Yan KK et al. (2014) Genome Biol "OrthoClust: an orthology-based network framework for clustering data across multiple species."

Cheng C, Wang D, Yan KK, Gerstein M, Zheng H, Rozowsky J

[Genome Biol, 2014]

Increasingly, high-dimensional genomics data are becoming available for many organisms.Here, we develop OrthoClust for simultaneously clustering data across multiple species. OrthoClust is a computational framework that integrates the co-association networks of individual species by utilizing the orthology relationships of genes between species. It outputs optimized modules that are fundamentally cross-species, which can either be conserved or species-specific. We demonstrate the application of OrthoClust using the RNA-Seq expression profiles of Caenorhabditis elegans and Drosophila melanogaster from the modENCODE consortium. A potential application of cross-species modules is to infer putative analogous functions of uncharacterized elements like non-coding RNAs based on guilt-by-association.

Tokumitsu H et al. (1999) J Biol Chem "Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain."

Takahashi N, Yano S, Eto K, Muramatsu M, Tokumitsu H, Soderling TR

[J Biol Chem, 1999]

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.

Y Kimura et al. (1999) International C. elegans Meeting "Ca2+/CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans"

K Eto, H Tokumitsu, Y Kimura, M Muramatsu

[International C. elegans Meeting, 1999]

In mammals, Ca 2+ /CaM protein kinases (CaM-K) is thought to be important for Ca 2+ -dependent signal transduction. This cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In the present study, we have identified CaM-KK and CaM-KI orthologue of C. elegans (named Ce CaM-KK and Ce CaM-KI, respectively) and analyzed their biochemical activities. Ce CaM-KK has two unusual features in the catalytic domain which is conserved between species, that is, a lack of acidic residues important for substrate recognition and an Arg-Pro rich insert (RP-domain) which is essential for the recognition and activation of CaM-KIV and CaM-KI. Indeed, Ce CaM-KK activated mammalian CaM-KIV in vitro . Ce CaM-KI is highly homologous to mammalian CaM-KI and is activated by Ce CaM-KK through phosphorylation of Thr179 in a Ca 2+ /CaM-dependent manner. Truncation at residue 295 in CeCaM-KI generates a constitutively active enzyme, suggesting that COOH-terminal at residue 295 contains autoinhibitory and CaM-binding domains. Unlike mammalian CaM-KI, Ce CaM-KI mainly localized in the nucleus of transfected mammalian cells by the virtue of the NH 2 -terminal six residues containing a functional nuclear localization signal. Taken together, these results suggest that CaM-KK/CaM-KI cascade in C. elegans is conserved and operated both in vitro and intact cells. Current research aims to the physiological functions of Ce CaM-KK and Ce CaM-KI cascade in vivo . Analyses for the expression patterns and isolation of mutant worms for both genes are underway.

Eto K et al. (1999) J Biol Chem "Ca(2+)/Calmodulin-dependent protein kinase cascade in Caenorhabditis elegans. Implication in transcriptional activation."

Kimura Y, Eto K, Tokumitsu H, Arai K, Takahashi N, Muramatsu M, Masuho Y

[J Biol Chem, 1999]

We have recently demonstrated that Caenorhabditis elegans Ca(2+)/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr(179) in a Ca(2+)/CaM-dependent manner, resulting in a 30-fold decrease in the K(m) of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH(2)-terminal six residues ((2)PLFKRR(7)) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca(2+)-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca(2+)-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

Yoshishige Kimura et al. (2001) International C. elegans Meeting "CaM-K signal cascade of C. elegans functionsin vivo"

Hiroshi Tokumitsu, Yoshishige Kimura, Masatoshi Hagiwawa

[International C. elegans Meeting, 2001]

Ca 2+ /CaM protein kinases (CaM-K) have significant roles for Ca 2+ -dependent signal transduction. CaM-K-mediated signal cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). A transcription factor cAMP-responsive element binding protein (CREB) is known to be activated by phosphorylation with downstream CaM-KIV. In this study, we identified C. elegans orthologues of mammalian CaM-KK (ckk-1 ), CaM-KI ( cmk-1 ) and CREB, and found that CaM-KK-CaM-KI-CREB cascade is conserved and operated both in vitro and in transfected cells. Furthermore, we investigated the physiological functions through this signal cascade by genetical approaches. The distribution of activated CREB was analyzed by using the transgenic worms that carries 4XCRE::gfp fusion gene. GFP fluorescence cannot be seen in normal development, however, transgenic worms carrying constitutive-active form of cmk-1 induces GFP-expression in small number of sensory neurons in head and tail. On the other hand, wild type and mutant cmk-1 at T179A did not show any CREB activation, suggesting the requirement of Ca 2+ mobilization and the upstream kinase which was identified as CaM-KK . In addition, CREB-deficeient worms show no GFP fluorescence reasonably. Interestingly, when the worms were maintained in starved condition, the number of GFP-positive neurons increased and the intensity of GFP fluorescence was dramatically enhanced. These results suggest that CaM-KK-CaM-KI-CREB cascade of C. elegans is conserved in vivo in the some sensory neurons and activated by the circumstantial stimuli.

