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.

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

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, Mg 2؉ 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 Mg 2؉ -rich diet, TRPM7-deficient DT40 B-lymphocytes show wild type cell growth if supplied with 5-10 mM Mg 2؉ 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 other's 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 Mg 2؉ 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 TRPM proteins are a recently identified subgroup of ion channels comprising eight members. They belong to the growing transient receptor potential (TRP) 2 superfamily of cationic channels (1-5). TRPM6 (ChaK2) and TRPM7 (TRP-PLIK, ChaK1, and LTRPC7) have been shown to be involved in regulating Mg 2ϩ homeostasis in vertebrates (6 -10). Both channels show the unique functional duality of being ion channels and kinases, since they include an active Thr/Ser kinase at their C terminus, which belongs to the atypical family of eukaryotic ␣-kinases (11, 12). Genomic studies revealed the existence of six ␣-kinases in mammals, including TRPM6 and TRPM7. These kinases show no sequence homology to conventional protein kinases. Structural analyses of the TRPM7 kinase domain revealed similarities to the fold of protein kinase A family members (13). TRPM6 was first identified by Ryazanova et al. (14), who screened for homologues of eukaryotic elongation factor 2 kinase, as well as by two other groups who showed that mutations in the TRPM6 gene loci are linked to an autosomal recessive form of familiar hypomagnesemia with secondary hypocalcemia (HSH), by performing a candidate gene approach (15,16 (19). In contrast, another group using imaging approaches reports that TRPM6 is only present at the cell surface when associating with TRPM7, leading to increased Mn 2ϩ entry as assessed by fura-2 fluorescence quenching measurements (20). TRPM7 was originally cloned by three different research groups; Clapham and co-workers (21) identified the TRPM7 kinase domain in a yeast two-hybrid screen using the C2 domain of phospholipase C␤ as a bait, and Ryazanova et al. (14) cloned TRPM7 as already de...

Guamanian amyotrophic lateral sclerosis (ALS-G) and parkinsonism dementia (PD-G) have been epidemiologically linked to an environment severely deficient in calcium (Ca 2؉ ) and magnesium (Mg 2؉ ). Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein containing both channel and kinase domains that has been proposed to be involved in the homeostatic regulation of intracellular Ca 2؉ , Mg 2؉ , and trace metal ion concentration. There is evidence that TRPM7 is constitutively active and that the number of available channels is dependent on intracellular free Mg 2؉ levels. We found a TRPM7 variant in a subset of ALS-G and PD-G patients that produces a protein with a missense mutation, T1482I. Recombinant T1482I TRPM7 exhibits the same kinase catalytic activity as WT TRPM7. However, heterologously expressed T1482I TRPM7 produces functional channels that show an increased sensitivity to inhibition by intracellular Mg 2؉ . Because the incidence of ALS-G and PD-G has been associated with prolonged exposure to an environment severely deficient in Ca 2؉ and Mg 2؉ , we propose that this variant TRPM7 allele confers a susceptibility genotype in such an environment. This study represents an initial attempt to address the important issue of gene-environment interactions in the etiology of these diseases.amyotrophic lateral sclerosis ͉ calcium ͉ gene-environment interactions ͉ phosphorylation ͉ parkinsonism dementia