Maxim V. Dorovkov

Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes

Double-stranded RNA interference (RNAi) is an effective method for disrupting expression of specific genes in Caenorhabditis elegans and other organisms. Applications of this reverse-genetics tool, however, are somewhat restricted in nematodes because introduced dsRNA is not stably inherited. Another difficulty is that RNAi disruption of late-acting genes has been generally less consistent than that of embryonically expressed genes, perhaps because the concentration of dsRNA becomes lower as cellular division proceeds or as developmental time advances. In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference. We sought to extend the applicability of RNAi by in vivo expression of heritable inverted-repeat (IR) genes. We assayed the efficacy of in vivo-driven RNAi in three situations for which heritable, inducible RNAi would be advantageous: (i) production of large numbers of animals deficient for gene activities required for viability or reproduction; (ii) generation of large populations of phenocopy mutants for biochemical analysis; and (iii) effective gene inactivation in the nervous system. We report that heritable IR genes confer potent and specific gene inactivation for each of these applications. We suggest that a similar strategy might be used to test for dsRNA interference effects in higher organisms in which it is feasible to construct transgenic animals, but impossible to directly or transiently introduce high concentrations of dsRNA.

Phosphorylation of Annexin I by TRPM7 Channel-Kinase

TRPM7 is an unusual bifunctional molecule consisting of a TRP ion channel fused to a protein kinase domain. It has been shown that TRPM7 plays a key role in the regulation of intracellular magnesium homeostasis as well as in anoxic neuronal death. TRPM7 channel has been characterized using electrophysiological techniques; however, the function of the kinase domain is not known and endogenous substrates for the kinase have not been reported previously. Here we have identified annexin 1 as a substrate for TRPM7 kinase. Phosphorylation of annexin 1 by TRPM7 kinase is stimulated by Ca2+ and is dramatically increased in extracts from cells overexpressing TRPM7. Phosphorylation of annexin 1 by TRPM7 kinase occurs at a conserved serine residue (Ser5) located within the N-terminal amphipathic α-helix of annexin 1. The N-terminal region plays a crucial role in interaction of annexin 1 with other proteins and membranes, and therefore, phosphorylation of annexin 1 at Ser5 by TRPM7 kinase may modulate function of annexin 1.

The Channel Kinases TRPM6 and TRPM7 Are Functionally Nonredundant

TRPM7 and its closest homologue, TRPM6, are the only known fusions of an ion channel pore with a kinase domain. Deletion of TRPM7 in DT40 B-lymphocytes causes growth arrest, Mg2+ deficiency, and cell death within 24–48 h. Amazingly, in analogy to TRPM6-deficient patients who can live a normal life if provided with a Mg2+-rich diet, TRPM7-deficient DT40 B-lymphocytes show wild type cell growth if supplied with 5–10 mm Mg2+ concentrations in their extracellular medium. Here we have investigated the functional relationship between TRPM6 and TRPM7. We show that TRPM7 deficiency in DT40 cells cannot be complemented by heterologously expressed TRPM6. Nevertheless, both channels can influence each others biological activity. Our data demonstrate that TRPM6 requires TRPM7 for surface expression in HEK-293 cells and also that TRPM6 is capable of cross-phosphorylating TRPM7 as assessed using a phosphothreonine-specific antibody but not vice versa. TRPM6 and TRPM7 coexpression studies in DT40 B-cells indicate that TRPM6 can modulate TRPM7 function. In conclusion, although TRPM6 and TRPM7 are closely related and deficiency in either one of these molecules severely affects Mg2+ homeostasis regulation, TRPM6 and TRPM7 do not appear to be functionally redundant but rather two unique and essential components of vertebrate ion homeostasis regulation.

The α‐kinases TRPM6 and TRPM7, but not eEF‐2 kinase, phosphorylate the assembly domain of myosin IIA, IIB and IIC

MINT‐6700314: GNA1 (uniprotkb:Q96EK6) and GNA1 (uniprotkb:Q96EK6) bind (MI:0407) by X-ray crystallography (MI:0114)

Novel Type of Signaling Molecules: Protein Kinases Covalently Linked with Ion Channels

Recently we identified a new class of protein kinases with a novel type of catalytic domain structurally and evolutionarily unrelated to the conventional eukaryotic protein kinases. This new class, which we named alpha-kinases, is represented by eukaryotic elongation factor-2 kinase and the Dictyosteliummyosin heavy chain kinases. Here we cloned, sequenced, and analyzed the tissue distribution of five new putative mammalian α-kinases: melanoma α-kinase, kidney α-kinase, heart α-kinase, skeletal muscle α-kinase, and lymphocyte α-kinase. All five are large proteins of more than 1000 amino acids with an α-kinase catalytic domain located in the carboxyterminal part. We expressed the catalytic domain of melanoma α-kinase in Escherichia coli, and found that it autophosphorylates at threonine residues, demonstrating that it is a genuine protein kinase. Unexpectedly, we found that long aminoterminal portions of melanoma and kidney α-kinases represent new members of the TRP ion channel family, which are thought to mediate the capacitative Ca2+entry in nonexcitable mammalian cells. This suggests that melanoma and kidney α-kinases, which represent a novel type of signaling molecule, are involved in the regulation of Ca2+influx in mammalian cells.

Phosphorylation of annexin A1 by TRPM7 kinase: a switch regulating the induction of an α-helix.

TRPM7 is an unusual bifunctional protein consisting of an α-kinase domain fused to a TRP ion channel. Previously, we have identified annexin A1 as a substrate for TRPM7 kinase and found that TRPM7 phosphorylates annexin A1 at Ser5 within the N-terminal α-helix. Annexin A1 is a Ca2+-dependent membrane binding protein, which has been implicated in membrane trafficking and reorganization. The N-terminal tail of annexin A1 can interact with either membranes or S100A11 protein, and it adopts the conformation of an amphipathic α-helix upon these interactions. Moreover, the existing evidence indicates that the formation of an α-helix is essential for these interactions. Here we show that phosphorylation at Ser5 prevents the N-terminal peptide of annexin A1 from adopting an α-helical conformation in the presence of membrane-mimetic micelles as well as phospholipid vesicles. We also show that phosphorylation at Ser5 dramatically weakens the binding of the peptide to S100A11. Our data suggest that phosphorylation at Ser5 regulates the interaction of annexin A1 with membranes as well as S100A11 protein.