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.

Sulston JE et al. (1992) Nature "The C. elegans genome sequencing project: a beginning."

Wilson RK, Metzstein MM, Ainscough R, Waterston RH, Coulson AR, Craxton M, Thomas K, Dear S, Qiu L, Staden R, Berks M, Halloran N, Thierry-Mieg J, Hillier L, Sulston JE, Du Z, Durbin RM, Hawkins TL, Green P

[Nature, 1992]

The long-term goal of this project is the elucidation of the complete sequence of the Caenorhabditis elegans genome. During the first year methods have been developed and a strategy implemented that is amenable to large-scale sequencing. The three cosmids sequenced in this initial phase are surprisingly rich in genes, many of which have mammalian homologues.AD - MRC Laboratory of Molecular Biology, Cambridge, UK.FAU - Sulston, JAU - Sulston JFAU - Du, ZAU - Du ZFAU - Thomas, KAU - Thomas KFAU - Wilson, RAU - Wilson RFAU - Hillier, LAU - Hillier LFAU - Staden, RAU - Staden RFAU - Halloran, NAU - Halloran NFAU - Green, PAU - Green PFAU - Thierry-Mieg, JAU - Thierry-Mieg JFAU - Qiu, LAU - Qiu LAU - et al.LA - engPT - Journal ArticleCY - ENGLANDTA - NatureJID - 0410462RN - 0 (Cosmids)SB - IM

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.

Kalb JM et al. (1997) International C. elegans Meeting "FKH-1 IS EXPRESSED IN THE PHARYNX, RECTUM AND SOMATIC GONAD"

Kalb JM, McGhee JD, Goszczynski B, Lau KK

[International C. elegans Meeting, 1997]

C. elegans fkh-1 is a member of the fork head/HNF-3 family of transcription factors. We have raised antibodies to a portion of FKH-1 common to the products of all three major fkh-1 transcripts. These antibodies react with most (possibly all) nuclei of the embryonic pharynx and eight nuclei in the rectal region. If FKH-1 protein can be detected at all in the intestine, it is transitory: this agrees with our previous in situ hybridization results. However, reporter constructs (with 7 kb of upstream DNA) express heavily in the gut, suggesting additional control elements must exist. FKH-1 remains detectable in the pharynx and rectum at all larval stages. We have begun to examine fkh-1 expression in mutants with abnormal pharynx development. FKH-1 appears to be expressed normally in pha-1 (e2123ts) embryos but can not be detected in pha-4 (q490) embryos. fkh-1 maps near pha-4 and the expression pattern of fkh-1 matches what one might expect the expression pattern of pha-4 to be (Susan Mango, personal communication), raising the possibility that fkh-1 and pha-4 may indeed be the same gene. FKH-1 antibodies also react with nuclei in larval distal tip cells and ventral uterine cells. Interestingly, a fkh-1::lacZ reporter fusion, which is also expressed in the somatic gonad, is not expressed in the somatic gonad of lin-15 (e1763) animals. We have begun to look at fkh-1 somatic gonad expression in other mutants in vulva and uterine development.

Kalb JM et al. (1998) West Coast Worm Meeting "pha-4, a fork head/HNF-3??? homolog, participates in multiple steps of pharynx organogenesis"

Kalb JM, McGhee JD, Lau KK, Goszczynski B, Okkema PG, Fukushige T

[West Coast Worm Meeting, 1998]