Bock Axelsen J et al. (2007) Biol Direct "Parameters of proteome evolution from histograms of amino-acid sequence identities of paralogous proteins."

Yan KK, Bock Axelsen J, Maslov S

[Biol Direct, 2007]

ABSTRACT: BACKGROUND: The evolution of the full repertoire of proteins encoded in a given genome is mostly driven by gene duplications, deletions, and sequence modifications of existing proteins. Indirect information about relative rates and other intrinsic parameters of these three basic processes is contained in the proteome-wide distribution of sequence identities of pairs of paralogous proteins. RESULTS: We introduce a simple mathematical framework based on a stochastic birth-and-death model that allows one to extract some of this information and apply it to the set of all pairs of paralogous proteins in H. pylori, E. coli, S. cerevisiae, C. elegans, D. melanogaster, and H. sapiens. It was found that the histogram of sequence identities $p$ generated by an all-to-all alignment of all protein sequences encoded in a genome is well fitted with a power-law form ~p;(-gamma) with the value of the exponent gamma around 4 for the majority of organisms used in this study. This implies that the intra-protein variability of substitution rates is best described by the Gamma-distribution with the exponent alpha ~ 0.33. Different features of the shape of such histograms allow us to quantify the ratio between the genome-wide average deletion/duplication rates and the amino-acid substitution rate. CONCLUSIONS: We separately measure the short-term (;;raw'''') duplication and deletion rates r*_dup, r*_del which include gene copies that will be removed soon after the duplication event and their dramatically reduced long-term counterparts r_dup, r_del. High deletion rate among recently duplicated proteins is consistent with a scenario in which they didn''t have enough time to significantly change their functional roles and thus are to a large degree disposable. Systematic trends of each of the four duplication/deletion rates with the total number of genes in the genome were analyzed. All but the deletion rate of recent duplicates r*_del were shown to systematically increase with N_genes. Abnormally flat shapes of sequence identity histograms observed for yeast and human are consistent with lineages leading to these organisms undergoing one or more whole-genome duplications. This interpretation is corroborated by our analysis of the genome of {Paramecium tetraurelia where the p;(-4) profile of the histogram is gradually restored by the successive removal of paralogs generated in its four known whole-genome duplication events. This article was reviewed by Eugene Koonin, Yuri Wolf (nominated by Eugene Koonin), David Krakauer, and Eugene Shakhnovich.

Maslov S et al. (2004) BMC Evol Biol "Upstream plasticity and downstream robustness in evolution of molecular networks."

Yan KK, Sneppen K, Eriksen KA, Maslov S

[BMC Evol Biol, 2004]

BACKGROUND: Gene duplication followed by the functional divergence of the resulting pair of paralogous proteins is a major force shaping molecular networks in living organisms. Recent species-wide data for protein-protein interactions and transcriptional regulations allow us to assess the effect of gene duplication on robustness and plasticity of these molecular networks. RESULTS: We demonstrate that the transcriptional regulation of duplicated genes in baker's yeast Saccharomyces cerevisiae diverges fast so that on average they lose 3% of common transcription factors for every 1% divergence of their amino acid sequences. The set of protein-protein interaction partners of their protein products changes at a slower rate exhibiting a broad plateau for amino acid sequence similarity above 70%. The stability of functional roles of duplicated genes at such relatively low sequence similarity is further corroborated by their ability to substitute for each other in single gene knockout experiments in yeast and RNAi experiments in a nematode worm Caenorhabditis elegans. We also quantified the divergence rate of physical interaction neighborhoods of paralogous proteins in a bacterium Helicobacter pylori and a fly Drosophila melanogaster. However, in the absence of system-wide data on transcription factors' binding in these organisms we could not compare this rate to that of transcriptional regulation of duplicated genes. CONCLUSIONS: For all molecular networks studied in this work we found that even the most distantly related paralogous proteins with amino acid sequence identities around 20% on average have more similar positions within a network than a randomly selected pair of proteins. For yeast we also found that the upstream regulation of genes evolves more rapidly than downstream functions of their protein products. This is in accordance with a view which puts regulatory changes as one of the main driving forces of the evolution. In this context a very important open question is to what extent our results obtained for homologous genes within a single species (paralogs) carries

Pop, C et al. (2005) Cell "The nematode death machine in 3D."

