Cardiac myosin is a substrate for zipper-interacting protein kinase (ZIPK)

Abstract

Zipper-interacting protein kinase (ZIPK) 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 and protein are abundant in heart tissue and isolated ventricular neonatal rat cardiac myocytes. An unbiased substrate search performed with purified ZIPK on heart homogenates led to the discovery of a prominent 20-kDa protein substrate identified as RLC of ventricular myosin. Biochemical analyses showed ZIPK phosphorylated cardiac RLC at Ser-15 with a V(max) value 2-fold greater than the value for smooth/nonmuscle RLC; cardiac RLC is a favorable biochemical substrate. Knockdown of ZIPK in cardiac myocytes by small interfering RNA significantly decreased the extent of RLC Ser-15 phosphorylation. Thus, ZIPK may act as a cardiac RLC kinase and thereby affect contractility.

FIGURE 1.

Expression of ZIPK mRNA and protein in tissues. A, quantitative comparison of ZIPK mRNA in mouse tissues by QPCR. Relative quantities are shown as ratios of amounts found in each tissue relative to the amount in cerebellum, after normalization to an internal control, mouse cyclophilin B. Bars ± S.E. represent the average of separate experiments using tissues from different wild-type C57 mice. Measurements were performed in triplicate (* = p < 0.05 and ** = p < 0.001 versus bladder), n = 5 for all except skeletal muscle (SK Muscle, n = 4). B, ZIPK protein expression in mouse tissues. Total tissue proteins (∼10 μg/lane) were separated by SDS-PAGE and ZIPK protein (▶) shown by Western blotting. C, Coomassie Blue-stained gradient gel shows equal loading of total protein. Mass markers (kDa) are indicated.

FIGURE 2.

Identification of cardiac RLC as a substrate for ZIPK. A, ZIPK substrate search in mouse bladder and heart tissue homogenates incubated with (+) or without (−) purified ZIPK. Upper panel, assay samples were subjected to electrophoresis on a 4–20% gradient gel and transferred to PVDF membrane. Total proteins on membrane are shown by MemCode PVDF stain (Pierce), and mass of markers (kDa) is indicated. Middle panel, proteins phosphorylated by ZIPK were visualized by autoradiography of membrane shown in the upper panel. Lower panel, RLC band presence in lanes with (+) or without (−) added kinase was confirmed subsequently by Western blot analysis of the same membrane used in the upper and middle panels. Specific antibodies used to detect cardiac (α-cRLC) and smooth muscle RLC (α-smRLC) are indicated. B, RLC in cardiac myosin is a substrate for ZIPK. Purified ZIPK (300 ng) was incubated with 0.5–4.25 μm purified myosin (molecular mass 520 kDa) for 3 min in 50 μl of assay buffer. Top panel, Gelcode Blue Safe (Pierce)-stained 4–20% gradient gel of assay samples resolved by SDS-PAGE. Mass markers (kDa) are indicated. Dominant proteins include myosin heavy chain (200 kDa), actin (45 kDa), myosin light chain 1 (24 kDa), and RLC (20 kDa). Bottom panel, autoradiograph of gel with + and − symbols depicting the presence or absence of kinase, respectively. C, purified recombinant human smooth muscle RLC and human cardiac RLC (1 μg each) were phosphorylated with ZIPK for the indicated minutes and resolved by urea/glycerol-PAGE. Non- (P0), mono- (P1), and di- (P2) phosphorylated forms are indicated. D, phosphorylated cardiac RLC was subjected to phosphorylation site identification by LC/MS/MS where greater than 90% of protein was analyzed, and the peptide shown contained the only phosphorylated residue. Peaks that correspond to specific b residues are darkened and labeled. The b6 residue that corresponds to Ser-15 is the only site phosphorylated. amu, atomic mass units. E, the rates of phosphorylation of cardiac RLC (n = 4), smooth muscle RLC (n = 5), and RLC peptide (n = 3) by ZIPK were measured at different substrate concentrations, and average K_m and V_max values with S.E. were determined from independent nonlinear regression analysis using GraphPad Prism software. F, Lineweaver-Burk plots of the average values are shown.

FIGURE 3.

Effect of ZIPK knockdown on cardiac RLC phosphorylation in intact myocytes. A, NRCM were transfected with 10 nm of negative control or ZIPK-targeted siRNA (siZIPK, ABI siRNA sequence number s64814) and harvested 72 h after transfection for Western blot analysis with antibodies against ZIPK, GAPDH (load control), phospho-cardiac RLC (RLC-P), smooth muscle myosin light chain kinase (smMLCK), and cardiac myosin light chain kinase (cMLCK). B, densitometric quantitation of samples treated with siRNA for amounts of ZIPK (open) and RLC-P (hatched) normalized to negative control-siRNA transfected sample median values (filled). **, p < 0.001, n = 3.