Mutations in the pha-4 gene block pharynx formation at the earliest stages.1 pha-4 is the same gene as fkh-1, which was previously cloned based on its homology to the Drosophila fork head and mammalian HNF-3??? genes.2 Antibodies raised against PHA-4 show the protein is present in the nuclei of all pharyngeal precursor cells beginning at the ~100 cell stage of embryogenesis and continues to be found in pharyngeal nuclei throughout the life cycle of the worm. In addition, PHA-4 is present in rectal nuclei (as expected; pha-4 embryos also have rectal defects) as well as in nuclei of the somatic gonad. pha-4 expression patterns in embryos mutant for maternal-effect genes that control the identity of pharynx-producing blastomeres suggest the pha-4 must lie downstream of the convergence of the distinct inductive and autonomous regulatory pathways determining the pharynx primordium; this is consistent with previous genetic epistasis experiments.1 pha-4 expression was also examined in pha-1 embryos; pharynx formation is blocked in pha-1 embryos at a slightly later stage than in pha-4 embryos. pha-4 expression is wild type in pha-1 embryos if assayed in embryos younger than 9 hours, but is not detected in older embryos, suggesting that pha-1 is required for the maintenance of PHA-4 levels. We find pha-4 activates expression of two pharynx-specific genes, myo-2 and ceh-22. The promoter of the myo-2 gene, which encodes pharyngeal myosin, contains an enhancer called the C subelement, which drives reporter gene expression in all pharyngeal cells. The C subelement contains a consensus binding site for HNF-3 transcription factors; in vitro transcribed/translated PHA-4 specifically binds to the C subelement as determined using an electrophoretic mobility shift assay. Furthermore, in heat-shocked embryos producing ectopic PHA-4 from a hsp16::pha-4 transgene, we find ectopic expression of a C subelement-driven reporter gene throughout the embryo and ectopic myo-2 produced in (probably) body wall muscle. CEH-22 is a homeodomain containing protein that binds to a pharyngeal-muscle specific enhancer in the myo-2 promoter; ectopic CEH-22 also produces ectopic myo-2.3 We find ectopic PHA-4 is capable of activating a ceh-22::lacZ reporter gene. We are currently testing if PHA-4 and CEH-22 act synergistically to activate myo-2 expression. We propose the detailed properties of the five different pharyngeal cell types are specified by successive waves of transcription factors. A transcription factor first produced in one particular cell cycle would cooperate with transcription factors produced in previous cycles to produce progressive increases in regulatory specificity. We propose that PHA-4 acts as an "organ identity factor" by participating in all transcriptional events, both early and late, during pharyngeal development. 1) Mango et al. (1994). Development 120, 3019-3031. 2) Azzaria et al. (1996). Dev. Biol. 178, 289-303. 3) Okkema et al. (1997). Development 124, 3965-3973. We thank Susan Mango for providing strains and discussing unpublished results.

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.

Kalb JM et al. (1998) Development "pha-4 is Ce-fkh-1, a fork head/HNF-3alpha,beta,gamma homolog that functions in organogenesis of the C. elegans pharynx."

McGhee JD, Lau KK, Moons D, Kalb JM, Fukushige T, Goszczynski B, Okkema PG

[Development, 1998]

The C. elegans Ce-fkh-1 gene has been cloned on the basis of its sequence similarity to the winged-helix DNA binding domain of the Drosophila fork head and mammalian HNF-3alpha,beta,gamma genes, and mutations in the zygotically active pha-4 gene have been shown to block formation of the pharynx (and rectum) at an early stage in embryogenesis. In the present paper, we show that Ce-fkh-1 and pha-4 are the same gene. We show that PHA-4 protein is present in nuclei of essentially all pharyngeal cells, of all five cell types. PHA-4 protein first appears close to the point at which a cell lineage will produce only pharyngeal cells, independently of cell type. We show that PHA-4 binds directly to a 'pan-pharyngeal enhancer element' previously identified in the promoter of the pharyngeal myosin myo-2 gene; in transgenic embryos, ectopic PHA-4 activates ectopic myo-2 expression. We also show that ectopic PHA-4 can activate ectopic expression of the ceh-22 gene, a pharyngeal-specific NK-2-type homeodomain protein previously shown to bind a muscle-specific enhancer near the PHA-4 binding site in the myo-2 promoter. We propose that it is the combination of pha-4 and regulatory molecules such as ceh-22 that produces the specific gene expression patterns during pharynx development. Overall, pha-4 can be described as an 'organ identity factor', completely necessary for organ formation, present in all cells of the organ from the earliest stages, capable of integrating upstream developmental pathways (in this case, the two distinct pathways that produce the anterior and posterior pharynx) and participating directly in the transcriptional regulation of organ specific genes. Finally, we note that the distribution of PHA-4 protein in C. elegans embryos is remarkably similar to the distribution of the fork head protein in Drosophila embryos: high levels in the foregut/pharynx and hindgut/rectum; low levels in the gut proper. Moreover, we show that pha-4 expression in the C. elegans gut is regulated by elt-2, a C. elegans gut-specific GATA-factor and possible homolog of the Drosophila gene serpent, which influences fork head expression in the fly gut. Overall, our results provide evidence for a highly conserved pathway regulating formation of the digestive tract in all (triploblastic) metazoa.