The MLK-related kinase (MRK) is a novel RhoC effector that mediates lysophosphatidic acid (LPA)-stimulated tumor cell invasion.

The small GTPase RhoC is overexpressed in many invasive tumors and is essential for metastasis. Despite its high structural homology to RhoA, RhoC appears to perform functions that are different from those controlled by RhoA. The identity of the signaling components that are differentially regulated by these two GTPases is only beginning to emerge. Here, we show that the MAP3K protein MRK directly binds to the GTP-bound forms of both RhoA and RhoC in vitro. However, siRNA-mediated depletion of MRK in cells phenocopies depletion of RhoC, rather than that of RhoA. MRK depletion, like that of RhoC, inhibits LPA-stimulated cell invasion, while depletion of RhoA increases invasion. We also show that active MRK enhances LPA-stimulated invasion, further supporting a role for MRK in the regulation of invasion. Depletion of either RhoC or MRK causes sustained myosin light chain phosphorylation after LPA stimulation. In addition, activation of MRK causes a reduction in myosin light chain phosphorylation. In contrast, as expected, depletion of RhoA inhibits myosin light chain phosphorylation. We also present evidence that both RhoC and MRK are required for LPA-induced stimulation of the p38 and ERK MAP kinases. In conclusion, we have identified MRK as a novel RhoC effector that controls LPA-stimulated cell invasion at least in part by regulating myosin dynamics, ERK and p38.

Identification of RIP1 kinase as a specific cellular target of necrostatins.

Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.

Human retinol dehydrogenase 13 (RDH13) is a mitochondrial short-chain dehydrogenase/reductase with a retinaldehyde reductase activity.

Retinol dehydrogenase 13 (RDH13) is a recently identified short-chain dehydrogenase/reductase related to microsomal retinoid oxidoreductase RDH11. In this study, we examined the distribution of RDH13 in human tissues, determined its subcellular localization and characterized the substrate and cofactor specificity of purified RDH13 in order to better understand its properties. The results of this study demonstrate that RDH13 exhibits a wide tissue distribution and, by contrast with other members of the RDH11-like group of short-chain dehydrogenases/reductases, is a mitochondrial rather than a microsomal protein. Protease protection assays suggest that RDH13 is localized on the outer side of the inner mitochondrial membrane. Kinetic analysis of the purified protein shows that RDH13 is catalytically active and recognizes retinoids as substrates. Similar to the microsomal RDHs, RDH11, RDH12 and RDH14, RDH13 exhibits a much lower Km value for NADPH than for NADH and has a greater catalytic efficiency in the reductive than in the oxidative direction. The localization of RDH13 at the entrance to the mitochondrial matrix suggests that it may function to protect mitochondria against oxidative stress associated with the highly reactive retinaldehyde produced from dietary beta-carotene.

Biochemical properties of purified human retinol dehydrogenase 12 (RDH12): catalytic efficiency toward retinoids and C9 aldehydes and effects of cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) on the oxidation and reduction of retinoids.

Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber's congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits approximately 2000-fold lower K(m) values for NADP(+) and NADPH than for NAD(+) and NADH and recognizes both retinoids and lipid peroxidation products (C(9) aldehydes) as substrates. The k(cat) values of RDH12 for retinaldehydes and C(9) aldehydes are similar, but the K(m) values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (k(cat)/K(m) approximately 900 min(-)(1) microM(-)(1)), followed by 11-cis-retinal (450 min(-)(1) mM(-)(1)) and 9-cis-retinal (100 min(-)(1) mM(-)(1)). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products.