Pop, C, Salvesen, GS

[Cell, 2005]

Regulated apoptosis is part of the development of the nematode Caenorhabditis elegans. In a recent paper in Nature, Yan et al. (2005) describe the in vitro reconstitution of the core components of the worm apoptotic pathway. Based on a structural analysis of the complex between the death activator CED-4 and the antiapoptotic protein CED-9, the authors explain the regulation of activity of CED-4. Intriguingly, CED-4 comprises a AAA+ type ATPase domain yet does not seem to need ATP hydrolysis for activity.

Kimura Y et al. (2000) Japanese Worm Meeting "CaM-K-mediated signal cascade is conserved in some neurons of C. elegans in vivo"

Kimura Y, Tokumitsu H, Eto K

[Japanese Worm Meeting, 2000]

Ca2+/CaM protein kinases (CaM-K) is kown to have important roles for Ca2+-dependent signal transduction. CaM-K-mediated signal cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In addition, a transcription factor cAMP-responsive element binding protein (CREB) is activated by phosphorylation with downstream CaM-KIV. We characterized C. elegans orthologues of CaM-KK (ckk-1) and CaM-KI (cmk-1) and found that CaM-KK-CaM-KI-CREB cascade is conserved and operated both in vitro and in transfected cells. CMK-1is highly homologous to mammalian CaM-KI and is activated by CKK-1 through phosphorylation of Thr179 in a Ca2+ /CaM-dependent manner. An activated CMK-1 can phosphorylate CREB protein and activate its transcription activity. We constructed ckk::gfp and cmk::gfp fusion genes and analyzed the expression patterns of both genes. A ckk-1::gfp reporter was expressed small number of head neruons after L3 stage. On the other hand, cmk-1::gfpwas seen in the most of the neurons from embryo to adult. To see the localization of activated CREB in vivo, we made a reporter worm that conains 4XCRE::gfp fusion gene. GFP expression cannot be seen in normal development but the expression in small number of neurons in head and tail can be induced by cAMP soaking. This induction can also occur by the expression of constitutively active form of CMK-1. Interestingly, this expression can be seen only after L3 stage and the expression was decreased or disappeared in adult. We also found that the constitutively active form of CMK-1 induced the malformaion of vulva and Egl phenotype. These results suggest that CaM-KI-CREB cascade of C. elegans is also conserved in vivo but it works only in small number of neurons.

Yoshishige Kimura et al. (2002) Mid-west Worm Meeting "A CaM-kinase cascade activates CRE-mediated transcription in neurons of Caenorhabditis elegans"

Keiko Gengyo-Ando, Hiroshi Tokumitsu, Masatoshi Hagiwara, Shohei MItani, Ryoji Kobayashi, Masa-aki Muramatsu, Ethan E Corcoran, Koh Eto, Jonathan H Freedman, Anthony R Means, Yoshishige Kimura

[Mid-west Worm Meeting, 2002]

Calcium (Ca2+ ) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+ /CaM-dependent protein kinases I and IV (CaM-Ks) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP-response element binding protein (CREB) has been proposed as a target for a CaM-K cascade involving not only CaM-KI or IV, but also an upstream kinase kinase that is also CaM-regulated (CaM-KK). We previously identified C.elegans orthologue of CaM-KI(cmk-1), CaM-KK(ckk-1) and CREB (crh-1) which constituted a signaling cadcade analogous to mammalian system. In order to examine the function of this signaling cascade in vivo, we generated transgenic worms carrying pCRE::GFP monitoring vector. Whereas, worms carrying only this vector showed weak fluorescence in small number of neurons, transgenic worms expressing either constitutively-active or Ca2+ -independent form of cmk-1 showed enhanced CRE-GFP expression. Furthermore, crh-1-deficeient worms exhibited extremely reduced GFP fluorescence, suggesting that crh-1 is a main activator of CRE-genes in C. elegans. Introduction Ca2+ -independent form of cmk-1 with T179A mutation did not induce the CRE::GFP fluorescence and also ckk-1-deficient worm expressing Ca2+ -independent form of cmk-1 did not show the enhanced GFP-expression, indicating that ckk-1 is absolutely required for cmk-1-mediated transcriptional activation through phosphorylation of crh-1. These results indicate that CaM-KK/CaM-K/CREB cascade is conserved and operated in the several neurons of C. elegans and plays an important role for regulation of Ca2+ -dependent gene expression in this organism.