The studies reported here assign ZIPK yet another role as a kinase for cardiac RLC in the heart

The studies reported here assign ZIPK yet another role as a kinase for cardiac RLC in the heart. 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. Keywords:Enzyme Kinetics, Heart, Myosin, Protein Kinases, Protein Phosphorylation, ZIPK, Myosin Light Chain == Introduction == Myosin is the molecular motor in the thick filament of the sarcomere that binds to actin thin filaments to initiate myocyte shortening and force development when Ca2+binds to troponin in the thin filament (1). Phosphorylation of sarcomeric proteins modulates this Ca2+-dependent contraction process, including phosphorylation of Ser-15 in cardiac myosin RLC3(24). The basal phosphorylation of RLC (4050%) in beating hearts is maintained by slow rates of phosphorylation and dephosphorylation and increases the Ca2+sensitivity of contraction (2,5). This modulatory role on contractile function is conferred through structural alterations that affect myosin cross bridges binding to actin thin filaments (610). Studies with transgenic and knock-out mice have shown that although RLC phosphorylation is not necessary for striated muscle development, it is necessary for optimal contractile performance (1013). Cardiac abnormalities arise from age-dependent increased load and depressed ventricular ejection in transgenic mice overexpressing a nonphosphorylatable cardiac RLC, and physiological and pathophysiological hypertrophic responses are attenuated in hearts with increased RLC phosphorylation, confirming the importance of phosphorylation in long term modulation of cardiac function (11,14,15). In addition, RLC phosphorylation appears to be involved in positive inotropic responses to long term -adrenergic stimulation (15). Although the importance of cardiac RLC phosphorylation is clear, the identity of its protein kinase(s) remains is not determined. The unique amino acid TCS PIM-1 4a (SMI-4a) sequence adjacent to the phosphorylation site makes cardiac RLC a distinct substrate from smooth/nonmuscle RLCs (16). Smooth muscle myosin light chain kinase (MLCK) is not a good kinase for cardiac RLC, and skeletal MLCK is not significantly present in cardiac myocytes (10,17,18). A novel cardiac muscle-specific myosin light chain kinase (cardiac MLCK) was recently identified (17,18). Its knockdown in NRCM did not completely ablate cardiac RLC phosphorylation, which raises the possibility of an alternate RLC kinase. ZIPK, also known as DAPK3, is a highly conserved Ca2+-independent serine/threonine protein kinase belonging to the death-associated protein kinase (DAPK) family (19). ZIPK is comprised of an N-terminal kinase domain (amino acids 13275) and a C-terminal domain of unknown function that contains a leucine TCS PIM-1 4a (SMI-4a) zipper and possibly an autoinhibitory loop (19,20). Northern blots of various tissues show that ZIPK is ubiquitously expressed, including in heart (21,22). Previous investigations have focused on its respective roles in apoptosis in nonmuscle cells and contraction in smooth muscle, where studies on the latter were stimulated TCS PIM-1 4a (SMI-4a) by identification of smooth muscle RLC and the regulatory subunit of smooth muscle myosin light chain phosphatase (MYPT1) as substrates for ZIPK (21,23,24). Therefore, we initiated studies to identify protein substrates for ZIPK in heart. We found that cardiac RLC is phosphorylated by ZIPK at Ser-15in vitroandin vivo, implicating a role for this TCS PIM-1 4a (SMI-4a) protein kinase in modulating cardiac contractility. == EXPERIMENTAL PROCEDURES == == == == == == Quantitative Real-time PCR == mRNA was prepared from C57 mouse tissues or NRCM by TRIzol and phenol-chloroform (Invitrogen) extraction, and cDNA was prepared using SuperScript III for QPCR (Invitrogen). Template cDNA (50 ng) was used in each 10-l reaction performed in triplicate with SYBR GreenER (Invitrogen) Vegfa in an ABI PRISM 7200 sequence detector (Applied Biosystems). Mouse cyclophilin B was used as an internal control. Relative tissue ZIPK TCS PIM-1 4a (SMI-4a) mRNA quantities were normalized to that measured from mouse urinary bladder. For knockdown, ZIPK mRNA amount was normalized to the amount obtained as negative control with scrambled siRNA-transfected NRCM. There were no differences in ZIPK mRNA amounts between untreated and negative control for NRCM. == Substrate Search == Wild-type C57 mouse tissues were homogenized in 30 weight to volume ratio (100 mmMOPS, pH 7.4, 300 mmNaCl, 10 mmEGTA, 1 mmdithiothreitol, 1% Triton X-100, protease inhibitor cocktail (Sigma)) and cleared by centrifugation (10,000 gfor 5 min at 4 C). The supernatant fraction was desalted using Sephadex G-25 mini-columns (PD-10, from GE Healthcare) to remove endogenous ATP and preincubated at 30 C for 20 min to dephosphorylate proteins. The processed supernatant fraction (25 l) was incubated with constitutively active ZIPK in a 50-l total assay mixture containing 10 mmMgCl2, 1 g of ZIPK, 0.5 l of phosphatase inhibitor cocktail I and II (Sigma), and 0.1 mmATP for 5 min at 30 C. The reaction was stopped by adding 10 l of 6 Laemmli sample buffer and.