and cardiac RLCs were good substrates, with a Vmax value two-fold greater for cardiac as compared to smooth/nonmuscle RLC. Moreover, ZIPK phosphorylated cardiac RLC at Ser15, the site responsible for modulating Ca2+ sensitivity of myofibrillar contraction. Knockdown of ZIPK in isolated neonatal cardiac myocytes by siRNA decreased the extent of RLC Ser15 phosphorylation. Localization studies using adenovirus- mediated overexpression of GFP-ZIPK in neonatal and adult rat cardiac myocytes showed it is in the nucleus, but also the cytoplasm where it may affect RLC phosphorylation. In adult cardiac myocytes, ZIPK appears to associate with myofilaments. ZIPK gene ablation specifically in mouse heart (ZKflox/Nkx2.5Cre) did not affect basal RLC phosphorylation, nor did it induce apparent pathological responses (assessed by histological analysis and heart weight measurements). Based on what is known about the DAPK family, we hypothesize that ZIPK function in the heart may be stress or death signal-dependent. Effects of physiological and pathophysiological stresses on hearts from ZKflox/Nkx2.5Cre mice are currently being investigated. Supported by NIH NHLBI (J. T. S.) and AHA Postdoctoral Fellowship (A.N.C.).