85, 79C87 [PubMed] [Google Scholar] 25. proteins. The processed supernatant fraction (25 l) was incubated with constitutively active ZIPK in Tos-PEG4-NH-Boc a 50-l total assay mixture containing 10 mm MgCl2, 1 g of ZIPK, 0.5 l of phosphatase inhibitor cocktail I Tos-PEG4-NH-Boc and II (Sigma), and 0.1 mm ATP for 5 min at 30 C. The reaction was stopped by adding 10 l of 6 Laemmli sample buffer and boiling for 5 min. Samples were resolved by 4C20% gradient SDS-PAGE and transferred to PVDF membrane, which was then fixed in 0.4% glutaraldehyde for 15 min at room temperature. After rinsing the membrane in phosphate-buffered saline, it was exposed to film overnight. The membrane was stained with MemCode (Pierce) to obtain an image of the transferred bands and confirm equal loading of samples. Membrane was destained and blotted with RLC antibodies (anti-cardiac RLC antibody 1:10,000, anti-smooth muscle RLC antibody Tos-PEG4-NH-Boc 1:5,000). Mouse Cardiac Myosin Purification Myosin was purified from C57 mouse ventricles using low salt precipitation steps at 4 C, similar to the original protocol by Murakami (25). Recombinant RLCs and ZIPK Purification Glutathione and shown in figures represent S.E. Significance of mRNA levels in tissues was determined by one-way analysis of variance followed by Dunnett’s multiple comparison test. Changes in ZIPK protein knockdown and RLC phosphorylation were analyzed by paired test. In all figure legends, = number of independent experiments performed. RESULTS ZIPK mRNA and Protein Are Expressed in the Heart QPCR results showed ZIPK mRNA was abundant in mouse striated muscles relative to the amount in urinary bladder smooth muscle (Fig. 1 S.E. represent the average of separate experiments using tissues from different wild-type C57 mice. Measurements were performed in triplicate (* = 0.05 and ** = 0.001 bladder), = 5 for all except skeletal muscle (= 4). and and labeled. The b6 residue that corresponds to Ser-15 is the only site phosphorylated. = 4), smooth muscle RLC (= 5), and RLC peptide (= 3) by ZIPK were measured at different substrate concentrations, and average and by ZIPK. Autoradiography of the Coomassie Blue-stained gel confirmed autophosphorylation of ZIPK and phosphorylation of cardiac RLC (Fig. 2and and and value of smooth muscle RLC was lower. value was significantly greater. ZIPK Protein Knockdown in NRCM Decreased Cardiac RLC Phosphorylation Transfection of NRCM with ZIPK siRNA resulted in significant reduction in ZIPK protein and phosphorylated cardiac RLC as compared with the negative control, with no significant changes in cardiac MLCK and smooth muscle MLCK, two other kinases that can phosphorylate cardiac RLC (Fig. 3 0.001, = 3. ZIPK Protein Overexpression in NRCM Increased Cardiac RLC Phosphorylation Overexpression of full-length ZIPK using adenoviral infection of NRCM resulted in a significant increase in phosphorylated FGF3 cardiac RLC as compared with the empty virus-infected control. Quantitation showed that ZIPK overexpression was accompanied by a 16 4% increase in cardiac RLC phosphorylation ( 0.02, = four independent experiments performed in triplicate). DISCUSSION ZIPK is a Ca2+-independent kinase with various substrates participating in diverse processes, ranging from promotion of apoptosis, to attenuation of inflammatory signaling, to regulation of contraction in smooth muscle (19, 21, 32, 33). The studies reported here assign ZIPK yet another role as a kinase for cardiac RLC in the heart. ZIPK message is abundant in cardiac muscle, in agreement with previous studies (21, 22). We show that ZIPK protein is present in both adult heart and NRCM. Cardiac RLC was identified as a primary substrate for ZIPK in an unbiased substrate search, and purified RLC had favorable biochemical substrate properties for the kinase. Importantly, ZIPK.